Abstract
Rennet, traditionally obtained from calves, is non-vegeterian and unethical due to the slaughter of unweaned animals. Chymosin is highly specific to the Phe105-Met106 bond of κ-casein and has low proteolytic activity. Microbial aspartic proteases can partly replace chymosin. However, recombinant DNA technology has allowed chymosin itself to be produced by bacteria, yeast, and molds. Not only rennet from calf, but from animals like goat kid, lamb, buffalo, camel, and others can be used in cheesemaking. Chymosins of these animals can be cloned and successfully expressed in microorganisms and can be employed in the production of novel as well as traditional cheese products from the milk of camel, goat, and even horse and donkey. This chapter outlines the recombinant DNA techniques applied over the past few years to improve the microbial production of recombinant rennet, from animals and plants.
TopBackground
The major milk protein is casein, constituting about 80% of the total milk proteins. The remaining 20% correspond to the so-called whey proteins, including lactalbumin, lactoglobulin and immunoglobulins. The caseins consist of α, β, κ, γ-forms and subforms of them with few amino acid variations. α-, and β-caseins are sensitive to calcium-mediated coagulation by the formation of colloidal calcium phosphate bridges (Coultate, 2002). The different casein proteins are clustered in the form of micelles, where κ–caseins aggregate such that the formation of colloidal calcium phosphate bridges between submicelles is limited due to its highly hydrophilic but non-calcium-binding C-terminal trisaccharide residues. Thereby, κ-casein acts as a micelle stabilizer.
In classical cheesemaking, milk is first coagulated into a smooth semi-solid curd, resembling yogurt, and is next cut in order to remove the whey. This type of milk coagulation is, generally, referred to as curdling to distinguish it from acid coagulation. Unlike yogurt formation which requires acid production and isoelectric point precipitation of caseins, curd can be formed at higher pH values by the action of enzymes. In particular one enzyme is important for curdling. This enzyme is called chymosin.
Chymosin cleaves a specific peptide bond of κ–casein, namely the bond between methionine (Met) 106 and phenylalanine (Phe) 105. This cleavage divides the κ–casein molecule into two peptides. The smaller peptide, named the glycomacropeptide fragment carries carbohydrates. Carbohydrates confer protection to caseins. When the glycopeptide is cleaved, the protective action of κ–casein is lost and this results in formation of calcium bridges destabilizing micelles.