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MODIFICATION OF MILK-CLOTTING ASPARTIC PROTEASES, CHYMOSIN AND MUCOR RENNIN
Beppu, Teruhiko Sasaki, Katsutoshi Suzuki, Junko Sasao, Yuko Yamashita, Takashi Nishiyama, Makoto Horinouchi, Sueharu
Beppu, Teruhiko
Sasaki, Katsutoshi
Suzuki, Junko
Sasao, Yuko
Yamashita, Takashi
Nishiyama, Makoto
Horinouchi, Sueharu
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Issue Date
1989
Submitted date
2023-11-03
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Abstract
Artificial mutagenesis of milk-clotting aspartic proteases, chymosin and a fungal aspartic protease from Mucor pusillus (MPR), was carried out by recombinant DNA techniques. The native and the modified chymosins were prepared by using the expression system of Escherichia coli and their activities were measured with aciddenatured haemoglobin and synthetic peptides. A marked change of substrate specificity with a change of Km or kcat was observed with the mutation of 17077) on’ the flap structure to Phe. Involvement of Lys(221) in determining the pH-activity profile was also suggested. Correctly processed but highly glycosylated MPR was secreted from yeast cells carrying the fungal gene. The decreased milk-clotting activity of the yeast MPR was improved by treatment with endoglycosidase H. Exchange of the Tyr residue on the flap and Trp(45) suggested that the hydrogen bonding between these residues is required for correct arrangement of the S1 subsite. X-ray crystallographic analysis of several aspartic proteases has revealed that 3-dimensional structures of these enzymes are very similar to each other (1). The molecules are bilobal, composing of two topologically similar domains rich in 6 -sheet structures. Their junction forms an extended substrate binding cleft and the two essential aspartyl residues reside at the bottom of the Cleft . A flexible flap region is located at the entrance of the cleft and a tyrosine residue on the flap as well as several hydrophobic residues in the adjacent region are involved to form the Sl subsite for substrate binding. In spite of these high similarity in tertiary structures, marked diversity of the catalytic activity and substrate specificity in these enzymes suggest that different sets of amino acid residues may be involved in their catalytic functions. Calf chymosin and a fungal aspartic protease produced by Mucor pusillus are characterized by their relatively high milk clotting activity and low proteolytic activity, which allow them to be used as milk coagulants in cheese industry. Mutagenesis of these two enzymes was performed by using the recombinant DNA technique to obtain information on the structure-function relationship in this characteristic group of the aspartic proteases.
Citation
Advances in protein design, 87 - 92
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Book chapter
conference paper
conference paper
Language
en
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Series/Report no.
GBF monographs ; Volume 12
ISSN
0930-4320
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ISBN
0895739534
3527280243
3527280243
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Attribution-NonCommercial-ShareAlike 4.0 International