title: [Increasing of product specificity of gamma-cyclodextrin by mutating the active domain of alpha-cyclodextrin glucanotransferase from Paenibacillus macerans sp. 602-1].
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.
authors.affiliation: National Engineering Laboratory for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Xie
authors.affiliation: National Engineering Laboratory for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yue
authors.affiliation: National Engineering Laboratory for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Binghong
authors.initials: B
authors.lastname: Song
authors.affiliation: National Engineering Laboratory for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Yapeng
authors.initials: Y
authors.lastname: Chao
authors.affiliation: National Engineering Laboratory for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Shijun
authors.initials: S
authors.lastname: Qian
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D056507:Paenibacillus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics; D047408:gamma-Cyclodextrins / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D047408:gamma-Cyclodextrins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; C023792:gamma-cyclodextrin