期刊
PROCESS BIOCHEMISTRY
卷 50, 期 10, 页码 1572-1580出版社
ELSEVIER SCI LTD
DOI: 10.1016/j.procbio.2015.06.005
关键词
Maltogenic amylase; Transglycosylation; Malto-oligosaccharide; Site-directed mutagenesis; Protein engineering
资金
- Malaysia Genome Institute, Ministry of Science, Technology and Innovation Malaysia [02-05-20-SF0007]
Malto-oligosaccharide synthesis using maltogenic amylase often struggles with product rehydrolyzation. The malto-oligosaccharide synthesis using a maltogenic amylase (MAGI) from Bacillus lehertsis G1 was enhanced using a structure-guided protein engineering approach. Mutations decreased the hydrolysis activity of the enzyme and caused various modulations in its transglycosylation properties. W359F, Y377F and M375I mutations caused a reduction in steric interference, an alteration of subsite occupation and an increase in internal flexibility to accommodate longer donor/acceptor molecules for transglycosylation, resulting in an increase in the transglycosylation to hydrolysis ratio of up to 4.0-fold. The increase in active site hydrophobicity that was caused from the W359F and M375I mutations reduced the concentration of maltotriose required for use as a donor/acceptor for transglycosylation to 100 mM and 50 mM, respectively, compared to the 200 mM needed for wild-type. An improvement of the transglycosylation to hydrolysis ratio by 4.2-fold was also demonstrated in each of the mutants. Interestingly, a reduction of steric interference and hydrolysis suppression was caused by the Y377F mutation and introduced a synergistic effect to produce malto-oligosaccharides with a higher degree of polymerization than wild-type. These findings showed that modification of the active site structure imposed various effects on MAGI activities during malto-oligosaccharide synthesis. (C) 2015 Published by Elsevier Ltd.
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