Chymosinexpressing clone

Figure 1. Making recombinant chymosin (see text for details).

Box 5—2. Making Calf Chymosin in Fermentors (Continued)

this case, chymosin—are not secreted but instead are packaged inside the cell in the form of inclusion bodies.Thus,to retrieve the chymosin,the cells had to be collected and disrupted, either mechanically or chemically, to release the chymosin-containing inclusion bodies.The latter, then, had to be solubilized to liberate active chymosin. Nonetheless, despite these awkward and expensive steps, genetically engineered chymosin won FDA approval and quickly gained acceptance in the marketplace (Flamm, 1991). Subsequently, expression systems for eukaryotic organisms, including Kluyveromyces lactis and Aspergillus nidulans, were developed that led to secreted product with high yield. Engineered chymosin is currently marketed worldwide by several major companies, including DSM Food Specialties (formerly Gist-Brocades), Chrs. Hansens, and Danisco.Among the production organisms currently used for these so-called recombinant chymosin products are K. lactis and Aspergillus niger.

Aside from the obvious cost advantage of the genetically engineered chymosins, other benefits were also realized.These products are derived from a fermentor, rather than calf stomachs, and are not subject to supply problems or price fluctuations.They can be classified as vegetarian and can more easily obtain kosher status. Importantly, the chymosin is relatively easy to purify and can be produced free of other proteases that are sometimes present in calf chymosin preparations. In fact, manufacturers claim that the engineered chymosin is 100% pure and has high specific activity.Thus, according to some experts, cheese made using these chymosin products has organoleptic properties as good as (if not better than) cheese made with calf chy-mosin. (There was also a contrary argument—that pure chymosin makes a less flavorful aged cheese, since non-specific proteolysis is absent.)

Currently, engineered chymosin sells for about half the cost of what calf chymosin sold for in the 1980s.Thus, it is not surprising that engineered chymosin has,for all practical purposes, entirely displaced calf chymosin in the United States. Calf chymosin has essentially become a niche product, with less than 10% of the coagulant market. However, in Europe, where many cheese manufacturers prefer or are required to produce cheese by traditional techniques, calf chymosin is more widely used. It is worth noting that unlike other foods produced via biotechnology, when engineered chymosin was introduced, there was little public concern or protest. Perhaps this was because the notion of a product produced by microorganisms in fermentors was more appealing to consumers than the image of calf stomach extracts.In addition, the FDA determined that no special labels would be required for cheese made with engineered chy-mosin, which they considered to be "not significantly different" from calf chymosin.

References van den Berg J.A., K.J. van der Laken,A.J. van Ooyen,T.C. Renniers, K. Rietveld,A. Schaap,A.J. Brake, R.J. Bishop, K. Schultz, D. Moyer, M. Richman, and J.R. Shuster. 1990. Kluyveromyces as a host for heterologous gene expression: expression and secretion of prochymosin. Bio/Technology 8:135-139. Dunn-Coleman, N.S., P. Bloebaum, R.M. Berka, E. Bodie, N. Robinson, G.Armstrong, M.Ward, M. Przetak, G.L. Carter, R. LaCost, L.J.Wilson, K.H. Kodama, E.F. Baliu, B. Bower, M. Lamsa, and H. Heinsohn. 1991. Commercial levels of chymosin production by Aspergillus. Bio/Technology 9:976-981. Flamm, E.L. 1991. How FDA approved chymosin: a case history. Bio/Technology 9:349-351. Marston, F.A.O., P.A. Lowe, M.T. Doel, J.M. Schoemaker, S. White, and S. Angal. 1984. Purification of calf prochymosin (prorennin) synthesized in Escherichia coli. Bio/Technology 2:800-804.

(i.e., whey), this process can result in enough precipitated whey protein to form a cheese. Examples of precipitated cheeses include Ricotta cheese, the Hispanic-style cheeses queso fresco and queso blanco, and Gjetost, a whey-derived cheese popular in Norway.

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