Reaction control and protein engineering of bacillus lehensis G1 maltogenic amylase for higher malto-oligosaccharide synthesis
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Date
2016
Authors
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Publisher
Universiti Teknologi Malaysia
Abstract
A multi-functional maltogenic amylase (MAG1) from alkaliphilic Bacillus lehensis G1 exhibited remarkable hydrolysis and transglycosylation activity to produce malto-oligosaccharides of various lengths. MAG1 demonstrated hydrolysis activity over wide range of substrates. Kinetic analysis revealed that the enzyme hydrolyzed small substrate more efficiently than the larger substrate. This was shown by lower Michaelis constant (Km) value and higher turnover number (kcat) and second order rate constant (kcat/Km) values for ß-cyclodextrin compared to that of soluble starch. Malto-oligosaccharide synthesis by transglycosylation activity of MAG1 faces problem of product re-hydrolyzation due to the hydrolysis activity of the enzyme. An equilibrium-control reaction approach has been successfully employed to improve malto-oligosaccharides production by decreasing hydrolysis activity. A yield of 38% transglycosylation products was obtained with the presence of malto-oligosaccharides longer than maltoheptaose. The addition of organic solvents demonstrated an increase in the transglycosylation-to-hydrolysis ratio from 1.29 to 2.15. The transglycosylation activity of MAG1 was also successfully enhanced by using structure-guided protein engineering approach. A molecular modeling and substrate docking was performed to study the structure-function relationship for rational design. A unique subsite structure which has not been reported in other maltogenic amylases was revealed and the information was used to design mutants that have active sites with reduced steric interference and higher hydrophobicity properties to increase the transglycosylation activity. Mutations decreased the hydrolysis activity of the enzyme and caused various modulations in its transglycosylation property. W359F, Y377F and M375I mutations caused reductions in steric interference and alteration of subsite occupation. In addition, the mutations increased internal flexibility to accommodate longer donor/acceptor molecule for transglycosylation, resulted in increased transglycosylation to hydrolysis ratio of up to 4.0-fold. The increase of the active site hydrophobicity from W359F and M375I mutations reduced concentration of maltotriose used as donor/acceptor for transglycosylation to 100 mM and 50 mM, respectively compared to 200 mM of the wild-type. The improvement of the transglycosylation to hydrolysis ratio by 4.3-fold was also demonstrated by both mutants. Interestingly, reductions of both steric interference and hydrolysis by Y377F and W359F mutations caused a synergistic effect to produce malto-oligosaccharides with higher degree of polymerization than the wild-type. These findings showed that the transglycosylation activity of MAG1 was successfully improved by controlling water activity and modification of the active site structure. The high transglycosylation activity of MAG1 and mutants offers a great advantage for synthesizing malto-oligosaccharides and rare carbohydrates
Description
Thesis (PhD. (Bioprocess Engineering))
Keywords
Protein engineering, Oligosaccharides—Synthesis, Hydrolysis