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 pre- cursor 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 <POjtYO I n /IIY1 AC 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.

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