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.
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页码:267 / 272
页数:6
相关论文
共 33 条
[11]  
Maniatis T., 1982, MOL CLONING
[12]   STRUCTURE AND POSSIBLE CATALYTIC RESIDUES OF TAKA-AMYLASE A [J].
MATSUURA, Y ;
KUSUNOKI, M ;
HARADA, W ;
KAKUDO, M .
JOURNAL OF BIOCHEMISTRY, 1984, 95 (03) :697-702
[13]   SECRETION OF BETA-LACTAMASE INTO THE PERIPLASM OF ESCHERICHIA-COLI - EVIDENCE FOR A DISTINCT RELEASE STEP ASSOCIATED WITH A CONFORMATIONAL CHANGE [J].
MINSKY, A ;
SUMMERS, RG ;
KNOWLES, JR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1986, 83 (12) :4180-4184
[14]  
NAKAJIMA R, 1986, APPL MICROBIOL BIOT, V23, P355
[15]   A GENERAL METHOD APPLICABLE TO SEARCH FOR SIMILARITIES IN AMINO ACID SEQUENCE OF 2 PROTEINS [J].
NEEDLEMAN, SB ;
WUNSCH, CD .
JOURNAL OF MOLECULAR BIOLOGY, 1970, 48 (03) :443-+
[16]   FACTORS AFFECTING TRANSFORMATION OF ESCHERICHIA-COLI STRAIN CHI-1776 BY PBR322 PLASMID DNA [J].
NORGARD, MV ;
KEEM, K ;
MONAHAN, JJ .
GENE, 1978, 3 (04) :279-292
[17]   PHAGE LAMBDA-PL PROMOTER CONTROLLED ALPHA-AMYLASE EXPRESSION IN ESCHERICHIA-COLI DURING FERMENTATION [J].
REINIKAINEN, P ;
LAHDE, M ;
KARP, M ;
SUOMINEN, I ;
MARKKANEN, P ;
MANTSALA, P .
BIOTECHNOLOGY LETTERS, 1988, 10 (03) :149-154
[18]   CONSERVED AMINO-ACID SEQUENCE DOMAINS IN ALPHA-AMYLASES FROM PLANTS, MAMMALS, AND BACTERIA [J].
ROGERS, JC .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1985, 128 (01) :470-476
[19]  
Smith T., 1981, ADV APPL MATH, V2, P482, DOI [DOI 10.1016/0196-8858(81)90046-4, 10.1016/0196-8858(81)90046-4]
[20]   Antibody-mediated selection of a Mycoplasma gallisepticum phenotype expressing variable proteins [J].
Gorton, TS ;
Geary, SJ .
FEMS MICROBIOLOGY LETTERS, 1997, 155 (01) :31-38