1. Background of the technology .

During the past decade, major advances have been made in the use of recombinant-DNA technology.

The technology has developed from a small fledgling scientific area to major industrial applications.

Recombinant-DNA technology has its origins in a number of different areas. These areas include

bacteriophage genetics and bacterial gene regulations, biochemistry of DNA synthesis and repair, the

synthesis and sequencing of oligonucleotides and protein gene cloning, virology and mammalian

embryology. Integration of these areas has resulted in the commercial production of biological

molecules from bacteria and yeast and the insertion of genes into a number of different animal types.

One of the easily identified areas where recombinant-DNA technology could be used was to produce

protein and other products which were prohibitively expensive to purify or synthesize using alternative

technologies. Compounds falling into this class include selected hormones, amino acids, vitamins and

proteins. The use of recombinant-DNA products in animal agriculture will result in sizable markets for

the companies. Potential or actual products include animal vaccines, vitamins and amino acids for feed

supplement, and hormonal agents that promote growth and/or repartitioning. The use of this

technology to produce vaccines to such long-standing problems as hoof and mouth disease and other

viral diseases will increase production efficiency.

The food industry has had a long history in using selected microorganisms to accomplish making

selected products, such as yeasts for fermentation (Wilson, 2012 ). Some of the potential uses of

recombinant-DNA technology to produce products for food or to be used by the food industry such as

Food Enzymes , Alcohol Fermentation , Peptide Sweeteners , Flavors and Fragrances, Modify Functional

Proteins, Vitamins and Amino Acids.

The technology to insert foreign DNA into the genome of animals has been available for some time

(Palmiter& Brinster ,2005). Investigators have been able to insert foreign DNA into the genome of a

number of animals, including Xenopus, Drosophila, mice, sea urchins and more recently, into rabbits,

pigs and sheep (Hammer et al., 2010). There are many areas where inserting a gene into an organism

would be of great benefit.

2. Recombinant Chymosin

Cheeses represent a traditional way of preserving a perishable foodstuff, milk. Cheese is made by

addition of milk to a starter culture of lactic acid bacteria which acidify the milk to about pH 5.5. In

addition, milk clotting enzymes called rennet are added, resulting in a coagulated protein gel trapping

other proteins and fat.

Traditionally , the rennet used is a preparation of several enzymes isolated from calf stomach. Modern

cheeses making, however, increasingly relies upon microbial sources of the most important enzyme, the

protease chymosin. This is now produced by recombinant micro-organisms

a. Milk Proteins

Milk consists of water, fat, protein, phosphate, lactose, citric acid and inorganics such as calcium

phosphate. The protein component of milk can be divided into two groups, the casein fraction and the

whey proteins:

Table 1 : the casein fraction and the whey proteins

The casein proteins are the ones that will form the curd during cheese making. Casein proteins tend not

to have a particularly compact globular structure and they tend to be rather susceptible to proteolysis.

As they are all phosphorylated, they bind the calcium content of the milk and existed in the form of

casein micelles.

b. Chymosin mechanism of action

Chymosin, commonly known as rennin, is the main milk-coagulating enzyme that consists of a single

polypeptide chain of 323 amino acids with intramolecular disulfide linkages. Preparations of calf rennet

contain two forms of chymosin, A and B. The only difference between chymosins A and B is one amino

acid in the polypeptide chain; the chymosin A contains an aspartic acid residue at position 286, whereas

the chymosin B contains a glycine residue at the same position.

Like other acidic proteases from the gastric juice, chymosin is secreted as an inactive precursor,

prochymosin. The precursor is stable at weakly alkaline pH whereas the active enzyme is rapidly

denatured at pH values above 7. Below pH 5 prochymosin is converted into chymosin by a limited

proteolysis during which a peptide segment is cleaved from the N-terminus. The proteolytic activity of

chymosin has optimum pH about pH 3-5.

The milk-clotting activity of chymosin is due to the proteolysis of the κ-casein. κ-Casein consists of two

parts: one part is hydrophobic and the other one is hydrophilic. In milk, this protein stabilizes the casein

micelles against aggregation. During milk-clotting, a Phe-Met bond is hydrolysed, the hydrophilic part of

the κ-casein is liberated and aggregation occurs

Figure 1 : 3D structure of chymosin

c. Microbes used in chymosin production

Three of the most used microbes are Aspergillus niger, Kluyveromices lactis and Escherichia coli. These

three species have really well known genetics and metabolic pathways, specially Aspergillus niger and

Escherichia coli, which make them very useful for recombination experiments like chymosin production.

Aspergillus is the name used for a genus of molds that reproduce only by asexual

means. Aspergillus species are common and widespread. They are among the most successful groups of

molds with important roles in natural ecosystems and the human economy( ). Aspergillus niger is one

of the most common species of this genu.

It is found in a large variety of environments, but especially on vegetation, decaying organic matter, and

soil. In humans, an Aspergillus niger infection is only a problem with people who have a weakened

immune system. It most commonly causes lung infections, but it can also spread to other organs and

open wounds

Kluyveromyces lactis is an ascomyceteous yeast (very similar to Saccharomyces cerevisiae) which has

the ability to assimilate lactose and convert it into lactic acid, in fact, it has been isolated from milk and

constitutes the predominant eukaryote during cheese production. K. lactis is used for the production of

beta-galactosidase and its fermentation properties are well understood. It shares most of the features

that make Saccharomyces cerevisiae the best eukaryotic model organism (the existence of well-

established genetics, the availability of fast and efficient transformation procedures and its efficient

homologous recombination machinery) and it is also important that this microbe is used in other stages

of the cheese production chain too.

Escherichia coli is a bacterium that is a common inhabitant of the human colon. It also lives in the

intestine of many other animals, wild as well as domestic. It is a gram negative, rod-shaped gamma

proteobacteria. It is the model organism for the study of bacteria. Normally, Escherichia coli does not

cause disease although some strains frequently cause diarrhea, and it is the most common cause of

urinary tract infections. Escherichia coli is one of the most thoroughly studied of all living things. It is a

favorite organism for genetic engineering since its genetics and metabolism are the best understood.

Another advantage of studying Escherichia coli is that it can be grown very easily and inexpensively in a

laboratory setting.

The enzymatic properties of recombinant E. coli chymosin are indistinguishable from those of native calf

chymosin ( ) which is also important because the efficiency of the process of cheese production is

the same.

References

Wilson, G.A. (2012). Genetic Engineering and Its Impact On the Food Industry. Proc. Recip. Meat Conf.

35:36.

Palmiter, R.D. and Brinster, R.L. (2005). Transgenic Mice. Cell 41 :343. Uncorking the Genes. Barron's.

May 5, 1986. p. 10.

Hammer, RE.; Pursel, V.G.; Rexford, C.E. Jr.; Wall, R.J.; Bolt, D.J.; Ebert, K.M.; Palmiter, R.D.; Brinster, R.L. (2010). Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315:680.

Biotechnology. The U.S. Department of Agriculture's Biotechnology Research Efforts. Oct. 1985. GAOIRCED-86-39BR.