Chymosin for Classroom Experiments an Introductionto the Nature & Action of Enzymes Ken Harewood Richard Hinman

How-To-Do-It

Chymosin for Classroom Experiments An Introductionto the Nature & Action of Enzymes Ken Harewood Richard Hinman

The Story of Chymosin in the lining of the calf stomach and about 4.5 with hydrochloric acid, the inserted that gene into E. coli and other same method used for centuries to When Little Miss Muffet sat on her common microorganismsusing recom- prepare rennin from the extractof calf Downloaded from http://online.ucpress.edu/abt/article-pdf/61/4/288/48912/4450673.pdf by guest on 28 September 2021 tuffet, her frugal repast of curds and binant DNA methods. The resulting stomachs. The old and new methods whey was coagulated milk-the first "/recombinant organism" was then are compared in Figure 2. intermediate in cheese-making. Her induced to produce copious quantities It is worth noting that the microor- meal was probably the product of the of prochymosin by the process of fer- ganisms were engineered to produce acidity from milk souring, the result mentation.A descriptionof the process prochymosin rather than chymosin of microbial action. Addition of acid for E. coli is given in Flamm 1991. itself because the genetic engineering such as vinegar also causes coagula- This first novel step was followed by strategy was much simpler and more tion. When the semi-liquid curd from conversion of the prochymosin to certain to produce a product identical these processes is freed from the liquid active chymosin (Figure 1) by adjust- to chymosin prepared in the tradi- whey by filtration, it can be used as ing the pH of the protein solution to tional way. the starting point for some cheeses. However, the best-tasting cheeses are made by using the enzyme rennin extractedfrom calf stomachsto convert milk to curds and whey. The calf- stomach extract was used for genera- tions in commercial cheese-making under the name rennet. Your grand- mother may have purchased the dried extract at the local grocery store for preparationof a pudding-type dessert called junket. The activeingredient in rennetextract is the enzyme rennin, now called chymosin. It has been traditionally obtainedin purest form from the stomachs of unweaned (milk-fed) calves. Rennet extract can also be obtained from the stomachs of adult cows and from other milk-producing species: goats, sheep, horses, zebra, and even camels. In these cases, however, the extractusually containsother enzymes, which produce bitter off-flavors. With the advent of genetic engineering it became possible to prepare pure chymosin without extractingcalf stomachs. Molecularbiologists isolated the gene for prochymosin, a protein precursor of chymosin itself, from cells

Ken Harewood is the Director of the Biomedical/Biotechnology Research Instituteat NorthCarolina Central Uni- versity, Durham, NC 27707. Please address article correspondence to Richard Hinman, Pfizer Central Figure 1. Chymosin and prochymosin, showing site of cleavage of prochymosin Research, Groton, CT 06340. by acid.

288 THEAMERICAN BIOLOGY TEACHER, VOLUME 61, NO. 4, APRIL1999 Old Method

DNiACODE FOR PRCYOI PROCHYMOSIN

New Method

Figure 2. Chymosin manufacturing methods.

Chymosin produced by fermenta- casein, which is therefore referred to human genome, gene therapy, the Downloaded from http://online.ucpress.edu/abt/article-pdf/61/4/288/48912/4450673.pdf by guest on 28 September 2021 tion was approved for sale by the as the natural substratefor chymosin. cloning of mammals, and the produc- U.S. Food and Drug Administration Made up of 169 amino acids with tion of genetically improved foods. in 1990. This was the first genetically a molecular weight of 19,000, kappa Many of the experiments outlined engineered food additive to receive casein is about half the size of chy- here can be carriedout at lower grade FDA approval. It is now manufactured mosin. Chymosin cleaves the long levels. Others, such as serial dilution, in specially designed facilities and sold kappa casein polymer in only one can be reserved for use in more throughout the United States as the place-between amino acids 105 and advanced classes. premiere enzyme for cheese manufac- 106 in the chain, forming two large ture. Chymosin produced by fermenta- protein pieces. Other proteases can Objectives tion is identical to chymosin produced react with casein and coagulate milk, by the stomach of the calf. Proof of but since they are less selective than The goal of these experiments is to this identity was the basis for FDA chymosin, they create more fragments, familiarize the student with some of clearance of fermentation-produced some of which produce off-flavors in the fundamental aspects of enzyme/ chymosin for food use (see Flamm cheese. The most common of these substrate interaction. Successful com- 1991). The genetically engineered prod- proteases is the enzyme pepsin, often pletion of these assignments will pro- uct has largely replaced the product found with chymosin in the stomachs vide valuable experience with the extracted from calf stomachs because of adult animals. For a survey of the properties of enzymes, including the the former is purer, less expensive, and many factorsinvolved in milk clotting, effect of concentration, temperature available in unlimited supply. see Berridge 1954. and pH on their activity; denaturation For a lively portrayal of the steps in of enzymes; and the nature and prop- the development of the manufacturing erties of proteases. method for fermentation-based chy- Classroom Use mosin, see the video titled, "Biotech- The protease chymosin is particu- Materials & Methods nology; Careers for the 21st Century," larly well suited for classroom use at available from NABT. all grade levels because it is safe, the * Chymax?(chymosin manufactured endpoint-curd formation-can be by fermentation' observed with the unaided eye, and * Milk-various fat levels How Does Chymosin experiments can be performedrapidly * Water (tap water can usually be Work? with simple equipment. Previously used) described experiments (Gill 1987; * White vinegar (5%acetic acid) The enzyme chymosin is made up Wheelock1971) used rennet (calf stom- * 5% sodium bicarbonatesolution of 323 amino acids (Figure 1) and has ach extract) or rennilase (a microbial * 500 ml beakers a molecular weight of 35,700. Some protease) as the enzyme source with * 50 ml beakers enzymes react with other proteins, the problem of considerablebatch-to- * Plastic droppers breaking a long chain into smaller batch variation in activity. Genetically * Pipets, graduatedin 0.5 and 1.0 ml chains. These enzymes are called pro- engineered chymosin is purer (clear volumes teases. Chymosin is a protease that liquid vs. red-brown solution) and * Waterbath reacts with casein protein in milk. gives more reproducible results. Ear- * Test tubes of 20 ml capacity Milk is an emulsion-a mixture of lier reports used complete clotting of * Universal pH paper two normally immiscible liquids, fat milk as an endpoint, which gives high- * Thermometer and water, held together by a natural ly variable results. The endpoint mea- * Ice emulsifying agent called casein, the surement described below is more * Timer name for a family of proteins. If casein reproducible.Finally, the use of geneti- is destroyed, the emulsion separates cally engineered chymosin broadens 'The commercialproduct, CHY-MAX,is into its immiscible components and the teaching arena considerably, pro- sold by Chris Hansen and Co., 222 State insoluble calcium caseinate precipi- viding a basis for classroomdiscussion Street, Milwaukee, WI. The liquid product tates as the curd. Chymosin reacts of recent technological advances that contains approximately 0.1% active chy- almost exclusively with one key mem- are interesting to teachers and stu- mosin. It should be stored in a refrigerator ber of the casein family called kappa dents, such as the sequencing of the to minimize loss of activity.

CHYMOSINEXPERIMENTS 289 General Purpose Protocol experimenter to also determine the 9. Let stand at room temperature effect of enzyme concentration on the (220 C) for 10 minutes. To ensure a coagulation time of speed of reaction. 10. At the end of 10 minutes, about 10 minutes, which is practical observe each tube for clotting for classroom use, aging milk in Student Activities and record your results in Table advance at room temperature for 6 to 2. Alternatively, observe and 8 hours is helpful when working at Qualitative Approach record the time for the curdling 20 to 250 C. Microbial action lowers process to reach the endpoint, the pH, facilitating coagulation. As an Label five test tubes 1 through 5. as described above. to aging, one can add two alternative Add 5 ml of regular, aged homoge- 11. Let all tubes stand overnight at drops of white vinegar diluted 1:4 (1% nized milk to each. Adjust the temper- room temperature. What do acetic acid) to 5 to 10 ml of milk. 300 a water bath. ature to 25 to using you observe? However, for higher temperatures (30 Add 2 drops of commercial enzyme to 370 C), aged milk is not necessary, solution to the first tube of milk, 4 to Prepare about 20 ml of the concen- reaction is very rapid. as the the second, 6 to the third, and 8 to tration of chymosin that clots milk the fourth. Do not put any enzyme in in about 10 minutes. This is called Endpoint Observation the fifth tube. Record the time for the the stock solution or standard to coagulate. milk in each tube to begin enzyme preparation. Reserve this for The endpoint should be the first table similar to Table 1 to Prepare a further experiments outlined below.

sight of a fine precipitate. Since this record your observations. For further Downloaded from http://online.ucpress.edu/abt/article-pdf/61/4/288/48912/4450673.pdf by guest on 28 September 2021 is a white precipitate against a white experiments, use the concentration of Conclusions background, some practice is required chymosin that begins to clot milk in to achieve reproducible results. Best about 10 minutes. 1. What conclusion can you draw results are obtained by tilting a test Prepare a graph of the data like about the effect of enzyme con- tube on its side and turning between Figure 3. centration on the speed of reac- the fingers. Viewing the test tube in tion with a fixed volume of milk? a bright light is helpful. The first sign Question 2. What is the concentration of chy- of coagulation is a thickening (cream- The fifth tube, containing no enzyme, mosin in Tube #7? ing) in the film along the side of the is called a negative control. Why do test tube. Particulate matter is we include this in the experiment? Students should be able to calculate observed soon thereafter. Once cur- the concentration in each dling begins, the process is essentially Quantitative Approach-Serial chymosin tube since each contains one-tenth as exponential and complete clotting Dilution much enzyme as the previous one. occurs rapidly. This process is espe- In this case the dilution at each step Commercial chymosin is about 0.1% cially rapid at higher temperatures (300 is 10-fold. to 37?) where substantially decreased active enzyme. concentrations must be used for a 5 1. Place seven test tubes in a test- to 10 minute endpoint. tube rack; number them 1 through 7. Complete clotting can also be used 10 as the endpoint (Gill 1987; Wheelock 2. Pipet 2.0 ml of distilled water but the results tend to be more into each tube. 1971), 8 - difficult to reproduce. Complete clot- 3. Add 0.5 ml of commercial ting will finally occur in all the experi- enzyme to tube #1 and mix ments described here, as the change gently. from fine precipitate to clot is rapid. 4. Transfer 0.5 ml of the mixture Observation of clotting may enhance from Tube #1 to Tube #2 and student interest as the first step in mix. making cheese. 5. Repeat the transfer process until Tube #7 is reached. 6. Discard the 0.5 ml that is Effect of Enzyme removed from Tube #7. 0 2 4 6 8 7. You should now have 2.0 ml in Concentration on each of the seven tubes. No. of drops of enzyme Chymosin Activity 8. Quickly add 8 ml of aged milk Figure 3. Sample graph. to each tube, mix gently. Preparing a Standard Enzyme Preparation (Stock Solution) Table 1. Record of coagulation time. Commercial chymosin solution can I TEST TUBE NO. DROPS OF ENZYME COAGULATIONTIME be used as received as the stock solution, but at this concentration chy- 1 2 mosin can coagulate milk very quickly at temperatures above 30? C. If neces- 2 4 sary, it should be diluted until the average time for milk clotting is about 3 6 10 minutes. 4 8 Two approaches for determining a useful working concentration are 5 0 described below. Both enable the

290 THEAMERICAN BIOLOGY TEACHER, VOLUME 61, NO. 4, APRIL1999 Table 2. Record of enzyme concentration and related observations. TUBE77 ENZYMECONCENTRATION |OBSERVATION 1j f 2 f

3 I. 4

5 Downloaded from http://online.ucpress.edu/abt/article-pdf/61/4/288/48912/4450673.pdf by guest on 28 September 2021

Temperature Effects on 4. Return the tube to the appropriate milk as the previous experiment. Every temperature and incubate until 5 minutes, add to one of the tubes Chymosin Activity the milk clots. the same number of drops of 600 C Enzyme activity is strongly influ- 5. Place all tubes into boiling water chymosin solution as used for the room enced by temperature. Each enzyme after clotting is observed. Record temperature experiment. Observe the exhibits maximum reaction rate at a results in Table 3. time for clotting to begin. The results, certain temperature. (Most of the when plotted, will produce a graph Questions enzymes in the human body work best like Figure 4. at body temperature [370 C]). How- The data can also be converted into 1. How does heat affect the clotting a form that enables construction of a ever, if the temperature is raised high time of chymosin? enough, the enzyme becomes dena- graph of time to clot vs. percent resid- 2. How do you explain the results ual enzyme activity. tured and loses all activity. In this observed in Tube #1? set of experiments these effects are 3. Why are the tubes placed in boil- investigated. ing water after clotting? The Effect of pH on Chymosin Activity Enzyme Denaturation Student Activities The activity (rate at which reaction Rate of Reaction If a solution of chymosin is main- takes place) of enzymes varies with tained at about 600 C, it will rapidly pH. Each enzyme has its greatest activ- 1. Pipet 8 ml of whole milk into lose activity. For this experiment, first ity at a certain pH, called the pH three test tubes labeled 1 through make one measurement with chymosin optimum. This set of experiments is 3. Hold Tube #1 on ice (40 C), and milk at room temperature, select- designed to investigate the change of Tube #2 at room temp (220 C), ing the number of drops of chymosin activity of chymosin with pH. Note and Tube #3 at 370 C (use a water that will clot milk in 5 minutes or that too much acid will curdle milk bath or just hold the tube under less. This short time is necessary for even in the absence of chymosin. Even your armpit). practical classroom use as the heated white vinegar (5% acetic acid) can 2. Allow each tube to adjust to the samples will take longer to react. cause some curdling. Therefore, we surrounding temperature (ap- Maintain about 2 ml of chymosin at recommend that vinegar be diluted 1:4 proximately 5 minutes). about 600 C in a water bath. Now set with water. About 5 drops of this 3. Quickly add 0.5 ml of the standard up six test tubes at room temperature, concentration in 5 ml of milk produces enzyme preparation to each tube. each containing the same amount of a pH of about 6.5.

Table 3. Record of temperature, clotting time and observations. TUBE TEMP CLOTTINGTIME OBSERVATION

CHYMOSINEXPERIMENTS 291 Since milk contains calcium and the 30 curd is primarily calcium caseinate, predict the effect of added calcium salts on the rate of reaction. Devise an experimentto test your hypothesis. 25 Additional Areas for Study ) 20 Proteases 0 Proteases have become a popular (0 subject in the media because protease <'5 inhibitors are among the most impor- 15t tant drugs in the fight against AIDS. In fact, proteases are importantto the cn / survival of a wide variety of disease- producing organisms, from malariato the common cold. The searchfor drugs to interferewith the action of proteases is an active area of research (see Seife 1997). Downloaded from http://online.ucpress.edu/abt/article-pdf/61/4/288/48912/4450673.pdf by guest on 28 September 2021

0 I Pro-enzymes 0 5 10 15 20 25 30 As describedabove, the actualprod- uct secreted by the cells in the calf stomachis prochymosin,which has no inherentenzymatic activity. It is called Observedtime for milkto dot a zymogen. (minutes) Pepsin, also found in the calf stomach (and, incidentally,the active ingre- dient in meat tenderizers),is also pro- Figure 4. Sample graph. duced as a zymogen, called pepsino- gen. There are many examples of precursor proteins in nature. Insulin Student Activities Explain in simple terms how tem- for the treatment of diabetes is pro- perature, pH and enzyme concentra- duced initially by cells of the pancreas 1. Add 10 ml of milk to each of tion affect the activity of chymosin as pro-insulin.Cleavage of the peptide three small beakers or test tubes with its naturalsubstrate. Do you think segment of pro-insulinreleases insulin. of 20 ml capacity. all enzymes would respond like Other examples of pro-proteins can 2. Add 1%acetic acid (white vinegar chymosin? be found in the literature (and on diluted 1:4) dropwise to the first the Web). beaker until the pH is adjusted to 5.0. Integration of Math & Science Genetic Engineering 3. Add a 5% solution of sodium The time course of enzymatic pro- The use of the product of genetic bicarbonatedropwise to the milk cesses lends itself very well to inte- engineering in these experiments in the second beaker until the pH grated exercises in math and science. affords the opportunityto discuss and reaches 8.0. The graphing exercises outlined above investigate the subject more broadly; 4. Measure the pH of the milk in deal with one variable, e.g. time vs. both technical aspects, such as gene the third beaker. temperature.For more advanced stu- splicing, and methods of moving genes 5. Transfer 8 ml of milk from each dents, two variablescan be considered from one cell to another; and ethical beaker to a clean test tube. such as enzyme concentration and considerations. 6. Add 2 ml of the standardenzyme temperature, e.g. "How can a dilute preparation to each tube and solution of enzyme be made to react mix gently. as rapidly as a more concentrated References 7. Incubate the tubes at room tem- one?" or "How can a cold solution of perature and record the clotting chymosin and milk be made to react Berridge, N.J. (1954). Rennin and the time as describedunder Endpoint as fast as a warm one?" Graphing clotting of milk. Advancesin Enzymol- Observation. exercises are appropriatehere. ogy, 15, 423-448. Flamm, E.L. (1991). How FDA approved Make a table like Table 3, substi- chymosin: A case history. Biotechnol- tuting pH for temperature. Additional Experiments for Students ogy, 9, 349-351. Questions Gill, J. & Saunders, T. (1987). Rennin-A Compare the curdling times for neglected enzyme? Journalof Biological 1. What is the pH of natural whole different types of milk: skim, 2% fat Education,21(4), 248-250. milk? free, "half-and-half," and powdered Seife, C. (1997). Blunting nature's swiss 2. Explain the effects of pH on chy- milk. Develop a hypothesis to explain army knife. Science, 277, 1602. mosin activity. the differences. Be sure to keep all Wheelock, J.V. (1971). Experiments on 3. What pH yields the highest other variables constant. Why is this the rennin coagulation of cows' milk. enzyme activity? Explain. important? Journalof BiologicalEducation, 5, 295-300.

292 THEAMERICAN BIOLOGY TEACHER, VOLUME 61, NO. 4, APRIL1999 Glossary Flower Press Polymer A long molecule arranged as a chain of many subunits. mer = unit poly = many Example: A-B-A-B-A-B-A-B- The best instructionalmaterials available Protein A polymer in which the subunits are amino acids. DNA The polymer of deoxyribonucleicacid, found in every living Garbage cell, which tells the cell to produce proteins. It is a chain MaryAppelhof w>e x0 of alternating sugar (deoxyribose) and phosphoric acid Manual describes groups with a segment called a base attached to each how to set up and yb. sugar unit: maintain a worm __ -S-P-S-P-S------composting system. All ages. 0-942256-10-7 B B B

where S = sugar, P = phosphoric acid, and B = nucleotide Worms Eat Our Garbage: Downloaded from http://online.ucpress.edu/abt/article-pdf/61/4/288/48912/4450673.pdf by guest on 28 September 2021 base. When DNA exists as a double helix, two chains are Classroom Activities for a wrapped around each other in reverse fashion with one Better Environment base from each chain paired with one from the other chain, Appelhof, harris, and Fenton hence the term "base pairs." Curriculum guide and inter- disciplinary activities teach Denaturation Drastic change in a protein's environment will result in a math, biology, language and change in its 3-dimensionalconfiguration and loss of activity. For example, heating an egg causes the clear portion to arts. turn white. It has coagulated, lost its original 3-dimensional Grades4-8 0-942256-05-0 structure, and its original properties, including color and solubility. Wormanial -in ts Video shows live earthwormsin natural habitats, hatching from a cocoon, mating. Studentswith even the largest lung ~All .~~~ ages. capacitiesmay now learn first hand aboutrespiratory volume thanks to 0-942256-09-3 the Phipps& Bird"' 9-liter, Student WetSpirometer. WormBin Creatures Alive Madeof lightweightyet durable Througha Microscope ;A;Rsli Highl Density11 Polyethylene (HDPE) Captivating video zeros in on plasticand aluminum, the spirometer the tiny organisms often seen permitsfive different respiratory q. - volumesto be determinedfrom in a worm bin. Springtails and Jt:3 * _ _ _ - measurementsobLained: mites, centipedes and sowbugs amuse, inform, and entertain. * TidalVolume (TV) ...... InspiratoryCapacity (IG) All ages. 1-884195-36-9 _ InspiratoryReserve Volume (IRV) _ ExpiratoryReserve Volume (ERV) The WormCafe: _ VitalCapacity (VC) Mid-scaleVermi- compo5ting of Lunchroom g3ig Wastes 21 breath. Binet Payne Eachporable and storable unit comes Detailed manual - completewith full instructions, disposable describes how a 0-942256-11-5 mouthpiecesand one-way plastic valve school saves for classmomuse. Also available sanitary $6000 a year recycling food A is thepopular Phipps & Bird""' 6liter, r-^TM StudentWet Spirometer. waste with redworms. Teachers. F"ormore information orto place an order, P0()Box 27324 lRichmond, VA23261-7324 contactyour science education apparatus Flower Press I 8009Th 7621 * I'ax804-254-2955 dealerorPhipps&Bird. 10332Shaver Rd., Kalamazoo, Ml 49024 w~wwphippsbirdcorn* [email protected] www.wormwoman.com616*327-0108

CHYMOSINEXPERIMENTS 293