Fusion of a family 20 carbohydrate-binding module (CBM20) with cyclodextrin glycosyltransferase of Geobacillus sp. CHB1 improves catalytic efficiency.
Cyclodextrin glycosyltransferase (CGTase) is an important industrial enzyme for production of cyclodextrins (CDs) from starch by intramolecular transglycosylation. CGTase consists of five domains labeled A to E. For optimizing catalytic activity of CGTase, CGTase of Geobacillus sp. was fused with the family 20 carbohydrate-binding module (CBM) of the Bacillus circulans strain 251 CGTase. The CBMbc251 that has a low binding free energy with maltohexaose, was selected by in silico design. Then the fusion enzyme, CGTΔE-CBMbc251, was constructed by fusing the CBMbc251 to the C-terminal region of CGTΔE. The fusion enzyme displayed an even greater enhancement of total α-cyclization activity (40.2%) and γ-cyclization activity (181.58%). Optimal reaction pH range was wilder and the thermal stability was better under 50 and 60 °C. Compared to the wild-type CGTase, the fusion enzyme showed a remarkable decrease in Km and a slight alteration in Vmax. The enhancement of soluble starch catalytic efficiency might be due to the changes of substrate binding ability in the critical substrate binding sites between the CBM and starch granule.
['Jia|Xianbo|X|http://orcid.org/0000-0002-0815-8713', 'Guo|Yonghua|Y|', 'Lin|Xinjian|X|', 'You|Minsheng|M|', 'Lin|Chenqiang|C|', 'Chen|Longjun|L|', 'Chen|Jichen|J|']
D001407:Bacillus D001665:Binding Sites D055162:Biocatalysis D002241:Carbohydrates D003198:Computer Simulation D003505:Cyclodextrins D056506:Geobacillus D005964:Glucosyltransferases D006863:Hydrogen-Ion Concentration D007700:Kinetics D008958:Models, Molecular D016297:Mutagenesis, Site-Directed D009844:Oligosaccharides D011487:Protein Conformation D011993:Recombinant Fusion Proteins D013213:Starch
cyclodextrin glycosyltransferase catalytic efficiency in silico design substrate affinity