Conversion of the maltogenic α-amylase Novamyl into a CGTase

被引:49
作者
Beier, L [1 ]
Svendsen, A [1 ]
Andersen, C [1 ]
Frandsen, TP [1 ]
Borchert, TV [1 ]
Cherry, JR [1 ]
机构
[1] Novo Nordisk AS, DK-2800 Lyngby, Denmark
来源
PROTEIN ENGINEERING | 2000年 / 13卷 / 07期
关键词
amylase; CGTase; product specificity; site-directed mutagenesis; structural homology;
D O I
10.1093/protein/13.7.509
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
Novamyl is a thermostable five-domain maltogenic alpha-amylase that shows sequence and structural homology with the cyclodextrin glycosyltransferases (CGTases), Comparing X-ray crystal structures of Novamyl and CGTases, two major differences in the active site cleft were observed: Novamyl contains a loop insertion consisting of five residues (residues 191-195) and the location of an aromatic residue known to be essential to obtain an efficient cyclization reaction. To convert Novamyl into a cyclodextrin (CD)producing enzyme, the loop was deleted and two substitutions, F188L and T189Y, were introduced. Unlike the parent Novamyl, the obtained variant is able to produce beta-CD and showed an overall conversion of starch to CD of 9%, compared with CGTases which are able to convert up to 40%. The lower conversion compared with the CGTase is probably due to additional differences in the active site cleft and in the starch-binding E domain. A variant with only the five-residue loop deleted was not able to form beta-CD.
引用
收藏
页码:509 / 513
页数:5
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