Increasing of product specificity of gamma-cyclodextrin by mutating the active domain of alpha-cyclodextrin glucanotransferase from Paenibacillus macerans sp. 602-1.
- Author:
Ting XIE
1
;
Yang YUE
1
;
Binghong SONG
1
;
Yapeng CHAO
1
;
Shijun QIAN
1
Author Information
1. National Engineering Laboratory for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
- Publication Type:Journal Article
- MeSH:
Escherichia coli;
genetics;
metabolism;
Glucosyltransferases;
genetics;
metabolism;
Mutant Proteins;
genetics;
metabolism;
Mutation;
Paenibacillus;
enzymology;
Recombinant Proteins;
genetics;
gamma-Cyclodextrins;
metabolism
- From:
Chinese Journal of Biotechnology
2013;29(9):1234-1244
- CountryChina
- Language:Chinese
-
Abstract:
We studied the mutation effect of subsites -3(Lys47), -7(146-152), and cyclization center (Tyr195) in active domain on product specificity of alpha-cyclodextrin glucanotransferase (alpha-CGTase) from Paenibacillus macerans sp. 602-1. The Lys47 was replaced by Thr47 and Tyr195 by Ile195, and the amino acids from 146 to 152 were replaced by Ile (named as delta6). All these mutant alpha-CGTases were actively expressed in E. coli BL21. Compared with the wild-type alpha-CGTase, the starch-degrading activities of all the mutant enzymes were declined. For mutant Y195I, the percentage of alpha-CD was decreased from 68% to 30%, and beta-CD was raised from 22.2% to 33.3%. Interestingly, gamma-CD was increased from 8.9% to 36.7% and became the main product, while the actual yield was increased from 0.4 g/L to 1.1 g/L. Mutant K47T and delta6 still produced alpha-CD as main product though the percentage of beta- and gamma-CD increased. Purified Y195I CGTase showed similar optimum temperature with the wild-type alpha-CGTase, but its optimum pH shifted from 5.0 to 6.0 with better pH stability. In summary, mutant Y195I CGTase has the potential to produce gamma-CD as the main product.
