SITE-DIRECTED MUTAGENESIS OF A THERMOSTABLE ALPHA-AMYLASE FROM BACILLUS-STEAROTHERMOPHILUS - PUTATIVE ROLE OF 3 CONSERVED RESIDUES

被引:73
作者
VIHINEN, M [1 ]
OLLIKKA, P [1 ]
NISKANEN, J [1 ]
MEYER, P [1 ]
SUOMINEN, I [1 ]
KARP, M [1 ]
HOLM, L [1 ]
KNOWLES, J [1 ]
MANTSALA, P [1 ]
机构
[1] VALT TEKNILLINEN TUTKIMUSKESKUS,BIOTECH LAB,SF-02150 ESPOO,FINLAND
关键词
D O I
10.1093/oxfordjournals.jbchem.a123037
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus α-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and temperature optima. Replacement of Asp-331 by Glu in the active site caused almost total inactiva-tion. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling. © 1990 COPYRIGHT, 1990 BY THE JOURNAL OF BIOCHEMISTRY.
引用
收藏
页码:267 / 272
页数:6
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