ANALYSIS OF MUTATIONS IN CYCLODEXTRIN GLUCANOTRANSFERASE FROM BACILLUS-STEAROTHERMOPHILUS WHICH AFFECT CYCLIZATION CHARACTERISTICS AND THERMOSTABILITY

被引:73
作者
FUJIWARA, S
KAKIHARA, H
SAKAGUCHI, K
IMANAKA, T
机构
[1] OSAKA UNIV,FAC ENGN,DEPT BIOTECHNOL,SUITA,OSAKA 565,JAPAN
[2] NIHON SHOKUHIN KAKO CO LTD,CHIYODA KU,TOKYO 100,JAPAN
关键词
D O I
10.1128/jb.174.22.7478-7481.1992
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) produces cyclodextrin from starch. The CGTase molecule is composed of four globular domains, A, B, C, and D. In order to gain better understanding of the amylolytic and cyclization mechanisms of CGTase, mutant CGTases were constructed from a CGTase gene (cgt1) of Bacillus stearothermophilus NO2. Cgt1-F191Y (Phe at position 191 was replaced by Tyr), Cgt1-F191Y-F255Y, Cgt1-W254V-F2551, Cgt1-W254V, and Cgt1-F255I were constructed for the analysis of the NH2-terminal region. It was revealed that amino acids surrounding a spiral amylose are important for cyclization characteristics and that hydrophobic amino acids just after the Glu catalytic site play an important role in the hydrolysis characteristics of the enzyme. Mutant CGTases Cgt1-T591F and Cgt1-W629F were also constructed to study the role of a second substrate-binding site in domain D, and it was suggested that substrate binding at both domains A and D stabilized the enzyme and optimized cyclodextrin production.
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页码:7478 / 7481
页数:4
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