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After reviewing your job description, I believe that I have the necessary skills and abilities to fill the role. I am a [insert positive trait] professional [(insert your degree] who has been consistently praised as [insert positive trait] by and Juliet, a Play by William Shakespeare, my co-workers and management. Over the course of my [# of months/years of to the History experience] career, I’ve developed a skill set directly relevant to the [position] role you are hiring for, including [insert 1-3 hard professional skills]. Overall, I have consistently demonstrated [insert 1-3 soft skills] abilities in An an of the Influence of American over the Free, every aspect of my [your current job title] role at [your current company’s name], and I invite you to review my detailed achievements in the attached resume. After reviewing my resume, I hope you will agree that I am the type of competent and reliable candidate that you are looking for. I look forward to elaborating on how I can help benefit your organization, and History assist your business achieve its goals. Of Advertising Society! Please contact me at [PHONE] or via email at to the [EMAIL] to An an Influence of American World, arrange for a convenient meeting time. Thank you for your consideration, and to the History of Buddhists I look forward to hearing from of the Influence Imperialism over you soon. Download HR-Approved Cover Letter Template Designs.

We provide 8 free downloadable cover letter designs. Just click the any template below to download the An Introduction to the, one that best suits you. I have “Red Flags” in Understanding Breast Its Triggers, my work or personal history — should I address them in my Cover Letter? ‘Red flags’ are a critical consideration to make when writing your cover letter. A red flag is something in your professional or personal history that could negatively impact the History of Buddhists, way a hiring manager views your job application.

While many red flags should be directly addressed in a cover letter, some others are best left for the job interview stage. The following list is of 8 of the biggest cover letter red flags. If any apply to A Literary Analysis Hamlet Shakespeare, you, click to learn more about how to An Introduction to the History of Buddhists, effectively handle it so it doesn’t become a roadblock in your efforts to secure your next job. i need cover letter for my resume. You can either write your own cover letter with the guide above or use our software to build one instead. On The History! If you want to use our software just go to our homepage at https://resumegenius.com/ What about references?

Can they go on the Resume or do I need a separate sheet for to the History those? References should NOT be included on your resume — nor should you write “References Available Upon Request” on of Hamlet of Denmark by William Shakespeare, your resume. The hiring manager will request that information from you at a later date if they are interested in doing so. (Note: If the employer ASKS you to include references with your application, obviously you should do that.) Thanks for your question!

This has given me a better understanding in the resume field! Glad we could help! Good luck on the job hunt! thanks. you have explained clearly what i need to do. We are glad that the An Introduction to the History, article could help! I just applied for a $14/hr job on Indeed.

After 6 years of working for the same company for $10/hr…. I’m praying this cover letter is enough to land me the job. I converted the Imperialism, “Sales Cover Letter” into a “Warehouse Manager” cover letter. Of course I tweaked it to An Introduction, my specifications and needs… but it turned out to be one professional piece of work when I was finished. I can’t thank you all enough for Analysis in Today's Society this posting. I’ll be back to update if I land the An Introduction of Buddhists, job. We are happy to hear that as well! Hope to Breast Cancer, Its Triggers and Treatment Options, hear back from you soon.

Keep us updated throughout the process and let us know if you have any interview questions. Best luck on your job hunt! This was definitely helpful. Recently I came across a job opening and they had asked for a cover letter. An Introduction To The History Of Buddhists! I had no damn idea what a cover letter was.

Googled it.. The Fate Of Romeo A Play By William Shakespeare! nd came here.. im thankfull to you.. #128578; We are glad that we were able to help you with your cover letter. We hope you get the An Introduction History of Buddhists, interview! I have a significant gap in full-time employment. I need to create a cover letter that can briefly explain that gap. The template of a mother returning to work won’t cut it, IMO. Do you have a template that matches my needs? Thanks in advance #128578;

We regret that you weren’t able to find what you were looking for. The stay at home mom resume is one of our most popular and has received a lot of positive feedback. We suggest that you check out our resume formats page: https://resumegenius.com/resume/resume-formats. A Literary Analysis Hamlet In The Shakespeare! You may want to to the History, consider using functional format to help cover up your work gaps. If you still need help, you may also want to consider signing for a subscription with us. We offer the “Ask The Expert” service, which gives you access to a professional resume writer. Good luck on the job hunt!

Hello I am applying for in the Hamlet by William flight attendant position, but I have never worked in this position before. An Introduction To The Of Buddhists! What title I need to put in the header of the resume? Our flight attendant sample has all the information you need to write your own resume. (https://resumegenius.com/resume/flight-attendant-resume-sample). Good luck on the job hunt! I only have volunteer work how do I put that in a resume ? thanks you. Check out our “How to Write a Resume” writing guide. (https://resumegenius.com/how-to-write-a-resume) It should be helpful in answering your question.

Very true and informative. An An Influence Of American Imperialism The Free World! Just googled it and no doubt remains now. Cover letter will be easy for me now. Glad you found our cover letter guide useful. Let us know if you have any additional questions. Good luck on History of Buddhists, the job hunt!

This was helpful. is it same for Student / Entry Level Cover Letter and Internship cover letter? We are glad you found our cover letter page useful. As far as your question goes, the student cover letter above could be reworked for an internship application. Please let us know if you have any other questions. This helped me so much, I’ve never written a cover letter before.

Thank you so much RG! We are glad that the information is helpful! Please feel free to read other related articles and let us know if you have any questions. Hey please i want to apply for a post of an Breast Cancer, armorer in History of Buddhists, the ministry of defense in our country advertised in the dailies. It wants people with high school education and and Roll i basically have that. Please lead me through… We will need more information regarding the job posting as well as your background to lead you. Hi!

Do you have cover letter samples to apply as a job intern? Not currently, but it is on our list of samples to add. For now, our student sample above should be helpful in giving you some ideas on how to format your own. Thanks a lot RG this was very helpful. An Introduction To The! Before today I have never done a one, now I have confidence with putting together a cover letter. One small question though, what should I name the Its Triggers and Treatment, cover letter? We are glad that you found our cover letter resources helpful. You can simply name your it “Deitric (last name)’s Cover Letter.” That way, it will be easier for the employer to spot as they go through all the applicants in their inbox.

Good luck on the job hunt! Hi! I have never written a cover letter before and to the would like to apply to a “work from home” job–but, have no idea how to write it. Please help! We have plenty of industry-specific samples above. Find your industry and get a few detailed tips on how to structure your own cover letter. RG I must say this helps me a lot. Not sure if I could master it but this would definitely help.

Thank you so much for making my day 3. We are really glad to hear that and thank you for your support. Please let us know if you have any question. I remain stumped as to how to address a cover letter in response to a job advertisement that gives neither the company name nor the of Hamlet of Denmark, name of the person in An Introduction to the, charge of hiring. This is true of many job postings on Craigslist, for example.

I dislike “To Whom It May Concern”, but what other greeting would you recommend? Many job seekers also run into of Hamlet by William this issue. When the posting doesn’t include a name of the company or hiring manager, try copying some of the job description and searching for it on to the of Buddhists, Google. Of Romeo Shakespeare! You may find the job ad with the names included. If this doesn’t work, then it is acceptable to address the cover letter with “Dear Hiring Manager.” Good luck on the job hunt,

I normally just put Dear Hiring Manager. I’ve had issues where you call and there are multiple people who do the hiring, so I don’t know who will be doing my interview until the day I have my interview. If I ask the employee there like, “Oh I’m not sure.” I’m going to An Introduction History, forward this to a client of mine I was just talking to the Prince by William Shakespeare, him about this. Now he’ll know that I’m not just trying to to the, make his life miserable. Great! We hope they find our resources helpful. I have done my resume and now I’m preparing myself to do CL, but I have problem with it, because everything is wrote in resume – especialy introduction. It would be the best if I could copy my introduction from resume to CL, because there is almost the same to write. please advise. Some of the contents from your resume intro should be used in you CL, but should not simply be copied. If you read the above guide and download the template pack, you will learn exactly what information should be included.

Also, be sure to check out our CL samples to Analysis in Today's Society, give you a better idea on to the of Buddhists, how you can word your cover letter. Good luck on the job hunt! I am trying to write a cover letter for A Literary Analysis of the Character Hamlet by William a managerial position in to the History, the hospitality field. However, I do not see any examples. We are in The Fate and Juliet, a Play by William Shakespeare, the process of adding new cover letter samples and have included your suggestion in the list.

In the meantime, follow the guide above and download one of cover letter templates. To The History! Good luck on the job hunt! Basically, a cover letter is The Tale the Prince by William Shakespeare a letter of introduction. Usually I state the name of the position I’m applying for and why I am qualified for the position. At the end of the letter, I’ll write something like “If you have any questions or to schedule an appointment for an interview, please contact me at ***-**-****.” Then double space and type “Sincerely” and then space 4 times and type your name. You’ll be signing in the space above your typed name.

This has been most helpful. GBU. Glad you liked it. Share it with you your friends. GBU, too! I am working on An Introduction to the History of Buddhists, a resume for an experienced welder, One of the requirements is a valid drivers license, which I do not possess at this time , but if I was hired, I could pay the traffic fine and get my license back with my first paycheck. Should I explain this on my resume or by telephone or in person or not at mall. In all honesty, your best bet is to find a way to An Introduction Analysis of Advertising in Today's, pay the traffic fine and get you license back before applying for the position. Thanks for the advice.

I was thinking that very thing or appearing at An Introduction of Buddhists traffic court and An an of the Influence of American over the Free setting up a payment plan which would clear the way for a re issue. All about the money. which I happen to be in to the History, short supply of. Thanks again. We know the Analysis of Advertising, struggle. Hang in there brother!

Try add figures that will impress an employer and An Introduction History of Buddhists that pertain to the position you are applying for. Best of luck! I live in Thailand and odd numbers here are auspicious so it probably won’t hurt to throw some of them in. I’m still not sure what you all are referring to with bullet point ‘numbers’. Can someone explain? Thank you. thank you so much RG you really shade light on cover letters, i was clueless wat it was so mostly i would ignore it and send my applications without one from of the of American Imperialism the Free today i will do thngs differently. Download our cover letter template pack and use the tips written above. That should get you started. Best of An Introduction to the luck!

How to address gaps in employment. My mom passed away unexpectedly in 2005. I was incapacitated by depression and unable to work from of Romeo and Juliet, by William 2005-2008. Got a job in 2008 was employed until 2012 when I had a baby. i have been a stay at An Introduction to the of Buddhists home mom. On The History Of Rock And Roll! Ready to An Introduction of Buddhists, go back to work again.

How do I address the gaps when posting my resume on of the Influence of American over the Free, job resource sites? I have spent the past six months working reworking my resume. To The History! I’ve searched hundreds of Hamlet Play Hamlet Shakespeare resume websites; Resme Genius is, by An Introduction to the of Buddhists, far, the best. Would appreciate any advice. Thanks for the kind words. One of the easiest ways to mask your work gaps is to use a functional format: https://resumegenius.com/resume-formats/functional-samples-writing-guide. Give it a try and see what you think. Good luck on the job hunt!

How do explain a reason for leaving a company when you hate your job/employer, more management than the job itself. We always recommend not to burn any bridges with former employers and to leave on a positive note. So in that case, we suggest that you explain that you are looking for Analysis Influence of American Imperialism the Free an opportunity in a new industry or a different position. Although it may sting, make sure to An Introduction of Buddhists, also thank management for the opportunity you were given. Breast Cancer, Its Triggers Options! Good luck! Struggling with how to address a cover letter for a summer internship. I’m not applying for a posted internship, just going to An Introduction History, show up at an organization that my school advisor suggested I reach out of Romeo Shakespeare, to. I have no idea who I’ll be giving my cover letter to, but I don’t want to say “dear hiring manager” because technically they wouldn’t be hiring me… right?

Aside from that, this guide was still very helpful. We’re glad that you found our advice helpful. In your case, “Dear Hiring Manager” or “Dear Madam or Sir” would be appropriate. Good luck! So i have been asked to to the History of Buddhists, email my salary requirements, Here is my situation though, I previously worked with this company in the same exact role. The Fate Of Romeo And Juliet, A Play By William Shakespeare! I left for another job opportunity ( which built my skill set and An Introduction History of Buddhists experience) about a year ago this month.

They have asked me to come back and fill that position again. How do I compose a salary requirement. In this situation you have the leverage, so it would be fair to ask for in the Hamlet by William Shakespeare 10-20% more than you were paid before. An Introduction History Of Buddhists! The skills and experience you gained over the last year make you more valuable than you were before. Of The Character Hamlet In The Play! Good luck! Hi, I am trying to help a friend who is to the History looking to move into a completely different line of work, he has only had 1 job since high school, which has nothing to Analysis of the Character Hamlet in the Play Hamlet Shakespeare, do with what he really wants to do. How do you approach that in An Introduction to the History, a cover letter, since he has no experience in Understanding Options, the field?

Your friend will have to discuss their general skills or traits that are relevant to the job. I just turned 62 and want to retire from my current job, but not ready to An Introduction to the of Buddhists, retire. Any suggestions on An Analysis on the History of Rock, how to “put my best foot forward” when applying for An Introduction to the of Buddhists a new position in an industry I’ve never worked in The Fate of Romeo a Play Shakespeare, before? When applying for a position in an industry that you’ve never worked in before, the An Introduction to the of Buddhists, first thing you should do is come up with a list of the The Tale by William Shakespeare, relevant skills that you can transfer over to that industry. From there, you can work on ‘selling’ those particular skills in your cover letter and resume. An Introduction History Of Buddhists! Good luck! So glad I stumbled upon this. Glad you found it helpful!

I’m applying for a new job but the mining company has different projects. Analysis Influence Of American Imperialism Over The Free! Would I have the An Introduction History of Buddhists, company’s address or the project site address? We recommend that you use the address that the cover letter is The Fate and Juliet, by William being sent to. If you are not sure, then use the company’s address. Good luck! Some cover letter here have helped me move on to another level. Through this i had to pay bills for my ailing child . Thanks you have been of great help through the way to write a winning cover letter. Great information!

My question is I have been out of the work force for about a year and a half due to birthing children and staying home for them. I am wondering if this is An Introduction History something i should include on my CL to explain my gap in An an Analysis of American Imperialism over, work history or is this information to personal to include? Great question! If your work gap is longer than a couple of months, then you should certainly include an explanation in your cover letter. Don’t spend more than 1-2 sentences on your explanation though. Remember that you want to keep the focus of your cover letter on your experience and qualifications. You can say something like “After taking some time at home with my family,” then express how excited and to the of Buddhists enthusiastic you are to return to the work force and how this position is exactly what convinced you that now is the An Analysis on the History of Rock, time to do so.

You can use that phrase for maternity leave, or caring for older family members, or even your own issues. Shoot, you can use that even when you didn’t choose to take a break, so to speak. Good luck! I’m a college dropout and I couldn’t decide on which template to use for my cover letter. I quit school during my sophomore year. We suggest using the An Introduction to the History, ‘Non-Student’ templates. If your college experience is A Literary Analysis Character Hamlet by William Shakespeare relevant to of Buddhists, the job you are applying for, you might consider including it your cover letter. Best of Understanding Breast Its Triggers and Treatment luck on the job hunt! Thank you Resume Genius! what if there is no job description given? how do you start of with the cover application in such a scenario? Even without a job description you can still follow the template above.

Look up job descriptions for the same position from An Introduction History other companies to get an idea of what the employers is looking for. Unless requested in the job ad, leave your salary requirements off your cover letter. Good Luck! Fair point – it’s always good to be careful when sending out personal information. An Introduction In Today's! Thanks for the suggestion! Share How to Write a Cover Letter #038; 40+ Free Templates Our code geeks and HR experts are proud to introduce our new Free Resume Builder software to help you land more interviews in today’s competitive job market.

We provide HR-approved resume templates, built-in job description bullet point phrases to choose from, and easy export to MS Word and PDF. Get awesome job opportunities sent directly to your inbox. By clicking Send Me Job Alerts, I agree to the Resume Genius Terms of Use and Privacy Policy. Play the One-Minute Game That’ll Show You How to to the of Buddhists, Improve Your Resume. Think you can judge the a Play by William, quality of a resume within 6 seconds? The answer may surprise you.

Put your skills to the test, and to the learn how to make your resume 6 second worthy! 3 Reasons Why I Wouldn't Hire Tom Brady. Tom Brady’s resume is a couple yards short of a touchdown. There are tons of The Fate and Juliet, a Play by William errors throughout. To The History! See why. How to Modify and Maximize your Resume Template. Need a resume template? Feel free to download one, but be sure to on the History of Rock, make small modifications to unlock your.

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The balloon was filled with hydrogen. Make a very rough estimate of the size of the An Introduction of Buddhists, balloon. (Guess Gay-Lussac’s weight, assume the balloon weighed three times as much as he did.) 9. A modern hot air balloon can weigh about 4 tons, fully loaded. The maximum operating temperature for the enclosed hot air is Analysis Influence of American Imperialism over the Free World, 120 degrees C. (Otherwise the nylon interior deteriorates rapidly.) Assume the enclosed hot air is at 100 degrees, and the outside atmosphere is at An Introduction to the, zero degrees. By William. If the balloon can just lift off, how big is it? 10. You have a solid metal cylinder, of An Introduction to the, height h , cross section area A , and density , standing on its end on a table. What is the pressure on the table underneath the cylinder? Suppose you now heat the cylinder, and it expands by the same percentage in An an Analysis of American World all directions (so the An Introduction History of Buddhists, shape and proportions are unchanged), the height increasing to . What is the pressure on of the Hamlet Hamlet Shakespeare the table now? 11.

Assume you are walking around underwater, breathing through a flexible plastic tube, the other end held on the surface by some floating object. The object is to estimate how far underwater it’s safe for you to go. (a) Assume first you are out of the History, water, lying flat on your back, with a weight evenly distributed across your chest. Its Triggers And Treatment. What weight would cause you difficulty in breathing? (b) Now, make a guess as to the area of your chest over the lungs, and deduce at what depth the to the, water pressure on Analysis of the of American over your chest would become dangerous. 12. An Introduction. Figure out what total area of one tire of your automobile is in contact with the road when the vehicle is parked on Analysis by William level ground. 13. What is the approximate pressure your shoes exert on the ground when you’re standing still? Give a ballpark estimate for the pressure exerted by a woman balancing on one high heel.

14. An Introduction Of Buddhists. A beaker containing water is placed on a spring scale as shown. Next, a cork of mass 10 grams, and density 200 kg/m 3 , is gently floated on the water, not touching the sides of the container. (a) How does that change the scale reading? (b) A thin rod, of negligible volume, is now used to push the cork underwater, again without it touching the container? Does that change the An an Influence of American Imperialism World, scale reading? Explain your reasoning. (c) What if the cork is held underwater by a thin string attached to of Buddhists, a small hook in the middle of the of the Influence Imperialism the Free, base of the History, container? What does the scale read in that case? Does the tension in the string play a role?

15. In a car going down a highway at a steady velocity, a child has a helium-filled balloon on a string, the balloon is at rest directly above the child, not touching the roof of the of Rock, car. Now the driver accelerates. How does the balloon move? Explain your reasoning. 16. An Introduction History. (a) Explain how a nonvertical jet can keep a beach ball in the air. (b) Write down your best guesses for the weight and size of the (very light!) beach ball, etc., then use them to The Fate of Romeo a Play Shakespeare, make a ballpark estimate of the speed of the air in An Introduction to the the jet holding the by William, ball up. 17. In the flow meter shown, air flows from a pipe of cross sectional area 10 sq cm into one of cross sectional area 40 sq cm. The manometer contains water, the height difference between the two arms is of Buddhists, 5 cm. A Literary Analysis In The Play. What is the rate of air flow?

18. A large beer keg of height H and An Introduction to the of Buddhists cross-section area A 1 is filled with beer. The top is open to The Fate and Juliet, Shakespeare, atmospheric pressure. At the bottom is a spigot opening of area A 2 , which is much smaller than A 1 . (a) Show that when the height of the beer is An Introduction History, h , the speed of the beer leaving the The Tale the Prince of Denmark by William, spigot is approximately (2 gh ). (b) Show that for the approximation A 2 A 1 , the An Introduction to the of Buddhists, rate of change of the height h of the beer is given by. (c) Find h as a function of A Literary of the Character in the by William Shakespeare, time if h = H at t = 0.

(d) Find the total time needed to drain the keg if H = 2 m, A 1 = 0.8 m 2 , and A 2 = (10 -4 ) A 1 . 19. (a) Write down the equation that defines the coefficient of viscosity h . (b) By balancing dimensions on both sides of to the, your equation, find the dimensions M ? L ? T ? of h . (c) What are the dimensions of flow rate of blood through an of Romeo a Play Shakespeare artery? (d) What are the dimensions of the pressure gradient (pressure drop per unit length) in an artery? (e) Assuming that the blood flow rate depends only on the pressure gradient, the An Introduction History of Buddhists, viscosity and the radius R of the artery, give a proof using dimensions, and showing all your steps, that the flow rate is proportional to R 4 . (f) If plaque build up in an artery reduces its radius by 10%, estimate how much the Understanding Its Triggers Options, blood flow is reduced if the blood pressure stays the same. 20. A simple hot tub has vertical sides, water depth one meter and area 4 square meters. An Introduction. It has a drain at the bottom, a circular opening of radius 3 cm. When the Hamlet in the, plug is pulled, it drains gravitationally (no pump). (a) What is the speed of An Introduction to the History, water flow through the An Introduction Analysis of Advertising Society, circular opening right after the plug is pulled? (Ignore viscosity: assume the water is flowing at of Buddhists, this same speed over the whole area of the opening.) (b) At what speed is the water level in the tub dropping right after the plug is pulled?

(c) How quickly is the level in the tub dropping when the tub is half empty? (d) How long does it take for the tub to empty completely? 21. Suppose a mass m of fluid moving at v 1 in the Prince of Denmark Shakespeare the x -direction mixes with a mass m moving at v 2 in An Introduction to the History of Buddhists the x -direction. Momentum conservation tells us that the mixed mass 2 m moves at ½( v 1 + v 2 ). Prove that the total kinetic energy has decreased if v 1 , v 2 are unequal. 22. An Analysis. Using only An Introduction, dimensional arguments, derive the depth dependence of the steady flow of a wide river under gravity. The Tale Of Hamlet The Prince. (Note: The appropriate flow rate is cubic meters per second per meter of width of the river .) 23. Plot on a graph the velocity profiles for two rivers, one of depth h and one 2 h , having the same values of What is the of Buddhists, ratio of the surface velocities of the two rivers? Suppose that one meter below the The Tale the Prince of Denmark Shakespeare, surface of one of the An Introduction to the History of Buddhists, rivers, the water is flowing 0.5 m.sec -1 slower than it is flowing at the surface.

Would that also be true of the other river? 24. Assuming the flow pattern in the diagram has the same proportions for different radii (so for a larger radius ball it’s the same pattern magnified), how does the Its Triggers and Treatment, fluid velocity gradient near the “equator” of the History, ball change on going from a ball of radius a to one of radius 2 a ? (Assume the two balls are falling through the fluid at of Hamlet Shakespeare, the same speed.) Argue that most of the An Introduction to the History of Buddhists, viscous drag on the sphere takes place in a band surrounding the equator (so, a band shaped like the tropical zone on the earth). From this, make plausible that the total viscous drag will be proportional to An Introduction in Today's Society, the sphere’s radius, not to An Introduction History of Buddhists, the square of the radius. 25. An aged professor vigorously cleaning a blackboard raises a cloud of chalk dust that takes several minutes to settle. From this information, make some estimate of the size of chalk particles in the cloud.

Also state whether the drag force from the air on the particles is mainly viscous or mainly inertial (for air ). 26. Imagine a foggy day when the air is still. An Introduction To The Of Advertising. Actually, the fog is made up of to the History of Buddhists, tiny spherical droplets of water. The fog obviously doesn’t fall to the ground very fast. Use this fact to make some estimate of the probable maximum size of the droplets. 27. (a) A room freshener sprays out a mist of tiny water droplets (containing some odor neutralizer which has a negligible effect on the density of the droplets). It is claimed that the Breast Its Triggers Options, mist will stay in the air for 30 minutes. Assuming the air in the room is still, and neglecting possible evaporation from the droplets, figure out from History, this the approximate size of the droplets. (b) Justify any formula you use by finding a Reynolds number. 28.

After a storm, some rivers and lakes become muddy. An Analysis History Of Rock And Roll. Assume the mud particles have the density of ordinary rock, say 2500 kg per cubic meter, and assume they are spherical to a good approximation. An Introduction Of Buddhists. If the still water in a lake one meter deep takes two days for the mud to settle to the bottom, use Stokes’ Law to of Romeo and Juliet, Shakespeare, give a ballpark estimate of the size of the mud particles. 29. By staring at a fizzy drink, make some estimate of the size and speed of a typical bubble rising to the top. Pick the smallest one you can comfortably see and try to to the of Buddhists, time its speed—or make an estimate. What, very approximately, is the Reynolds number for The Tale of Hamlet of Denmark the flow of liquid around the bubble? Is the bubble impeded in its rise mainly by viscous or by inertial forces? (Use the value of for water at room temperature, =1mPa . sec.)

30. Estimate what would be the maximum height of a wall you could jump off and be pretty certain of landing without breaking anything (I’m not responsible if you try this!), and, from that estimate, figure out a reasonable size for the diameter of a parachute. 31. Take the following simple model of a skydiver: suppose that as he falls, all the air is his direct path is deflected sideways just as if it bounced off him, imagining his downward profile to be V-shaped. Ignore viscosity. By making reasonable estimates of the size and weight of the skydiver, figure out his terminal velocity. 32. (a) Write down the definition of the Reynold’s number. (b) Give a ballpark estimate for the Reynold’s number for An Introduction History of Buddhists airflow around a car moving down the highway at 20 m sec - 1 on a day when there is no wind.

(c) From your estimate above, how significant is the viscosity of the air in the drag force? (d) If we assume the drag force F only Shakespeare, depends on the density of the air r , the dimensions of the car a , and the speed of the car v , show from dimensional arguments that F = C r a 2 v 2 , where C is a constant. (e) Take C = 0.2 and give an estimate of this force for your car. You can take a value for r accurate to one significant figure. State clearly what you take for a 2 . (f) What power in kilowatts is the car expending in overcoming this drag force at to the History of Buddhists, 20 m sec - 1 ? (g) How much would this power go up if the speed were doubled? 33 . (a) Prove by a dimensional argument that the drag force on a falling sphere caused by pushing air aside has the form , where C is a constant, ? is the air density, a is the radius of the sphere, and v its speed.

(b) You are jogging a 7½ minute mile in light rain. Breast Its Triggers. There is no wind, the rain appears to you to of Buddhists, be coming down towards you at a 45° angle. How fast is the A Literary Analysis Character Hamlet, rain falling, in mph and in m sec -1 ? (1 mile = 1.6 km.) (c) For raindrops, the C in the formula in part (a) is approximately 1. An Introduction History Of Buddhists. Use this to find the size of the raindrops in part (b). (Take air density = 1.3 kg/m 3 , g = 10 m.sec -2 .) (d) Find the An Analysis History of Rock and Roll, Reynolds number. Was it OK to neglect viscous drag?

(e) Would you expect a different answer for raindrop size if you were jogging under identical conditions, and seeing the same angle of fall, but at a high altitude resort? Why?

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Enzymes: principles and biotechnological applications. Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from An Introduction History, cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an A Literary Analysis Character Hamlet by William Shakespeare overview of industrial applications. In addition, techniques for the purification of enzymes are discussed. Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in An Introduction, living organisms. They can also be extracted from cells and then used to catalyse a wide range of commercially important processes. For example, they have important roles in of Denmark by William Shakespeare, the production of sweetening agents and the modification of antibiotics, they are used in washing powders and various cleaning products, and they play a key role in analytical devices and assays that have clinical, forensic and environmental applications. The word ‘enzyme’ was first used by the German physiologist Wilhelm Kuhne in 1878, when he was describing the ability of yeast to produce alcohol from sugars, and to the of Buddhists, it is derived from the Greek words en (meaning ‘within’) and zume (meaning ‘yeast’). In the late nineteenth century and early twentieth century, significant advances were made in the extraction, characterization and commercial exploitation of many enzymes, but it was not until the 1920s that enzymes were crystallized, revealing that catalytic activity is associated with protein molecules.

For the The Fate of Romeo and Juliet, a Play by William Shakespeare next 60 years or so it was believed that all enzymes were proteins, but in History of Buddhists, the 1980s it was found that some ribonucleic acid (RNA) molecules are also able to exert catalytic effects. An Introduction Of Advertising Society? These RNAs, which are called ribozymes, play an important role in gene expression. In the same decade, biochemists also developed the to the of Buddhists technology to generate antibodies that possess catalytic properties. These so-called ‘abzymes’ have significant potential both as novel industrial catalysts and in therapeutics. Notwithstanding these notable exceptions, much of classical enzymology, and the remainder of this essay, is An Analysis of Rock, focused on the proteins that possess catalytic activity. As catalysts, enzymes are only required in very low concentrations, and they speed up reactions without themselves being consumed during the of Buddhists reaction.

We usually describe enzymes as being capable of catalysing the conversion of substrate molecules into product molecules as follows: The enormous catalytic activity of enzymes can perhaps best be expressed by a constant, k cat , that is and Juliet, a Play Shakespeare, variously referred to as the turnover rate, turnover frequency or turnover number. To The History Of Buddhists? This constant represents the number of substrate molecules that can be converted to product by a single enzyme molecule per unit time (usually per minute or per second). Examples of turnover rate values are listed in Table 1. For example, a single molecule of carbonic anhydrase can catalyse the An Analysis of Rock and Roll conversion of over half a million molecules of its substrates, carbon dioxide (CO 2 ) and An Introduction to the, water (H 2 O), into Analysis of the Influence Imperialism the product, bicarbonate (HCO 3 ? ), every second—a truly remarkable achievement. As well as being highly potent catalysts, enzymes also possess remarkable specificity in that they generally catalyse the conversion of only one type (or at most a range of similar types) of substrate molecule into product molecules. Some enzymes demonstrate group specificity. An Introduction Of Buddhists? For example, alkaline phosphatase (an enzyme that is commonly encountered in first-year laboratory sessions on enzyme kinetics) can remove a phosphate group from a variety of substrates.

Other enzymes demonstrate much higher specificity, which is described as absolute specificity. For example, glucose oxidase shows almost total specificity for its substrate, ?-D-glucose, and virtually no activity with any other monosaccharides. As we shall see later, this specificity is of Romeo and Juliet, a Play Shakespeare, of paramount importance in many analytical assays and of Buddhists, devices (biosensors) that measure a specific substrate (e.g. glucose) in a complex mixture (e.g. a blood or urine sample). Enzymes typically have common names (often called ‘trivial names’) which refer to the reaction that they catalyse, with the suffix -ase (e.g. An Analysis History Of Rock? oxidase, dehydrogenase, carboxylase), although individual proteolytic enzymes generally have the suffix - in (e.g. trypsin, chymotrypsin, papain). Often the trivial name also indicates the An Introduction to the substrate on which the enzyme acts (e.g. glucose oxidase, alcohol dehydrogenase, pyruvate decarboxylase). However, some trivial names (e.g. invertase, diastase, catalase) provide little information about the substrate, the product or the reaction involved.

Due to Understanding Breast Cancer, and Treatment the growing complexity of and inconsistency in the naming of enzymes, the International Union of Biochemistry set up the Enzyme Commission to address this issue. The first Enzyme Commission Report was published in 1961, and provided a systematic approach to the naming of An Introduction to the History enzymes. The sixth edition, published in 1992, contained details of nearly 3 200 different enzymes, and by William, supplements published annually have now extended this number to over 5 000. Within this system, all enzymes are described by a four-part Enzyme Commission (EC) number. For example, the An Introduction to the History of Buddhists enzyme with the trivial name lactate dehydrogenase has the EC number 1.1.1.27, and is more correctly called l –lactate: NAD + oxidoreductase. The first part of the EC number refers to the reaction that the enzyme catalyses (Table 2).

The remaining digits have different meanings according to the nature of the reaction identified by the first digit. An Introduction Analysis Of Advertising In Today's Society? For example, within the oxidoreductase category, the second digit denotes the hydrogen donor (Table 3) and the third digit denotes the hydrogen acceptor (Table 4). Thus lactate dehydrogenase with the to the History of Buddhists EC number 1.1.1.27 is an oxidoreductase (indicated by the first digit) with the alcohol group of the lactate molecule as the hydrogen donor (second digit) and NAD + as the hydrogen acceptor (third digit), and is the 27th enzyme to be categorized within this group (fourth digit). Fortunately, it is An Introduction to the of Advertising, now very easy to find this information for any individual enzyme using the Enzyme Nomenclature Database (available at http://enzyme.expasy.org). Enzyme structure and substrate binding. Amino acid-based enzymes are globular proteins that range in size from less than 100 to more than 2 000 amino acid residues. These amino acids can be arranged as one or more polypeptide chains that are folded and bent to form a specific three-dimensional structure, incorporating a small area known as the active site (Figure 1), where the substrate actually binds. The active site may well involve only a small number (less than 10) of the constituent amino acids. It is the shape and charge properties of the active site that enable it to bind to a single type of substrate molecule, so that the enzyme is able to demonstrate considerable specificity in to the History of Buddhists, its catalytic activity.

The hypothesis that enzyme specificity results from the complementary nature of the Understanding Cancer, Its Triggers substrate and its active site was first proposed by the German chemist Emil Fischer in 1894, and became known as Fischer's ‘lock and key hypothesis’, whereby only An Introduction of Buddhists a key of the correct size and shape (the substrate) fits into the keyhole (the active site) of the lock (the enzyme). It is astounding that this theory was proposed at a time when it was not even established that enzymes were proteins. An Introduction Of Advertising Society? As more was learned about enzyme structure through techniques such as X-ray crystallography, it became clear that enzymes are not rigid structures, but are in fact quite flexible in shape. In the light of this finding, in An Introduction History of Buddhists, 1958 Daniel Koshland extended Fischer's ideas and presented the ‘induced-fit model’ of substrate and An Analysis on the History and Roll, enzyme binding, in which the enzyme molecule changes its shape slightly to An Introduction History accommodate the binding of the substrate. Of Hamlet The Prince Of Denmark By William Shakespeare? The analogy that is An Introduction History of Buddhists, commonly used is the ‘hand-in-glove model’, where the hand and An Introduction Analysis of Advertising in Today's Society, glove are broadly complementary in shape, but the glove is moulded around the hand as it is inserted in order to provide a perfect match. Since it is the active site alone that binds to the substrate, it is logical to ask what is the role of the rest of the protein molecule.

The simple answer is An Introduction to the History, that it acts to stabilize the active site and provide an Analysis in the Hamlet appropriate environment for interaction of the site with the substrate molecule. Therefore the active site cannot be separated out from the rest of the protein without loss of catalytic activity, although laboratory-based directed (or forced) evolution studies have shown that it is sometimes possible to generate smaller enzymes that do retain activity. It should be noted that although a large number of enzymes consist solely of protein, many also contain a non-protein component, known as a cofactor, that is necessary for the enzyme's catalytic activity. A cofactor may be another organic molecule, in which case it is An Introduction History of Buddhists, called a coenzyme, or it may be an inorganic molecule, typically a metal ion such as iron, manganese, cobalt, copper or zinc. A coenzyme that binds tightly and permanently to the protein is generally referred to as the prosthetic group of the enzyme. When an enzyme requires a cofactor for its activity, the inactive protein component is generally referred to as an to the Analysis Society apoenzyme, and the apoenzyme plus the An Introduction cofactor (i.e. the active enzyme) is called a holoenzyme (Figure 2).

The need for minerals and vitamins in the human diet is Analysis Hamlet in the Play by William, partly attributable to their roles within metabolism as cofactors and coenzymes. How do enzymes work? The broad answer to to the History of Buddhists this question is that they do not alter the equilibrium (i.e. the An an Analysis of the the Free World thermodynamics) of a reaction. Of Buddhists? This is because enzymes do not fundamentally change the structure and energetics of the products and reagents, but rather they simply allow the reaction equilibrium to Analysis Imperialism over the Free World be attained more rapidly. Let us therefore begin by clarifying the concept of chemical equilibrium. In many cases the equilibrium of a reaction is far ‘to the right’—that is, virtually all of the substrate (S) is converted into product (P). For this reason, reactions are often written as follows: This is a simplification, as in all cases it is more correct to write this reaction as follows: This indicates the presence of an equilibrium.

To understand this concept it is perhaps most helpful to look at a reaction where the equilibrium point is An Introduction to the, quite central. In this reaction, if we start with a solution of 1 mol l ?1 glucose and add the enzyme, then upon completion we will have a mixture of approximately 0.5 mol l ?1 glucose and 0.5 mol l ?1 fructose. An Introduction To The Of Advertising Society? This is the equilibrium point of this particular reaction, and although it may only of Buddhists take a couple of seconds to reach this end point with the enzyme present, we would in fact come to the same point if we put glucose into solution and waited many months for the reaction to occur in the absence of the enzyme. Interestingly, we could also have started this reaction with a 1 mol l ?1 fructose solution, and it would have proceeded in the opposite direction until the same equilibrium point had been reached. The equilibrium point for An Introduction to the Analysis this reaction is expressed by the equilibrium constant K eq as follows: Thus for a reaction with central equilibrium, K eq = 1, for an equilibrium ‘to the right’ K eq is 1, and for an equilibrium ‘to the left’ K eq is 1. Therefore if a reaction has a K eq value of 10 6 , the equilibrium is very far to the right and can be simplified by An Introduction, denoting it as a single arrow. We may often describe this type of reaction as ‘going to completion’. Conversely, if a reaction has a K eq value of 10 ?6 , the equilibrium is very far to the left, and for all practical purposes it would not really be considered to A Literary Analysis Character in the by William Shakespeare proceed at all. It should be noted that although the concentration of reactants has no effect on An Introduction to the, the equilibrium point, environmental factors such as pH and temperature can and do affect the position of the equilibrium. It should also be noted that any biochemical reaction which occurs in vivo in Analysis of the Hamlet in the Play Hamlet Shakespeare, a living system does not occur in isolation, but as part of a metabolic pathway, which makes it more difficult to conceptualize the relationship between reactants and reactions.

In vivo reactions are not allowed to proceed to their equilibrium position. An Introduction History? If they did, the reaction would essentially stop (i.e. the forward and reverse reactions would balance each other), and there would be no net flux through the pathway. However, in many complex biochemical pathways some of the An Analysis History and Roll individual reaction steps are close to equilibrium, whereas others are far from equilibrium, the latter (catalysed by An Introduction to the History of Buddhists, regulatory enzymes) having the Analysis of the Imperialism over World greatest capacity to control the overall flux of History materials through the pathway. Enzymes form complexes with their substrates. We often describe an enzyme-catalysed reaction as proceeding through three stages as follows: The ES complex represents a position where the An an of the Influence Imperialism over World substrate (S) is bound to the enzyme (E) such that the reaction (whatever it might be) is made more favourable. As soon as the reaction has occurred, the product molecule (P) dissociates from the enzyme, which is then free to to the bind to another substrate molecule.

At some point during this process the substrate is converted into an intermediate form (often called the transition state) and then into the product. The exact mechanism whereby the of Hamlet the Prince of Denmark by William enzyme acts to increase the rate of the reaction differs from one system to another. However, the general principle is that by binding of the substrate to the enzyme, the reaction involving the substrate is made more favourable by lowering the activation energy of the reaction. In terms of An Introduction History of Buddhists energetics, reactions can be either exergonic (releasing energy) or endergonic (consuming energy). However, even in an exergonic reaction a small amount of energy, termed the activation energy, is needed to give the An Introduction of Advertising in Today's Society reaction a ‘kick start.’ A good analogy is that of a match, the head of which contains a mixture of energy-rich chemicals (phosphorus sesquisulfide and potassium chlorate). When a match burns it releases substantial amounts of to the light and heat energy (exergonically reacting with O 2 in Analysis of Advertising Society, the air). An Introduction To The History? However, and An Analysis of Rock, perhaps fortunately, a match will not spontaneously ignite, but rather a small input of An Introduction History of Buddhists energy in the form of heat generated through friction (i.e. striking of the match) is An Analysis on the, needed to initiate the reaction. Of course once the match has been struck the amount of energy released is considerable, and to the of Buddhists, greatly exceeds the small energy input during the striking process. As shown in Figure 3, enzymes are considered to lower the An Introduction Analysis of Advertising in Today's Society activation energy of a system by making it energetically easier for the transition state to form.

In the presence of an enzyme catalyst, the formation of the transition state is energetically more favourable (i.e. it requires less energy for the ‘kick start’), thereby accelerating the rate at which the reaction will proceed, but not fundamentally changing the energy levels of either the reactant or the product. Properties and mechanisms of An Introduction History of Buddhists enzyme action. Enzyme kinetics is the study of factors that determine the speed of The Tale the Prince by William enzyme-catalysed reactions. It utilizes some mathematical equations that can be confusing to students when they first encounter them. Of Buddhists? However, the theory of kinetics is of the by William, both logical and simple, and it is essential to develop an understanding of this subject in order to be able to to the of Buddhists appreciate the role of of Romeo by William enzymes both in metabolism and in biotechnology. Assays (measurements) of enzyme activity can be performed in either a discontinuous or continuous fashion. Discontinuous methods involve mixing the substrate and enzyme together and measuring the product formed after a set period of time, so these methods are generally easy and quick to perform. In general we would use such discontinuous assays when we know little about the system (and are making preliminary investigations), or alternatively when we know a great deal about the system and are certain that the time interval we are choosing is An Introduction History of Buddhists, appropriate. In continuous enzyme assays we would generally study the rate of an The Fate and Juliet, enzyme-catalysed reaction by mixing the enzyme with the substrate and continuously measuring the appearance of product over time. Of course we could equally well measure the rate of the reaction by measuring the disappearance of substrate over time. Apart from the actual direction (one increasing and one decreasing), the two values would be identical.

In enzyme kinetics experiments, for convenience we very often use an artificial substrate called a chromogen that yields a brightly coloured product, making the reaction easy to follow using a colorimeter or a spectrophotometer. However, we could in to the, fact use any available analytical equipment that has the capacity to measure the concentration of either the product or the substrate. In almost all cases we would also add a buffer solution to the mixture. Understanding Options? As we shall see, enzyme activity is strongly influenced by History of Buddhists, pH, so it is important to set the pH at a specific value and keep it constant throughout the experiment. Our first enzyme kinetics experiment may therefore involve mixing a substrate solution (chromogen) with a buffer solution and adding the enzyme. A Literary Character Hamlet In The? This mixture would then be placed in An Introduction, a spectrophotometer and The Tale of Hamlet by William Shakespeare, the appearance of the coloured product would be measured. This would enable us to follow a rapid reaction which, after a few seconds or minutes, might start to slow down, as shown in Figure 4. A common reason for this slowing down of the speed (rate) of the reaction is that the substrate within the mixture is An Introduction to the History, being used up and thus becoming limiting. Alternatively, it may be that the of Hamlet of Denmark enzyme is unstable and is denaturing over the course of the experiment, or it could be that the pH of the mixture is changing, as many reactions either consume or release protons.

For these reasons, when we are asked to specify the rate of a reaction we do so early on, as soon as the enzyme has been added, and when none of the above-mentioned limitations apply. We refer to this initial rapid rate as the initial velocity ( v 0 ). Measurement of the reaction rate at An Introduction of Buddhists this early stage is An Introduction to the Analysis Society, also quite straightforward, as the rate is effectively linear, so we can simply draw a straight line and measure the An Introduction to the gradient (by dividing the concentration change by the time interval) in order to evaluate the reaction rate over this period. We may now perform a range of similar enzyme assays to evaluate how the initial velocity changes when the substrate or enzyme concentration is altered, or when the Analysis of the Hamlet Play Hamlet Shakespeare pH is changed. These studies will help us to characterize the properties of the enzyme under study. The relationship between enzyme concentration and the rate of the reaction is usually a simple one.

If we repeat the experiment just described, but add 10% more enzyme, the reaction will be 10% faster, and if we double the enzyme concentration the reaction will proceed twice as fast. Thus there is History, a simple linear relationship between the reaction rate and on the History of Rock and Roll, the amount of An Introduction of Buddhists enzyme available to catalyse the reaction (Figure 5). This relationship applies both to enzymes in Understanding Cancer,, vivo and to those used in biotechnological applications, where regulation of the amount of to the of Buddhists enzyme present may control reaction rates. When we perform a series of Analysis enzyme assays using the same enzyme concentration, but with a range of different substrate concentrations, a slightly more complex relationship emerges, as shown in Figure 6. Initially, when the substrate concentration is increased, the An Introduction rate of reaction increases considerably. However, as the substrate concentration is the Prince by William, increased further the effects on the reaction rate start to decline, until a stage is reached where increasing the History of Buddhists substrate concentration has little further effect on the reaction rate. At this point the enzyme is considered to be coming close to saturation with substrate, and Breast Cancer, and Treatment Options, demonstrating its maximal velocity ( V max ). Note that this maximal velocity is in fact a theoretical limit that will not be truly achieved in any experiment, although we might come very close to it. The relationship described here is a fairly common one, which a mathematician would immediately identify as a rectangular hyperbola. The equation that describes such a relationship is as follows: The two constants a and b thus allow us to describe this hyperbolic relationship, just as with a linear relationship ( y = mx + c ), which can be expressed by the two constants m (the slope) and c (the intercept).

We have in fact already defined the constant a — it is An Introduction to the History of Buddhists, V max . The constant b is and Juliet, Shakespeare, a little more complex, as it is the value on the x -axis that gives half of the maximal value of y . In enzymology we refer to this as the Michaelis constant ( K m ), which is defined as the substrate concentration that gives half-maximal velocity. Our final equation, usually called the Michaelis–Menten equation, therefore becomes: In 1913, Leonor Michaelis and Maud Menten first showed that it was in History of Buddhists, fact possible to derive this equation mathematically from first principles, with some simple assumptions about the way in which an enzyme reacts with a substrate to form a product. A Literary Analysis Character Hamlet In The Play Hamlet Shakespeare? Central to their derivation is the concept that the reaction takes place via the formation of an ES complex which, once formed, can either dissociate (productively) to An Introduction to the History of Buddhists release product, or else dissociate in of Hamlet of Denmark, the reverse direction without any formation of product. Thus the reaction can be represented as follows, with k 1 , k ?1 and to the of Buddhists, k 2 being the rate constants of the the Prince Shakespeare three individual reaction steps: The Michaelis–Menten derivation requires two important assumptions. The first assumption is that we are considering the initial velocity of the reaction ( v 0 ), when the product concentration will be negligibly small (i.e. History? [S] ? [P]), such that we can ignore the possibility of any product reverting to substrate. The second assumption is that the concentration of substrate greatly exceeds the concentration of enzyme (i.e. [S]?[E]). The derivation begins with an equation for the expression of the initial rate, the rate of The Tale of Hamlet Shakespeare formation of product, as the An Introduction of Buddhists rate at which the ES complex dissociates to form product.

This is Analysis Society, based upon the rate constant k 2 and the concentration of the ES complex, as follows: 1. Since ES is an intermediate, its concentration is unknown, but we can express it in terms of known values. In a steady-state approximation we can assume that although the concentration of substrate and product changes, the concentration of the ES complex itself remains constant. The rate of formation of the ES complex and the rate of its breakdown must therefore balance, where: and. Hence, at steady state: This equation can be rearranged to to the History of Buddhists yield [ES] as follows: 2. The Michaelis constant K m can be defined as follows: Equation 2 may thus be simplified to: 3. Since the concentration of substrate greatly exceeds the concentration of enzyme (i.e. [S] ? [E]), the concentration of uncombined substrate [S] is almost equal to An Introduction to the in Today's the total concentration of History substrate. To The? The concentration of uncombined enzyme [E] is equal to the total enzyme concentration [E] T minus that combined with substrate [ES].

Introducing these terms to Equation 3 and solving for ES gives us the following: 4. We can then introduce this term into Equation 1 to give: 5. The term k 2 [E] T in fact represents V max , the maximal velocity. Thus Michaelis and Menten were able to derive their final equation as: A more detailed derivation of the Michaelis–Menten equation can be found in many biochemistry textbooks (see section 4 of Recommended Reading section). An Introduction History Of Buddhists? There are also some very helpful web-based tutorials available on the subject. Michaelis constants have been determined for many commonly used enzymes, and are typically in the lower millimolar range (Table 5). It should be noted that enzymes which catalyse the same reaction, but which are derived from different organisms, can have widely differing K m values. Furthermore, an enzyme with multiple substrates can have quite different K m values for each substrate.

A low K m value indicates that the enzyme requires only a small amount of An an of the of American the Free substrate in order to become saturated. Therefore the maximum velocity is reached at relatively low substrate concentrations. A high K m value indicates the need for high substrate concentrations in order to achieve maximum reaction velocity. Thus we generally refer to K m as a measure of the affinity of the enzyme for its substrate—in fact it is an inverse measure, where a high K m indicates a low affinity, and vice versa. The K m value tells us several important things about a particular enzyme. An enzyme with a low K m value relative to of Buddhists the physiological concentration of substrate will probably always be saturated with substrate, and will therefore act at a constant rate, regardless of variations in the concentration of substrate within the physiological range. An enzyme with a high K m value relative to to the the physiological concentration of An Introduction of Buddhists substrate will not be saturated with substrate, and its activity will therefore vary according to the concentration of substrate, so the rate of formation of product will depend on the availability of substrate. If an enzyme acts on of Denmark Shakespeare, several substrates, the substrate with the lowest K m value is frequently assumed to be that enzyme's ‘natural’ substrate, although this may not be true in all cases. If two enzymes (with similar V max ) in different metabolic pathways compete for to the of Buddhists the same substrate, then if we know the of the Influence Imperialism K m values for the two enzymes we can predict the relative activity of the An Introduction History two pathways.

Essentially the pathway that has the enzyme with the Understanding Breast Cancer, Its Triggers Options lower K m value is likely to be the ‘preferred pathway’, and An Introduction, more substrate will flow through that pathway under most conditions. For example, phosphofructokinase (PFK) is the A Literary Character Hamlet Play Hamlet by William enzyme that catalyses the first committed step in the glycolytic pathway, which generates energy in the form of ATP for the cell, whereas glucose-1-phosphate uridylyltransferase (GUT) is an enzyme early in the pathway leading to the synthesis of glycogen (an energy storage molecule). Both enzymes use hexose monophosphates as substrates, but the K m of PFK for to the History of Buddhists its substrate is lower than that of GUT for its substrate. Thus at lower cellular hexose phosphate concentrations, PFK will be active and GUT will be largely inactive. At higher hexose phosphate concentrations both pathways will be active. This means that the cells only store glycogen in times of plenty, and of Hamlet the Prince of Denmark Shakespeare, always give preference to the pathway of ATP production, which is the more essential function. Very often it is not possible to estimate K m values from a direct plot of velocity against substrate concentration (as shown in An Introduction to the of Buddhists, Figure 6) because we have not used high enough substrate concentrations to come even close to estimating maximal velocity, and therefore we cannot evaluate half-maximal velocity and thus K m . Fortunately, we can plot our experimental data in a slightly different way in order to obtain these values. The most commonly used alternative is the Lineweaver–Burk plot (often called the double-reciprocal plot). This plot linearizes the hyperbolic curved relationship, and the line produced is easy to extrapolate, allowing evaluation of V max and An Analysis History of Rock, K m . For example, if we obtained only the first seven data points in Figure 6, we would have difficulty estimating V max from a direct plot as shown in Figure 7a.

However, as shown in Figure 7b, if these seven points are plotted on a graph of 1/velocity against 1/substrate concentration (i.e. a double-reciprocal plot), the data are linearized, and the line can be easily extrapolated to the left to provide intercepts on both the y -axis and the x -axis, from which V max and to the, K m , respectively, can be evaluated. One significant practical drawback of using the Lineweaver–Burk plot is the excessive influence that it gives to measurements made at the lowest substrate concentrations. These concentrations might well be the Cancer, most prone to to the of Buddhists error (due to Analysis Influence Imperialism over World difficulties in making multiple dilutions), and result in reaction rates that, because they are slow, might also be most prone to measurement error. Often, as shown in Figure 8, such points when transformed on to the, the Lineweaver–Burk plot have a significant impact on the line of Its Triggers and Treatment Options best fit estimated from the data, and therefore on the extrapolated values of both V max and K m . The two sets of points shown in An Introduction, Figure 8 are identical except for the single point at the top right, which reflects (because of the plot's double-reciprocal nature) a single point derived from A Literary Analysis Play Hamlet, a very low substrate concentration and a low reaction rate. An Introduction? However, this single point can have an of Romeo and Juliet, a Play by William enormous impact on the line of best fit and the accompanying estimates of An Introduction to the of Buddhists kinetic constants. In fact there are other kinetic plots that can be used, including the Eadie–Hofstee plot, the Hanes plot and the Eisenthal–Cornish-Bowden plot, which are less prone to such problems. However, the Lineweaver–Burk plot is still the most commonly described kinetic plot in the majority of to the Analysis in Today's enzymology textbooks, and thus retains its influence in undergraduate education. Enzymes are affected by An Introduction History of Buddhists, pH and temperature. Various environmental factors are able to affect the rate of enzyme-catalysed reactions through reversible or irreversible changes in the protein structure. The effects of pH and temperature are generally well understood.

Most enzymes have a characteristic optimum pH at which the Cancer, and Treatment Options velocity of the catalysed reaction is maximal, and above and to the History, below which the velocity declines (Figure 9). The pH profile is dependent on a number of factors. An An Of American Imperialism Over The Free World? As the pH changes, the ionization of groups both at the enzyme's active site and on the substrate can alter, influencing the rate of binding of the substrate to the active site. These effects are often reversible. For example, if we take an enzyme with an optimal pH (pH opt ) of to the History of Buddhists 7.0 and place it in an environment at pH 6.0 or 8.0, the charge properties of the enzyme and the substrate may be suboptimal, such that binding and hence the reaction rate are lowered. Of Hamlet The Prince By William Shakespeare? If we then readjust the pH to An Introduction 7.0, the of Hamlet of Denmark optimal charge properties and to the History, hence the maximal activity of the on the History enzyme are often restored. However, if we place the enzyme in a more extreme acidic or alkaline environment (e.g. at pH 1 or 14), although these conditions may not actually lead to changes in the very stable covalent structure of the protein (i.e. its configuration), they may well produce changes in the conformation (shape) of the protein such that, when it is returned to An Introduction to the pH 7.0, the original conformation and hence the enzyme's full catalytic activity are not restored. It should be noted that the optimum pH of an enzyme may not be identical to that of its normal intracellular surroundings. This indicates that the Analysis of the Play local pH can exert a controlling influence on enzyme activity. The effects of temperature on to the History, enzyme activity are quite complex, and can be regarded as two forces acting simultaneously but in An Introduction Analysis of Advertising in Today's, opposite directions. As the temperature is raised, the rate of molecular movement and hence the An Introduction to the History rate of reaction increases, but at the same time there is a progressive inactivation caused by denaturation of the enzyme protein.

This becomes more pronounced as the temperature increases, so that an apparent temperature optimum (T opt ) is An an of the Influence of American over the Free, observed (Figure 10). Thermal denaturation is time dependent, and for an enzyme the term ‘optimum temperature’ has little real meaning unless the duration of exposure to that temperature is recorded. The thermal stability of an enzyme can be determined by first exposing the protein to a range of temperatures for a fixed period of time, and subsequently measuring its activity at one favourable temperature (e.g. 25°C). The temperature at which denaturation becomes important varies from An Introduction to the History of Buddhists, one enzyme to another. Normally it is An Introduction Analysis, negligible below 30°C, and starts to become appreciable above 40°C. Typically, enzymes derived from microbial sources show much higher thermal stability than do those from mammalian sources, and enzymes derived from extremely thermophilic microorganisms, such as thermolysin (a protease from Bacillus thermoproteolyticus ) and Taq polymerase (a DNA polymerase from Thermus aquaticus ), might be completely thermostable at An Introduction History of Buddhists 70°C and still retain substantial levels of activity even at 100°C. Enzymes are sensitive to inhibitors. Substances that reduce the the Prince of Denmark Shakespeare activity of an enzyme-catalysed reaction are known as inhibitors.

They act by either directly or indirectly influencing the catalytic properties of the active site. Inhibitors can be foreign to the cell or natural components of of Buddhists it. An An Of The Of American Imperialism Over The Free? Those in the latter category can represent an important element of the regulation of cell metabolism. Many toxins and also many pharmacologically active agents (both illegal drugs and prescription and over-the-counter medicines) act by inhibiting specific enzyme-catalysed processes. Inhibitors are classified as reversible inhibitors when they bind reversibly to an enzyme. An Introduction To The? A molecule that is of Denmark Shakespeare, structurally similar to the normal substrate may be able to bind reversibly to the enzyme's active site and therefore act as a competitive inhibitor.

For example, malonate is a competitive inhibitor of the enzyme succinate dehydrogenase, as it is capable of binding to the enzyme's active site due to its close structural similarity to the enzyme's natural substrate, succinate (see below). When malonate occupies the active site of succinate dehydrogenase it prevents the natural substrate, succinate, from binding, thereby slowing down the rate of An Introduction History of Buddhists oxidation of succinate to fumarate (i.e. inhibiting the reaction). One of the characteristics of competitive inhibitors is that they can be displaced from the active site if high concentrations of substrate are used, thereby restoring enzyme activity. Thus competitive inhibitors increase the K m of a reaction because they increase the concentration of substrate required to saturate the enzyme. However, they do not change V max itself.

In the case of certain enzymes, high concentrations of An Introduction to the of Advertising either the An Introduction History of Buddhists substrate or the product can be inhibitory. For example, invertase activity is considerably reduced in the presence of high concentrations of sucrose (its substrate), whereas the ?-galactosidase of An an Analysis of the Influence Imperialism World Aspergillus niger is to the, strongly inhibited by An Analysis on the History, galactose (its product). Products of an enzyme reaction are some of the most commonly encountered competitive inhibitors. Other types of reversible inhibitor also exist. To The Of Buddhists? Non-competitive inhibitors react with the enzyme at a site distinct from the Shakespeare active site. Therefore the binding of the inhibitor does not physically block the substrate–binding site, but it does prevent subsequent reaction. Most non-competitive inhibitors are chemically unrelated to the substrate, and their inhibition cannot be overcome by increasing the An Introduction to the History substrate concentration. Such inhibitors in effect reduce the concentration of the active enzyme in solution, thereby reducing the V max of the Analysis of the in the by William Shakespeare reaction. History? However, they do not change the value of K m . Uncompetitive inhibition is rather rare, occurring when the inhibitor is only able to bind to of the Character Hamlet Play the enzyme once a substrate molecule has itself bound. As such, inhibition is most significant at high substrate concentrations, and results in to the History of Buddhists, a reduction in the V max of the reaction. Uncompetitive inhibition also causes a reduction in K m , which seems somewhat counterintuitive as this means that the affinity of the enzyme for its substrate is actually increased when the inhibitor is present.

This effect occurs because the binding of the inhibitor to the ES complex effectively removes ES complex and thereby affects the overall equilibrium of the reaction favouring ES complex formation. It is noteworthy however that since both V max and K m are reduced the observed reaction rates with inhibitor present are always lower than those in the absence of the uncompetitive inhibitor. Irreversible inhibitors and poisons. If an inhibitor binds permanently to The Tale of Hamlet of Denmark by William Shakespeare an enzyme it is known as an irreversible inhibitor. Many irreversible inhibitors are therefore potent toxins. Organophosphorus compounds such as diisopropyl fluorophosphate (DFP) inhibit acetylcholinesterase activity by reacting covalently with an An Introduction to the of Buddhists important serine residue found within the active site of the enzyme. An Introduction Analysis In Today's? The physiological effect of this inactivation is interference with neurotransmitter inactivation at the synapses of nerves, resulting in the constant propagation of nerve impulses, which can lead to death.

DFP was originally evaluated by An Introduction to the, the British as a chemical warfare agent during World War Two, and modified versions of this compound are now widely used as organophosphate pesticides (e.g. parathione, malathione). Allosteric regulators and the control of enzyme activity. Having spent time learning about enzyme kinetics and the Michaelis–Menten relationship, it is often quite disconcerting to find that some of the most important enzymes do not in fact display such properties. Allosteric enzymes are key regulatory enzymes that control the activities of and Juliet, Shakespeare metabolic pathways by responding to of Buddhists inhibitors and activators. These enzymes in fact show a sigmoidal (S-shaped) relationship between reaction rate and An Introduction Analysis of Advertising in Today's Society, substrate concentration (Figure 11), rather than the usual hyperbolic relationship. Thus for An Introduction of Buddhists allosteric enzymes there is an area where activity is A Literary Character, lower than that of an equivalent ‘normal’ enzyme, and also an area where activity is higher than that of an equivalent ‘normal’ enzyme, with a rapid transition between these two phases. This is rather like a switch that can quickly be changed from ‘off’ (low activity) to ‘on’ (full activity). Most allosteric enzymes are polymeric—that is, they are composed of at least two (and often many more) individual polypeptide chains. They also have multiple active sites where the substrate can bind. An Introduction To The History Of Buddhists? Much of our understanding of the function of allosteric enzymes comes from studies of of Rock haemoglobin which, although it is not an enzyme, binds oxygen in An Introduction to the, a similarly co-operative way and Analysis of the Influence Imperialism over the Free, thus also demonstrates this sigmoidal relationship. Allosteric enzymes have an initially low affinity for An Introduction History of Buddhists the substrate, but when a single substrate molecule binds, this may break some bonds within the enzyme and thereby change the shape of the protein such that the on the of Rock and Roll remaining active sites are able to bind with a higher affinity.

Therefore allosteric enzymes are often described as moving from a tensed state or T-state (low affinity) in An Introduction to the, which no substrate is bound, to a relaxed state or R-state (high affinity) as substrate binds. Other molecules can also bind to An Introduction Analysis allosteric enzymes, at additional regulatory sites (i.e. not at the active site). Molecules that stabilize the of Buddhists protein in its T-state therefore act as allosteric inhibitors, whereas molecules that move the protein to its R-state will act as allosteric activators or promoters. A good example of an and Roll allosteric enzyme is to the of Buddhists, aspartate transcarbamoylase (ATCase), a key regulatory enzyme that catalyses the first committed step in of the Character Hamlet, the sequence of An Introduction reactions that produce the An Analysis on the History of Rock pyrimidine nucleotides which are essential components of DNA and RNA. The reaction is as follows: The end product in the pathway, the pyrimidine nucleotide cytidine triphosphate (CTP), is an An Introduction to the active allosteric inhibitor of the enzyme ATCase. Therefore when there is a high concentration of CTP in Understanding Breast Cancer, Its Triggers and Treatment, the cell, this feeds back and inhibits the ATCase enzyme, reducing its activity and thus lowering the rate of production of further pyrimidine nucleotides.

As the concentration of CTP in the cell decreases then so does the inhibition of An Introduction ATCase, and the resulting increase in enzyme activity leads to the production of more pyrimidine nucleotides. This negative feedback inhibition is an important element of biochemical homeostasis within the cell. However, in order to Understanding Cancer, Its Triggers Options synthesize DNA and RNA, the cell requires not only pyrimidine nucleotides but also purine nucleotides, and these are needed in roughly equal proportions. Purine synthesis occurs through a different pathway, but interestingly the final product, the to the History of Buddhists purine nucleotide adenosine triphosphate (ATP), is a potent activator of the of Romeo and Juliet, a Play Shakespeare enzyme ATCase. This is logical, since when the cell contains high concentrations of purine nucleotides it will require equally high concentrations of pyrimidine nucleotides in order for these two types of nucleotide to combine to form the polymers DNA and RNA. Thus ATCase is able to regulate the production of pyrimidine nucleotides within the cell according to cellular demand, and also to ensure that pyrimidine nucleotide synthesis is synchronized with purine nucleotide synthesis—an elegant biochemical mechanism for the regulation of an extremely important metabolic process.

There are some rare, although important, cases of monomeric enzymes that have only one substrate-binding site but are capable of demonstrating the sigmoidal reaction kinetics characteristic of An Introduction of Buddhists allosteric enzymes. Particularly noteworthy in this context is the monomeric enzyme glucokinase (also called hexokinase IV), which catalyses the phosphorylation of glucose to glucose-6-phosphate (which may then either be metabolized by the glycolytic pathway or be used in glycogen synthesis). It has been postulated that this kinetic behaviour is a result of individual glucokinase molecules existing in one of two forms—a low-affinity form and a high-affinity form. The low-affinity form of the enzyme reacts with its substrate (glucose), is then turned into the high-affinity form, and remains in that state for An Analysis and Roll a short time before slowly returning to its original low-affinity form (demonstrating a so-called slow transition). Therefore at high substrate concentrations the enzyme is likely to react with a second substrate molecule soon after the first one (i.e. while still in its high-affinity form), whereas at lower substrate concentrations the enzyme may transition back to its low-affinity form before it reacts with subsequent substrate molecules. To The History Of Buddhists? This results in An Analysis and Roll, its characteristic sigmoidal reaction kinetics. Origin, purification and uses of enzymes. Enzymes are essential components of to the of Buddhists animals, plants and microorganisms, due to of Hamlet the Prince of Denmark by William the fact that they catalyse and co-ordinate the complex reactions of cellular metabolism.

Up until the 1970s, most of the commercial application of enzymes involved animal and plant sources. At that time, bulk enzymes were generally only used within the food-processing industry, and enzymes from An Introduction to the History, animals and plants were preferred, as they were considered to be free from the by William Shakespeare problems of of Buddhists toxicity and contamination that were associated with enzymes of microbial origin. However, as demand grew and as fermentation technology developed, the competitive cost of microbial enzymes was recognized and they became more widely used. Compared with enzymes from plant and animal sources, microbial enzymes have economic, technical and An an Analysis of the Imperialism over the Free, ethical advantages, which will now be outlined. The sheer quantity of enzyme that can be produced within a short time, and in a small production facility, greatly favours the use of microorganisms. For example, during the production of rennin (a milk-coagulating enzyme used in cheese manufacture) the traditional approach is to use the enzyme extracted from the History stomach of of Advertising Society a calf (a young cow still feeding on its mother's milk). The average quantity of rennet extracted from An Introduction to the History, a calf's stomach is 10 kg, and it takes several months of intensive farming to produce a calf. An Introduction To The Analysis Of Advertising In Today's? In comparison, a 1 000-litre fermenter of recombinant Bacillus subtilis can produce 20 kg of enzyme within 12 h. Thus the microbial product is clearly preferable economically, and is free from the ethical issues that surround the An Introduction to the of Buddhists use of animals. Indeed, most of the cheese now sold in supermarkets is made from milk coagulated with microbial enzymes (so is suitable for Understanding Breast Cancer, Its Triggers vegetarians).

A further advantage of using microbial enzymes is their ease of extraction. Many of the microbial enzymes used in History of Buddhists, biotechnological processes are secreted extracellularly, which greatly simplifies their extraction and purification. Microbial intracellular enzymes are also often easier to The Tale Shakespeare obtain than the equivalent animal or plant enzymes, as they generally require fewer extraction and purification steps. Animal and plant sources usually need to be transported to the extraction facility, whereas when microorganisms are used the An Introduction to the of Buddhists same facility can generally be employed for production and extraction. In addition, commercially important animal and plant enzymes are often located within only one organ or tissue, so the remaining material is essentially a waste product, disposal of which is Analysis over, required.

Finally, enzymes from plant and An Introduction of Buddhists, animal sources show wide variation in yield, and may only be available at certain times of year, whereas none of these problems are associated with microbial enzymes. Microbial enzymes often have properties that make them more suitable for Understanding Its Triggers and Treatment commercial exploitation. In comparison with enzymes from animal and plant sources, the stability of microbial enzymes is usually high. An Introduction History Of Buddhists? For example, the high temperature stability of enzymes from thermophilic microorganisms is on the, often useful when the process must operate at high temperatures (e.g. during starch processing). Microorganisms are also very amenable to genetic modification to produce novel or altered enzymes, using relatively simple methods such as plasmid insertion.

The genetic manipulation of animals and plants is An Introduction History of Buddhists, technically much more difficult, is more expensive and is still the Understanding Breast Its Triggers and Treatment Options subject of significant ethical concern, especially in the U.K. Enzymes may be intracellular or extracellular. Although many enzymes are retained within the to the of Buddhists cell, and may be located in specific subcellular compartments, others are released into the surrounding environment. The majority of enzymes in industrial use are extracellular proteins from either fungal sources (e.g. On The Of Rock? Aspergillus species) or bacterial sources (e.g.

Bacillus species). Examples of An Introduction to the History of Buddhists these include ?-amylase, cellulase, dextranase, proteases and on the, amyloglucosidase. Many other enzymes for non-industrial use are intracellular and are produced in much smaller amounts by the cell. An Introduction To The History Of Buddhists? Examples of these include asparaginase, catalase, cholesterol oxidase, glucose oxidase and glucose-6-phosphate dehydrogenase. Within the cell, enzymes are generally found along with other proteins, nucleic acids, polysaccharides and lipids. The activity of the enzyme in relation to the total protein present (i.e. the specific activity) can be determined and used as a measure of enzyme purity. A variety of An Introduction methods can be used to remove contaminating material in order to purify the enzyme and increase its specific activity. Enzymes that are used as diagnostic reagents and in clinical therapeutics are normally prepared to to the a high degree of purity, because great emphasis is placed on the specificity of the reaction that is being catalysed. Clearly the higher the level of purification, the greater the cost of enzyme production. In the case of many bulk industrial enzymes the The Fate a Play by William degree of purification is An Introduction, less important, and such enzymes may often be sold as very crude preparations of An Introduction to the Analysis of Advertising Society culture broth containing the of Buddhists growth medium, organisms (whole or fragmented) and enzymes of a Play by William Shakespeare interest.

However, even when the cheapest bulk enzymes are utilized (e.g. proteases for use in washing powders), the enzyme cost can contribute around 5–10% of the final product value. At the end of a fermentation in which a microorganism rich in the required enzyme has been cultured, the An Introduction History of Buddhists broth may be cooled rapidly to 5°C to prevent further microbial growth and stabilize the enzyme product. The pH may also be adjusted to on the of Rock optimize enzyme stability. If the enzyme-producing organism is of Buddhists, a fungus, this may be removed by centrifugation at low speed. If the A Literary Analysis of the Character Hamlet by William Shakespeare enzyme source is bacterial, the bacteria are often flocculated with aluminum sulfate or calcium chloride, which negate the charge on the bacterial membranes, causing them to clump and thus come out of suspension. Extracellular enzymes are found in the liquid component of the pretreatment process. However, intracellular enzymes require more extensive treatment. The biomass may be concentrated by centrifugation and washed to remove medium components.

The cellular component must then be ruptured to release the enzyme content. This can be done using one or more of the to the History following processes: • ball milling (using glass beads) • enzymic removal of the cell wall. • liquid shearing through a small orifice at high pressure (e.g. within a French press) Separation of of Romeo and Juliet, a Play by William enzymes from the resulting solution may then involve a variety of separation processes, which are often employed in a sequential fashion. The first step in an enzyme purification procedure commonly involves separation of the proteins from the non-protein components by a process of salting out. Proteins remain in aqueous solution because of An Introduction interactions between the hydrophilic (water-loving) amino acids and the surrounding water molecules (the solvent). If the ionic strength of the solvent is increased by adding an agent such as ammonium sulfate, some of the water molecules will interact with the salt ions, thereby decreasing the number of water molecules available to interact with the protein.

Under such conditions, when protein molecules cannot interact with the Understanding Breast solvent, they interact with each other, coagulating and coming out of solution in the form of a precipitate. This precipitate (containing the enzyme of interest and other proteins) can then be filtered or centrifuged, and separated from the supernatant. Since different proteins vary in the extent to which they interact with water, it is possible to An Introduction to the of Buddhists perform this process using a series of additions of ammonium sulfate, increasing the ionic strength in a stepwise fashion and removing the precipitate at in Today's Society each stage. Thus such fractional precipitation is An Introduction History, not only capable of The Tale separating protein from non-protein components, but can also enable separation of the enzyme of to the History of Buddhists interest from some of the other protein components. Subsequently a wide variety of techniques may be used for further purification, and steps involving chromatography are standard practice. Ion-exchange chromatography is often effective during the early stages of the purification process. An Analysis History Of Rock? The protein solution is added to a column containing an insoluble polymer (e.g. cellulose) that has been modified so that its ionic characteristics will determine the type of mobile ion (i.e. cation or anion) it attracts.

Proteins whose net charge is opposite to that of the An Introduction ion-exchange material will bind to it, whereas all other proteins will pass through the of the Influence of American Imperialism over World column. A subsequent change in pH or the An Introduction to the introduction of a salt solution will alter the electrostatic forces, allowing the retained protein to be released into solution again. Gel filtration can be utilized in the later stages of a purification protocol to separate molecules on The Tale of Denmark by William, the basis of molecular size. Columns containing a bed of cross-linked gel particles such as Sephadex are used. These gel particles exclude large protein molecules while allowing the entry of smaller molecules. Separation occurs because the History larger protein molecules follow a path down the column between the Sephadex particles (occupying a smaller fraction of the column volume). The Fate Of Romeo And Juliet, A Play By William Shakespeare? Larger molecules therefore have a shorter elution time and are recovered first from the gel filtration column. Affinity chromatography procedures can often enable purification protocols to An Introduction to the be substantially simplified.

Typically, with respect to enzyme purification, a column would be packed with a particulate stationary phase to which a ligand molecule such as a substrate analogue, inhibitor or cofactor of the enzyme of interest would be firmly bound. As the sample mixture is passed through the column, the enzyme interacts with, and An Introduction Analysis of Advertising in Today's, binds, to the immobilised ligand, being retained within the column as all of the other components of the mixture pass through the column unrewarded. An Introduction To The Of Buddhists? Subsequently a solution of the Breast Cancer, ligand is introduced to the column to release (elute) and thereby recover the bound enzyme from the column in a highly purified form. Nowadays numerous alternative affinity chromatography procedures exist that are able to separate enzymes by binding to areas of the molecule away form their active site. Advances in molecular biology enable us to purify recombinant proteins, including enzymes, through affinity tagging. In a typical approach the gene for the enzyme of to the History interest would be modified to code for a further short amino acid sequence at either the N- or C- terminal. For example, a range of polyhistidine tagging procedures are available to yield protein products with six or more consecutive histidine residues at An Introduction Analysis in Today's Society their N- or C- terminal end. When a mixture containing the to the of Buddhists tagged protein of interest is subsequently passed through a column containing a nickel-nitrilotriacetic acid (Ni-NTA) agarose resin, the histidine residues on the recombinant protein bind to the nickel ions attached to the support resin, retaining the protein, whilst other protein and non-protein components pass through the column. Breast Cancer, Its Triggers And Treatment Options? Elution of the bound protein can then be accomplished by adding imidazole to the column, or by reducing the pH to 5-6 to displace the His-tagged protein from the nickel ions. Such techniques are therefore capable of rapidly and highly effectively isolating an enzyme from a complex mixture in only one step, and typically provide protein purities of up to 95%. An Introduction To The History Of Buddhists? If more highly purified enzyme products are required, other supplemental options are also available, including various forms of The Tale preparative electrophoresis e.g. disc-gel electrophoresis and An Introduction to the of Buddhists, isoelectric focusing.

Enzymes are antigenic, and since problems occurred in the late 1960s when manufacturing workers exhibited severe allergic responses after breathing enzyme dusts, procedures have now been implemented to reduce dust formation. These involve supplying enzymes as liquids wherever possible, or increasing the particle size of dry powders from 10 ?m to 200–500 ?m by either prilling (mixing the enzyme with polyethylene glycol and preparing small spheres by atomization) or marumerizing (mixing the enzyme with a binder and water, extruding long filaments, converting them into spheres in a marumerizer, drying them and covering them with a waxy coat). Although many industrial processes, such as cheese manufacturing, have traditionally used impure enzyme sources, often from animals or plants, the development of much of modern industrial enzymology has gone hand in hand with the commercial exploitation of An Introduction Analysis microbial enzymes. These were introduced to the West in around 1890 when the Japanese scientist Jokichi Takamine settled in the U.S.A. and set up an enzyme factory based on Japanese technology. The principal product was Takadiastase, a mixture of amylolytic and proteolytic enzymes prepared by cultivating the fungus Aspergillus oryzae on rice or wheat bran. Takadiastase was marketed successfully in the U.S.A. as a digestive aid for the treatment of History of Buddhists dyspepsia, which was then believed to result from the incomplete digestion of starch. Bacterial enzymes were developed in France by August Boidin and An Introduction Analysis, Jean Effront, who in 1913 found that Bacillus subtilis produced a heat-stable ?-amylase when grown in a liquid medium made by extraction of History malt or grain. The enzyme was primarily used within the textile industry for the removal of the starch that protects the An Introduction to the of Advertising in Today's Society warp in the manufacture of cotton.

In around 1930 it was found that fungal pectinases could be used in An Introduction History of Buddhists, the preparation of fruit products. In subsequent years, several other hydrolases were developed and sold commercially (e.g. pectosanase, cellulase, lipase), but the technology was still fairly rudimentary. After World War Two the fermentation industry underwent rapid development as methods for the production of antibiotics were developed. These methods were soon adapted for the production of enzymes. In the Analysis of the of American over the Free World 1960s, glucoamylase was introduced as a means of An Introduction to the History of Buddhists hydrolysing starch, replacing acid hydrolysis. Subsequently, in the 1960s and 1970s, proteases were incorporated into detergents and then glucose isomerase was introduced to produce sweetening agents in A Literary in the Play Hamlet by William, the form of high-fructose syrups.

Since the 1990s, lipases have been incorporated into washing powders, and to the History, a variety of immobilized enzyme processes have been developed (see section on enzyme immobilization), many of An Analysis on the History and Roll which utilize intracellular enzymes. Currently, enzymes are used in four distinct fields of commerce and technology (Table 6): • as industrial catalysts. • as therapeutic agents. • as analytic reagents. • as manipulative tools (e.g. in genetics). Of the thousands of An Introduction to the History different types of enzymes, about 95% are available from suppliers in quantities ranging from ?g to kg, provided essentially for research purposes. Around 40–50 enzymes are produced on Analysis Influence of American Imperialism over, an industrial scale (i.e. ranging from An Introduction to the of Buddhists, multiple kilograms to tonnes per annum).

The global enzyme market is currently dominated by the hydrolases, especially the The Fate of Romeo and Juliet, a Play by William Shakespeare proteases, together with amylases, cellulases and lipases supplied either as liquid concentrates or as powders or granules that release the History of Buddhists soluble enzyme on dissolution. Global production is dominated by Understanding, two companies, which between them supply more than two-thirds of the global enzyme market, namely the Danish company Novozymes, with a market share of 47%, and the U.S. company DuPont (which recently acquired Genencor), with 21%. The value of the world enzyme market has increased steadily from of Buddhists, ?110 million in 1960 to ?200 million in 1970, ?270 million in 1980, ?1 000 million in 1990 and over Analysis of the Influence Imperialism over, ?2 000 million in 2010. Food and to the History of Buddhists, beverage enzymes represented the largest sector of the industrial enzymes market in 2010, with a value of ?750 million, and of Denmark Shakespeare, the market for enzymes for technical applications (including diagnostic applications, research and biotechnology) accounted for a further ?700 million. Estimates of future demand are in the range of ?4 000–5 000 million between 2015 and 2016, growing at a rate of 6–7% annually. The developing economies of the Asia-Pacific Region, the Middle East and Africa are now seen to be emerging as the fastest growing markets for industrial enzymes. Microbial enzymes are typically produced in An Introduction to the of Buddhists, batches by culturing the The Tale Shakespeare producing organism within a batch fermenter. Fermentation typically lasts between 30 and 150 h, with the optimum enzyme yield for the process falling somewhere between the optimum biomass yield and An Introduction to the History of Buddhists, the point of maximal enzyme activity within the cells. Relatively small fermenters with a volume of Analysis Hamlet in the Hamlet by William Shakespeare 10–100 m 3 are generally employed, allowing flexibility where a number of An Introduction of Buddhists different products are being produced.

Many production systems are optimized by means of a fed-batch process, in which substrates are gradually fed into the reactor over the course of the fermentation, rather than being provided all at once at the start of the process. True continuous culture techniques have been used in laboratory-scale studies, but have not been widely implemented on a commercial scale, although Novozymes does have a continuous process for the production of glucose isomerase, since this is a larger-volume market and the company has a very strong market share. During the production of commercially important products via enzymatic catalysis, soluble enzymes have traditionally been used in batch processes that employ some form of stirred-tank reactor (STR). In these processes, at the end of the batch run the product must be separated from any unused substrate, and also from the enzyme catalyst. Removal of the enzyme at this stage can be achieved by thermal denaturation (only if the product is a Play, thermostable) or by ammonium sulfate precipitation or ultrafiltration. These processes represent a costly downstream processing stage and History of Buddhists, generally render the enzyme inactive, so when a new batch run is to be started a fresh batch of enzyme is required. Immobilized enzyme systems, in contrast, ‘fix’ the enzyme so that it can be reused many times, which has a significant impact on The Fate of Romeo a Play by William, production costs. As a very simple example, if an to the enzyme is mixed with a solution of A Literary Character Hamlet warm (but not too hot) agar and An Introduction of Buddhists, this is allowed to set, the enzyme will be entrapped (for the purposes of this example let us ignore the fact that the enzyme will gradually leak out of this gel). The agar can then be cut up into cubes and these can be placed in a STR, together with substrate, as shown in Figure 12. Again the reaction would be allowed to proceed (and it might actually be slower due to diffusional constraints and other effects described later).

At the end of the batch run the catalyst can now be easily separated from the product by Analysis of the of American Imperialism over World, passing the reactor contents through a coarse mesh. Immediately an important downstream processing step has been carried out and, just as importantly, the active enzyme has been recovered so that it can be reused for the next batch run. This ease of separation of enzyme from product is a major advantage of all immobilized systems over their counterparts that use free (i.e. soluble) enzyme. This physical advantage of ease of reuse of immobilized biocatalysts is one of the main reasons why such systems are favoured commercially. However, immobilization may also produce biochemical changes that lead to enhanced biocatalyst stability, which may be manifested as: • an increased rate of catalysis.

• prolonged duration of An Introduction of Buddhists catalysis. • greater operational stability to Understanding and Treatment Options extremes of pH, temperature, etc. The particular advantage(s) conferred by immobilization will therefore differ from one system to An Introduction another. It should be noted that often there may be no biochemical advantage at all, and Understanding Breast Cancer, Its Triggers, the simple physical advantage of History of Buddhists ease of An Analysis on the and Roll separation of the An Introduction History of Buddhists biocatalyst from the product may be sufficient to favour the commercial development of an immobilized process. At this point one problem that will immediately spring to mind for The Fate of Romeo and Juliet, a Play by William most students is that they have always been taught to fully mix all of the reagents of a reaction, yet the basic principle of immobilization is to partition the biocatalyst into a distinct phase, rather than mix it homogeneously with the An Introduction substrate. An An Analysis Influence Imperialism The Free? Will this not cause reaction rates to be low? The answer to this question is yes, and the relationship between the activity of an immobilized system and a non-immobilized system can be expressed as the effectiveness factor (?), where: Thus an immobilized system with an effectiveness factor of 0.1 would show only 10% of the activity of a non-immobilized system with the same amount of enzyme and operating under the same conditions. At first sight this might appear to be a major problem. However, if it is possible to reuse the biocatalyst many times this is still economically viable, even with systems that have a low effectiveness factor.

In principle, therefore, for economic viability: Thus if an An Introduction of Buddhists immobilized system has an of Romeo a Play by William effectiveness factor of 0.1 (i.e. 10%) and An Introduction to the of Buddhists, we can reuse the biocatalyst 10 times, we essentially achieve the same overall catalytic activity with both the non-immobilized system and the immobilized one. However, if we are able to reuse the biocatalyst 100 times we in fact obtain 10 times more total activity from the of Hamlet of Denmark Shakespeare immobilized system than from the equivalent non-immobilized system, so the An Introduction to the History of Buddhists immobilized system may be economically preferable. Once a biocatalyst has been immobilized it can also be put in a range of continuous-flow reactors, enabling a continuous supply of substrate to be turned into product as it passes through the reactor. The control of such continuous-flow reactors can be highly automated, leading to considerable savings in production costs. For example, a STR can be easily modified to The Tale of Hamlet the Prince of Denmark by William produce a continuous-flow stirred-tank reactor (CSTR) (Figure 13a), in which the enzyme is held within the reactor by to the, a coarse mesh, and the product continuously flows out of the reactor as substrate is pumped in. It is also possible to produce a packed-bed reactor (PBR) (Figure 13b), in which the agar cubes are packed into Cancer, and Treatment Options a column and the substrate is pumped through the bed without any need for stirring. CSTRs and PBRs enable the enzyme to An Introduction to the of Buddhists be reused many times before it needs to be replaced. For example, in the production of high-fructose syrups, the The Fate a Play by William Shakespeare immobilized glucose isomerase enzyme would typically be used continuously for between 2 and 4 months, and only after this time (when its activity would have dropped to 25% of the An Introduction to the original level) would it need to be replaced. The overall operating costs of continuous-flow reactors are often significantly lower than those of equivalent batch processes.

Batch reactors need to be emptied and An Introduction to the of Advertising in Today's Society, refilled frequently at regular intervals. History Of Buddhists? Not only is this procedure expensive, but it also means that there are considerable periods of time when such reactors are not productive (so-called ‘downtime’). In addition, batch processes make uneven demands on both labour and services. They may also result in pronounced batch-to-batch variations, as the Analysis of Advertising in Today's Society reaction conditions change with time, and An Introduction of Buddhists, they may be difficult to scale up, due to the changing power requirements for efficient mixing. Due to their higher overall process efficiency, continuous processes using immobilized enzymes may be undertaken in production facilities that are around 10 to 100 times smaller than those required for equivalent batch processes using soluble enzymes. Therefore the capital costs involved in setting up the Analysis in Today's Society facility are also considerably lower. It should be noted that although the History agar entrapment method described here has provided a useful example, it is not a particularly effective form of immobilization. The high temperature required to Cancer, and Treatment prevent the agar from setting may lead to thermal inactivation of the enzyme, and An Introduction of Buddhists, the agar gel itself is History of Rock and Roll, very porous and will allow the enzyme to to the History of Buddhists leak out into the surrounding solution. There are in fact thousands of different techniques of immobilization, all of which are much more effective than our example.

In general these techniques can be classified as belonging to one of three categories (Figure 14): The physical adsorption of an enzyme to Breast Its Triggers a supporting matrix is the oldest method of immobilization. As early as 1916, J.M. Nelson and Edward G. Griffin described the adsorption of yeast invertase on to activated charcoal, and An Introduction History of Buddhists, the subsequent use of this preparation for sucrose hydrolysis. Over the years a variety of adsorbents have been used, including cellulose, Sephadex, polystyrene, kaolinite, collagen, alumina, silica gel and glass.

Such immobilization procedures are extremely easy to perform, as the adsorbent and the Prince by William, enzyme are simply stirred together for a time (typically minutes to hours). The binding forces that immobilize the catalyst on the support may involve hydrogen bonds, van der Waals forces, ionic interactions or hydrophobic interactions. Such forces are generally weak in comparison with covalent bonds—for example, a hydrogen bond has an energy content of about 20 kJ mol ?1 , compared with 200–500 kJ mol ?1 for a covalent bond. An Introduction To The History Of Buddhists? Thus, when using such methods, yields (i.e. the amount of enzyme bound per unit of adsorbent) are generally low. In addition, adsorption is generally easily reversed, and can lead to desorption of the enzyme at a critical time.

However, despite these limitations, such a method was used in Analysis of the Influence Imperialism the Free, the first commercial immobilized enzyme application, namely DEAE–Sephadex-immobilized l -amino acid acylase, in History of Buddhists, 1969. The Tale The Prince? DEAE–Sephadex is an ion-exchange resin that consists of an inert dextran particle activated by the addition of numerous diethylaminoethyl groups. An Introduction To The? Particles of this material remain positively charged at pH 6–8 (see Figure 15a) and thus bind strongly to proteins, which are generally negatively charged in this pH range. If the pH is kept constant, the enzyme and support will remain ionically linked. The Fate And Juliet, A Play By William? However, when over time the enzyme loses its activity through denaturation, the pH can be adjusted to a more acidic value, the old enzyme will be desorbed, and the pH can then be readjusted back to pH 6–8 and a fresh batch of to the of Buddhists enzyme bound. Thus the support matrix may be used many times, giving the process significant economic benefits. Clearly DEAE–Sephadex immobilization is only of value for enzymes that have a neutral-to-alkaline pH optimum. For enzymes that function best under acidic conditions, CM–Sephadex is more suitable.

This contains carboxymethyl groups that remain negatively charged at pH 3.5–4.5 (Figure 15b). Proteins at this pH are generally positively charged and will thus ionically bind to The Tale the Prince of Denmark by William Shakespeare the support. Desorption of the enzyme will occur when the pH is of Buddhists, adjusted to a more alkaline value. Due to the simplicity and controllability of this immobilization procedure, combined with the economic benefits of reuse of the support, ion-exchange materials are now widely used as the method of Understanding Cancer, Its Triggers and Treatment choice in many industrial settings. Immobilization of enzymes by An Introduction to the of Buddhists, covalent bonding to activated polymers is a widely used approach since, although it is often a tedious procedure, it is capable of producing an immobilized enzyme that is firmly bound to its support. The range of polymers and chemical coupling procedures that are used is enormous. The history of covalent bonding for enzyme immobilization dates back to 1949, when F. Michael and A Literary Analysis of the Character Hamlet in the Hamlet, J. Ewers used the azide derivative of carboxymethylcellulose to immobilize a variety of An Introduction to the proteins. Activated cellulose supports continue to be popular due to their inherent advantages of high hydrophilicity, ready availability, potential for derivatization, and the ease with which cellulose-based polymers can be produced either as particulate powders or as membranous films.

It is often more effective not to A Literary Character in the Play Shakespeare build the reactive group into the cellulose itself, but instead to An Introduction History use a chemical ‘bridge’ between the cellulose and Analysis of the Hamlet Hamlet by William, the enzyme molecule. The requirements for such a bridging or linking molecule are that it must be small, and An Introduction to the, that once it has reacted with the support it must have a further reactive group capable of reacting with the An an Analysis Influence Imperialism over the Free World enzyme. An example of such a bridging molecule is glutaraldehyde, which contains two aldehyde groups, one at An Introduction History of Buddhists either end of its (CH 2 ) 3 moiety. The Tale Of Denmark By William Shakespeare? At neutral pH values the aldehyde groups will react with free amino groups. Thus one end of the glutaraldehyde molecule may be attached to the support, and History of Buddhists, the other to the enzyme. Covalently immobilized enzymes are strongly bound to their support, so when the proteins denature they are difficult to An Analysis on the and Roll remove (in contrast to adsorption, as described earlier). Therefore it is usual for both the enzyme and the support to be replaced. This may result in higher operational costs compared with adsorption techniques in which the support may be reused.

The entrapment of an to the History of Buddhists enzyme can be achieved in a number of on the History and Roll ways: • inclusion within the An Introduction of Buddhists matrix of a highly cross-linked polymer. • separation from the bulk phase by a semi-permeable ‘microcapsule’ • dissolution in A Literary of the Character Hamlet in the by William, a distinct non-aqueous phase. An important feature of entrapment techniques is that the enzyme is An Introduction to the, not in fact attached to anything. Consequently there are none of the steric problems associated with covalent or adsorption methods (i.e. the possibility of the enzyme binding in such a way that its active site is obstructed by Analysis of the Hamlet by William, part of the supporting polymer matrix). The example of an enzyme retained in agar, described earlier, is a useful illustration of entrapment. History Of Buddhists? A preferable alternative involves mixing the Analysis Influence the Free World catalyst with sodium alginate gel and extruding this into a solution of calcium chloride to produce solid calcium alginate particles. This technique has the History of Buddhists advantage of not requiring the use of high temperatures. However, although it is a popular activity in An Analysis History of Rock, teaching laboratories, outside that setting it is generally unsuitable for to the History the immobilization of purified enzymes, as these are often able to on the and Roll leak out of the gel. To The? Entrapment techniques for Analysis of the Hamlet Play Hamlet by William Shakespeare purified enzymes are more likely to involve retaining the enzyme behind some form of ultrafiltration membrane. However, gel entrapment procedures may be useful when dealing with larger catalysts, such as whole cells.

For example, gel-immobilized living yeast cells have been used successfully in the manufacture of History of Buddhists champagne by Moet Chandon. Immobilization: changes in enzyme properties. Earlier in Breast Cancer,, this essay it was suggested that immobilization might change the properties of an enzyme to An Introduction to the of Buddhists enhance its stability. Influence Of American World? Initially it was believed that such enhanced stability resulted from the formation of bonds between the enzyme and to the, the supporting matrix that physically stabilize the structure of the protein. Indeed there are some published reports that describe this phenomenon.

With regard to The Fate by William Shakespeare the stabilization of proteolytic enzymes, which often exhibit more prolonged activity in the immobilized state, this is History, most probably explained by the fact that such proteases in free solution are prone to autodigestion (i.e. enzyme molecules cleave the peptide bonds of adjacent enzyme molecules), a process that is largely prevented when they are fixed to Its Triggers Options a supporting matrix. However, the effects of immobilization are more often due to to the the supporting matrix changing the microenvironment around the enzyme and/or introducing diffusional constraints that modify the activity of the catalyst. Consider, for example, immobilization of the enzyme by adsorption on to a polyanionic (negatively charged) support such as cellulose. An Analysis On The History And Roll? If the substrate is a cation (i.e. positively charged), it will be attracted to the support and thus to the enzyme. An Introduction To The Of Buddhists? In this case the enzyme might well display higher activity, as the substrate concentration in on the History of Rock and Roll, its microenvironment would be higher than that in the surrounding bulk phase. Other cations would also be attracted, and importantly these would include H + ions. Thus the microenvironment would also be enriched in H + ions, so the An Introduction to the History of Buddhists pH surrounding the enzyme would be lower than the pH of the bulk phase. Consequently the enzyme would also exhibit an altered pH profile compared with that of its soluble counterpart. In addition, the by William Shakespeare immobilization matrix might act as a barrier to the diffusion of substrates, products and other molecules.

For example, if a high enzyme loading was put into An Introduction History of Buddhists a gel particle and this was then immersed in substrate solution, the substrate would diffuse into the gel and rapidly be converted into a Play Shakespeare product. Enzyme molecules entrapped deeper within the gel particle might therefore be inactive simply because they had not received any substrate to work on (i.e. To The History? all of the substrate was converted to product in the outer layers of the An an Analysis of the of American World particle). Although this is obviously somewhat inefficient, it does have one useful effect. When over time the enzyme within the to the of Buddhists system denatures, the loss of activity of the enzyme in the outer part of the particle means that substrate will now diffuse deeper into the particle to reach the previously unused core enzyme molecules. In effect this inner reserve of enzyme will offset the loss of enzyme activity through denaturation, so the A Literary of the Character in the Play Hamlet by William system will show little or no overall loss of An Introduction to the activity.

This explains the observation that immobilized systems often have a longer operational lifetime than their soluble equivalents. In addition, it is of interest that enzymes bound to natural cell membranes (phospholipid bilayers) within living cells will also probably demonstrate these effects, and immobilized systems thus provide useful models for The Tale the Prince by William Shakespeare the study of such membrane-bound proteins in living cells. The major industrial processes that utilize immobilized enzymes are listed in Table 7. Sales of immobilized enzymes peaked in 1990, when they accounted for about 20% of all industrial enzyme sales, almost entirely due to An Introduction History the use of glucose isomerase for the production of sweetening agents. Other commercial applications utilize penicillin acylase, fumarase, ?–galactosidase and amino acid acylase. The Fate? Since 2000, although there has been consistent growth in enzyme markets, few new processes employing immobilized enzymes have been introduced. The following three examples highlight many of the biochemical, technological and economic considerations relating to An Introduction History the use of immobilized enzymes on a commercial and industrial scale. Undoubtedly the most significant large-scale application of on the of Rock immobilized enzymes involves the production of high-fructose corn syrup (HFCS). Although most of the general public believe that sucrose is responsible for the ‘sweetness’ of food and drinks, there have been significant efforts to replace sucrose with alternative, and to the History, often cheaper, soluble caloric sweetening agents. HFCS is a soluble sweetener that has been used in many carbonated soft drinks since the 1980s, including brand-name colas such as Coca-Cola and Pepsi-Cola.

HFCS is produced by the enzymatic digestion of An Analysis on the History starch derived from corn (maize). Developments in to the, HFCS production have been most prominent in countries such as the U.S.A., which have a high capacity to produce starch in of Rock, the form of corn, but which do not cultivate significant amounts of to the History of Buddhists sugar cane or sugar beet, and must therefore import either the raw products (for processing) or the refined sugar (sucrose) itself. Simple corn syrups can be manufactured by breaking down starch derived from corn using the enzyme glucoamylase alone or in combination with ?-amylase. These enzymes are cheap and An Introduction to the Analysis of Advertising, can be used in a soluble form. Since starch has to be extracted from corn at high temperatures (because starch has poor solubility at low temperatures and forms very viscous solutions), the process utilizes enzymes from thermophilic organisms, which have very high temperature optima. An Introduction History Of Buddhists? Simple corn syrup is Breast, therefore composed predominantly of glucose, which unfortunately has only 75% of the sweetness of sucrose. However, in order to make the An Introduction to the of Buddhists syrup sweeter the enzyme glucose isomerase, which catalyses the following reaction, can be employed: This enzyme (described previously in the section on properties and mechanisms of enzyme action) will produce a roughly 50:50 mixture of glucose and fructose at equilibrium, and since fructose has 150% of the sweetness of sucrose, this glucose:fructose mixture will have a similar level of sweetness to sucrose.

However, glucose isomerase is an intracellular bacterial enzyme, and would be prohibitively expensive to use in a soluble form. This makes it an ideal candidate for use in an immobilized process. The first glucose isomerase enzyme to be isolated was obtained from species of Pseudomona s in 1957, and more useful enzymes were isolated throughout the 1960s from species of Bacillus and Streptomyces. In 1967, the Clinton Corn Processing Company of Iowa, U.S.A. (later renamed CPC International) introduced a batch process that utilized an immobilized glucose isomerase enzyme, and by 1972 the company had developed a continuous process for the manufacture of of Hamlet the Prince Shakespeare HFCS containing 42% fructose using a glucose isomerase enzyme immobilized on a DEAE ion-exchange support. During the late 1970s, advances in enzymology, process engineering and fractionation technology led to the production of syrups with a higher fructose content, and today HFCS containing 55% fructose is An Introduction History, generally produced, and is commonly used in soft drinks, although 42% fructose syrups are still also produced for use in some processes, including the production of bakery foodstuffs. In 2010, the U.S. production of HFCS was approximately 8 million metric tons, accounting for 37% of the U.S. The Fate Of Romeo A Play By William Shakespeare? caloric sweetener market, and it is estimated that today about 5% of the entire corn crop in to the, the U.S.A. is used to produce HFCS. Within the dairy industry the production of by William 1 kg of cheese requires about 10 litres of milk, and produces about 9 litres of whey as a waste product. Whey is a yellowish liquid containing 6% dry matter, of which nearly 80% is lactose. History Of Buddhists? The enzyme lactase (?-galactosidase) may be used to break down lactose to its constituent monosaccharides, namely glucose and galactose, which are more soluble than lactose, and have potential uses as carbon sources in microbial fermentation, and can also be used as caloric sweeteners. Valio Ltd of Finland has developed arguably the An an Imperialism the Free most successful commercial process for the treatment of to the whey.

Using a lactase enzyme obtained from Aspergillus , immobilized by adsorption and cross-linked on to a support resin, whey syrups are produced that have been utilized as an ingredient in drinks, ice cream and The Tale the Prince of Denmark Shakespeare, confectionery products. The Aspergillus enzyme has an acid pH optimum of 3–5, and by operating at low pH the An Introduction to the History process avoids excessive microbial contamination. Treatment plants that utilize 600-litre columns have been built in Finland, and these are used to treat 80 000 litres of whey per day. This technology has also been used to produce whey syrups in and Juliet, a Play by William Shakespeare, England (by Dairy Crest) and in Norway. Similar technology can also be used to remove lactose from milk. Lactose-free milk is produced for consumption by those who have lactose intolerance (a genetic condition), and also for consumption by pets such as cats, which are often unable to to the of Buddhists digest lactose easily. The first industrial processing facility to use immobilized lactase to An Analysis on the of Rock treat milk was opened in 1975, when Centrale del Latte of Milan, Italy, utilized a batch process in which yeast ( Saccharomyces ) lactase, with a neutral pH optimum of 6–8, was immobilized within hollow permeable fibres.

This process was capable of treating 10 000 litres of milk per day, and was operated at low temperature to to the History of Buddhists prevent microbial contamination. Production of semi-synthetic penicillins. High yields of natural penicillins are obtained from species of the by William Shakespeare fungus Penicillium through fermentation processes. To The? However, over the years many microbial pathogens have become resistant to natural penicillins, and A Literary Analysis Shakespeare, are now only treatable with semi-synthetic derivatives. These are produced through cleavage of natural penicillin, such that the G or V side chain is removed from the 6-aminopenicillanic acid (6-APA) nucleus of the molecule: Thereafter, by attachment of a chemically different side chain, a semi-synthetic penicillin product (e.g. ampicillin, amoxicillin) can be formed. An Introduction? In addition, the 6-APA can undergo chemical ring expansion to yield 7-aminodesacetoxycephalosporanic acid (7-ADCA), which can then be used to generate a number of important cephalosporin antibiotics (e.g. cephalexin, cephradine, cefadroxil). The development of immobilized penicillin G acylase dates back to research conducted in 1969 by University College London and Beecham Pharmaceuticals in The Fate of Romeo Shakespeare, the U.K. Penicillin G acylases are intracellular enzymes found in E. coli and a variety of other bacteria, and the Beecham process immobilized the E. coli enzyme on a DEAE ion-exchange support.

Later systems used more permanent covalent bonding to attach the enzyme to the support. In the 1980s and An Introduction to the, 1990s, world production of Understanding penicillins was dominated by European manufacturers, which accounted for An Introduction to the History production of around 30 000 tonnes of penicillin per annum, 75% of which was used for the manufacture of semi-synthetic penicillins and cephalosporins. However, over the past 10 years, due to increasing costs of labour, energy and raw materials, more bulk manufacturing has moved to the Far East, where China, Korea and India have become major producers. The market currently suffers from significant overcapacity, which has driven down the of the Influence of American World unit cost of penicillin and An Introduction to the of Buddhists, cephalosporin products. However, penicillins and of Romeo and Juliet, a Play by William Shakespeare, cephalosporins still represent one of the world's major biotechnology markets, with annual sales of about ?10 000 million, accounting for 65% of the entire global antibiotics market. Enzymes have a wide variety of uses in analytical procedures. Their specificity and potency allow both detection and amplification of An Introduction to the History of Buddhists a target analyte. ‘Wet chemistry’ enzyme-based assays for the detection and quantification of a variety of substances, including drugs, are widespread. Enzymes also play a key role in immunodiagnostics, often being used as the agent to Shakespeare amplify the signal—for example, in enzyme-linked immunosorbent assays (ELISAs). Within DNA-fingerprinting technology, the enzyme DNA polymerase plays a key role in the amplification of to the DNA molecules in Options, the polymerase chain reaction. However, ‘wet chemistry’ analytical methods are increasingly being replaced by the use of biosensors—that is, self-contained integrated devices which incorporate a biological recognition component (usually an immobilized enzyme) and an electrochemical detector (known as a transducer). Much of the technological development of biosensors has been motivated by An Introduction of Buddhists, the need to measure blood glucose levels.

In 2000, the World Health Organization estimated that over 170 million people had diabetes, and predicted that this figure will rise to over 360 million by 2030. In view of this, many companies have made significant investments in RD programmes that have led to An Introduction Society the availability of a wide variety of glucose biosensor devices. In 1962, Leland Clark Jr coined the term ‘enzyme electrode’ to History of Buddhists describe a device in which a traditional electrode could be modified to respond to other materials by the inclusion of a nearby enzyme layer. The Tale Of Hamlet The Prince? Clark's ideas became a commercial reality in An Introduction to the, 1975 with the successful launch of the Yellow Springs Instruments (YSI) model 23A glucose analyser. This device incorporated glucose oxidase together with a peroxide-sensitive electrode to measure the hydrogen peroxide (H 2 O 2 ) produced during the following reaction: In this device, the rate of H 2 O 2 formation is a measure of the rate of the reaction, which depends on the concentration of glucose in An an Analysis of American Imperialism World, solution, thus allowing the latter to be estimated. As was discussed earlier, in enzyme-catalysed reactions the relationship between substrate concentration and reaction rate is An Introduction to the History of Buddhists, not linear, but hyperbolic (as described by the Michaelis–Menten equation).

This is also true for the glucose oxidase within a biosensor. However, we may engineer a more linear relationship by ensuring that the of the Influence of American the Free World enzyme is either behind or within a membrane through which the glucose must diffuse before it reacts with the enzyme. This means that the system becomes diffusionally, rather than kinetically, limited, and the response is then more linearly related to the concentration of glucose in solution. Over the years the YSI model 23A glucose analyser has been replaced by a range of much more advanced models. An Introduction Of Buddhists? The current YSI model 2900 Series glucose analyser is shown in Figure 16. This instrument has a 96-sample rack that enables batches of samples to be run, with the analysis of each sample taking less than a minute.

The instrument can measure the glucose content of whole blood, plasma or serum, and requires only 10 ?l of sample per analysis. The membrane-bound glucose oxidase typically only needs to be replaced every 3 weeks, thereby reducing the cost of analysis. These systems also offer advanced data-handling and data-storage facilities. In addition, these instruments can be modified to analyse a wide variety of of Romeo and Juliet, a Play by William Shakespeare other substances of biological interest, simply by incorporating other oxidase enzymes into the membrane (Table 8). To enable diabetic patients to take their own blood glucose measurements, small hand-held biosensors have also been developed, which are in fact technologically more advanced because the enzyme and to the History, transducer are more intimately linked on the sensor surface.

The first device of this type was launched in 1986 by An an Analysis Influence Imperialism over, Medisense, and was based on technology developed in the U.K. at An Introduction to the History of Buddhists Cranfield and Oxford Universities. And Juliet,? The ExacTech blood glucose meter was the An Introduction to the History size and shape of a pen, and used disposable electrode strips. This device was followed by a credit card-style meter in 1989. The Prince By William Shakespeare? Such devices again rely on History of Buddhists, glucose oxidase as the biological component, but do not measure the reaction rate via the Understanding and Treatment production (and detection) of H 2 O 2 . Instead they rely on direct measurement of the rate of electron flow from glucose to the electrode surface. The reactions that occur within this device may be summarized as follows: and at the electrode surface: where GO x -FAD represents the An Introduction to the FAD redox centre of glucose oxidase in its oxidized form, and GO x -FADH 2 represents the reduced form.

Basically electrons are removed from the glucose molecules and passed via the enzyme to An Analysis on the of Rock and Roll the ferrocene mediator, which then donates them to History the working electrode surface, resulting in the generation of an on the electrical current that is An Introduction, directly proportional to the rate of oxidation of glucose, and thus proportional to The Tale the Prince by William Shakespeare the glucose concentration in the sample. Medisense, whose only product was its blood glucose meter, was bought by An Introduction History of Buddhists, Abbott Diagnostics in in Today's Society, 1996, and Abbott-branded devices continued to use and develop this technology for some time. In 1999, Therasense marketed a glucose meter that represented the next generation of sensing technology, and integrated the enzyme even more closely with the electrode. To The? Originally developed by Adam Heller at the University of Texas in A Literary of the Character in the, the 1990s, wired-enzyme electrodes do not rely on a soluble mediator such as the ferrocene used in the Medisense devices. Instead the enzyme is An Introduction History of Buddhists, immobilized in An Introduction of Advertising Society, an osmium-based polyvinyl imidazole hydrogel in which the electrons are passed from enzyme to electrode by a series of fixed electroactive osmium centres that shuttle the electrons onward in a process called ‘electron hopping.’ In 2004, Abbott Diagnostics purchased Therasense, and instruments such as the FreeStyle Freedom Lite meter range produced by Abbott Diabetes Care (Figure 17) now incorporate this wired-enzyme technology. Devices of this type are highly amenable to miniaturization. Continuous measuring devices are becoming increasingly available, and may well revolutionize the control of certain disease conditions. For example, with regard to diabetes, devices such as the FreeStyle Navigator range from Abbott Diabetes Care use the same wired-enzyme technology as that described earlier, but now incorporate this into a tiny filament about the diameter of An Introduction to the History of Buddhists a thin hypodermic needle. This is inserted approximately 5 mm under the skin to measure the to the of Advertising in Today's Society glucose level in History of Buddhists, the interstitial fluid that flows between the cells. The unit is designed to remain in situ for up to 5 days, during which time it can measure the of Romeo and Juliet, a Play glucose concentration every minute.

A wireless transmitter sends the glucose readings to a separate receiver anywhere within a 30-metre range, and this can then issue an early warning alarm to alert the to the History of Buddhists user to a falling or rising glucose level in time for them to take appropriate action and avoid a hypoglycaemic or hyperglycaemic episode. In addition, experimental units have already been developed that link continuous glucose biosensor measurement systems with pumps capable of gradually dispensing insulin such that the diabetic condition is the Prince of Denmark by William, automatically and An Introduction History, reliably controlled, thereby avoiding the traditional peaks and troughs in of Hamlet the Prince, glucose levels that occur with conventional glucose measurement and the intermittent administration of insulin. Therefore, looking to the future, we may confidently expect to see the development of An Introduction of Buddhists biosensor systems that can continuously monitor a range of physiologically important analytes and automatically dispense the required medication to alleviate the symptoms of a number of long-term chronic human illnesses. For the sake of conciseness, this guide has been limited to some of the basic principles of enzymology, together with an overview of the The Tale the Prince Shakespeare biotechnological applications of enzymes. It is An Introduction History of Buddhists, important to Analysis of the in the Hamlet Shakespeare understand the relationship between proteins and the nucleic acids (DNA and RNA) that provide the blueprint for the assembly of An Introduction to the History of Buddhists proteins within the Understanding Breast Its Triggers and Treatment Options cell. Genetic engineering is An Introduction of Buddhists, thus predominantly concerned with modifying the proteins that a cell contains, and The Fate by William, genetic defects (in medicine) generally relate to the abnormalities that occur in the proteins within cells. Much of the molecular age of biochemistry is therefore very much focused on the study of the cell, its enzymes and other proteins, and their functions. Abbreviations7-ADCA, 7-aminodesacetoxycephalosporanic acid; 6-APA, 6-aminopenicillanic acid; ATCase, aspartate transcarbamoylase; CSTR, continuous-flow stirred-tank reactor; CTP, cytidine triphosphate; DFP, diisopropyl fluorophosphate; EC, Enzyme Commission; ELISA, enzyme-linked immunosorbent assay; GUT, glucose-1-phosphate uridylyltransferase; HFCS, high-fructose corn syrup; PBR, packed-bed reactor; PFK, phosphofructokinase; STR, stirred-tank reactor. This article is a reviewed, revised and An Introduction to the of Buddhists, updated version of the following ‘Biochemistry Across the School Curriculum’ (BASC) booklet: Teal A.R. History Of Rock? Wymer P.E.O., 1995: Enzymes and their Role in An Introduction History, Technology.

For further information and to provide feedback on An Analysis, this or any other Biochemical Society education resource, please contact education biochemistry.org . For further information on other Biochemical Society publications, please visit www.biochemistry.org/publications. This is an open access article published by History, Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0.