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Process Biochemistry
Vol. 50 (10), 2015, Pages: 1572–1580

Modulation of transglycosylation and improved malto-oligosaccharide synthesis by protein engineering of maltogenic amylase from Bacillus lehensis G1

Nor Hasmaliana Abdul Manas, Mohd Anuar Jonet, Abdul Munir Abdul Murad, Nor Muhammad Mahadi, Rosli Md. Illias

Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, Skudai, Johor, Malaysia.


Malto-oligosaccharide synthesis using maltogenic amylase often struggles with product re-hydrolyzation. The malto-oligosaccharide synthesis using a maltogenic amylase (MAG1) from Bacillus lehensis 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 MAG1 activities during malto-oligosaccharide synthesis.

Graphical abstract

Keywords: Maltogenic amylase; Transglycosylation; Malto-oligosaccharide; Site-directed mutagenesis; Protein engineering.

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