Site-Directed Mutagenic Engineering of a Bifidobacterium Amylosucrase toward Greater Efficiency of Turanose Synthesis

Abstract

The aim of this study was to establish one of the most efficient biocatalytic processes for turanose production by applying a robust Bifidobacterium thermophilum (BtAS) mutant developed through site-directed mutagenesis. A gene encoding the amylosucrase of B. thermophilum (BtAS) was cloned and used as a mutagenesis template. Among the BtAS variants generated by the site-directed point mutation, four different single-point mutants (P200R, V202I, Y265F, and Y414F) were selected to create double-point mutants, among which BtAS(Y414F/P200R) displayed the greatest turanose productivity without losing the thermostability of native BtAS. The turanose yield of BtAS(Y414F/P200R) reached 89.3% at 50 degrees C after 6 h with 1.0 M sucrose + 1.0 M fructose. BtAS(Y414F/P200R) produced significantly more turanose than BtAS-wild type (WT) by 2 times and completed the reaction faster by another 2 times. Thus, turanose productivity (82.0 g/(L h)) by BtAS(Y414F/P200R) was highly improved from 28.1 g/(L h) of BtAS-WT with 2.0 M sucrose + 0.75 M fructose.