Heterologous expression and site-directed mutagenesis of the enzyme chymosin

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Enzymes from fish processing waste materials and their commercial applications

Enzyme technology is an indispensable part of today's industrial processes and, hence, the global market for industrial enzymes continues to expand. The manufacturing of enzymes has become the cash cow of many businesses. However, the use of enzymes for industrial processes adds considerable cost to the final product. Further, several drawbacks associated with conventional enzymes have increased the need for alternatives with competitive advantages. Marine enzymes with habitat-related unique physicochemical characteristics seem ideal candidates and fish processing leftovers have been recognized as a cheap and promising source for unique marine enzymes. In this chapter, fish processing leftover-derived enzymes and their potential applications are discussed

Solvent binding analysis and computational alanine scanning of the bovine chymosin-bovine κ-casein complex using molecular integral equation theory

We demonstrate that the relative binding thermodynamics of single-point mutants of a model protein-peptide complex (the bovine chymosin-bovine κ-casein complex) can be calculated accurately and efficiently using molecular integral equation theory. The results are shown to be in good overall agreement with those obtained using implicit continuum solvation models. Unlike the implicit continuum models, however, molecular integral equation theory provides useful information about the distribution of solvent density. We find that experimentally observed water-binding sites on the surface of bovine chymosin can be identified quickly and accurately from the density distribution functions computed by molecular integral equation theory. The bovine chymosin-bovine κ-casein complex is of industrial interest because bovine chymosin is widely used to cleave bovine κ-casein and to initiate milk clotting in the manufacturing of processed dairy products. The results are interpreted in light of the recent discovery that camel chymosin is a more efficient clotting agent than bovine chymosin for bovine milk

Bovine chymosin: a computational study of recognition and binding of bovine kappa-casein

Bovine chymosin is an aspartic protease that selectively cleaves the milk protein kappa-casein. The enzyme is widely used to promote milk clotting in cheese manufacturing. We have developed models of residues 97-112 of bovine kappa-casein complexed with bovine chymosin, using ligand docking, conformational search algorithms, and molecular dynamics simulations. In agreement with limited experimental evidence, the model suggests that the substrate binds in an extended conformation with charged residues on either side of the scissile bond playing an important role in stabilizing the binding pose. Lys111 and Lys112 are observed to bind to the N-terminal domain of chymosin displacing a conserved water molecule. A cluster of histidine and proline residues (His98-Pro99-His100-Pro101-His102) in kappa-casein binds to the C-terminal domain of the protein, where a neighboring conserved arginine residue (Arg97) is found to be important for stabilizing the binding pose. The catalytic site (including the catalytic water molecule) is stable in the starting conformation of the previously proposed general acid/base catalytic mechanism for 18 ns of molecular dynamics simulations

Progress in the field of aspartic proteinases in cheese manufacturing: structures, functions, catalytic mechanism, inhibition, and engineering

International audienceAspartic proteinases are an important class of proteinases which are widely used as milk-coagulating agents in industrial cheese production. They are available from a wide range of sources including mammals, plants, and microorganisms. Various attempts have been made in order to get insights into enzyme structure/function relationships for designing improved biocatalysts. This review provides an overview of historical background and recent achievements on the classification and structural characteristics of such enzymes as related to their functional properties, mechanism of catalysis, pH, and temperature dependence, substrate specificities, mechanism of inhibition, enzyme engineering, and technological applications with the focus on cheese manufacturing