Site-specific mutations of calf chymosin B which influence milk-clotting activity

被引:9
|
作者
Chitpinityol, S [1 ]
Goode, D [1 ]
Crabbe, MJC [1 ]
机构
[1] Univ Reading, Sch Anim & Microbial Sci, Div Cell & Mol Biol, Reading RG6 6AJ, Berks, England
关键词
D O I
10.1016/S0308-8146(97)00204-5
中图分类号
O69 [应用化学];
学科分类号
081704 ;
摘要
Zymogen forms of wild type and three mutant calf chymosin B enzymes were heterologously expressed in Escherichia coli under control of a T7 promoter as inclusion bodies. The chaperone-like protein, alpha-crystallin, was used as a possible aid to unfolding. Prochymosin formed a complex with the chaperone-like protein alpha-crystallin, before and after folding; after activation, free chymosin was recovered without bound alpha-crystallin. Following solubilisation, refolding and activation, steady-state kinetic comparisons were determined using the synthetic substrate Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe. The mutation by deletion of 34 residues from the C-terminus (PC289) caused the loss of stability of the mature enzyme after activation. Insertion of histidine-glycine residues at the C-terminus produced a mutant (PC + 2) with a lower k(cat) (2.52 s(-1) compared to 18.9 s(-1) for the recombinant wild type) and k(cat)/K-m (3.8 mM(-1) s(-1) compared to 49.7 mM(-1) s(-1) for the recombinant wild type), suggesting functional involvement of this region. Exchange of threonine 77 on the flap of the enzyme for an aspartyl residue (T77D - pepsin numbering) caused little change in k(cat) or k(cat)/ K-m values. Both PC + 2 and T77D mutants showed reduced milk clotting activity (151.5 U mg(-1) and 303 U mg(-1), respectively) compared to the recombinant wild type enzyme (909 U mg(-1)), and reductions in the C/P (milk-clotting activity over proteolytic activity) ratios (3.0 and 3.03, respectively) compared to the recombinant wild type (6.09). (C) 1998 Elsevier Science Ltd. All rights reserved.
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页码:133 / 139
页数:7
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