Abstract:To improve the hydrolysis efficiency toward konjac powder, the mannanase from Rhizomucor miehei was successfully modified through directed evolution. After two rounds of screening, a mutant (RmMan5AM2) with improved catalytic efficiency was obtained. The optimal pH of the mutant was pH 7.0, and its optimal temperature was 65 ℃, which was increased by 10 ℃ compared with RmMan5A. RmMan5AM2 inherited the high specific activity of RmMan5A. Its specific activity toward locust bean gum was 10 371.4 U/mg. At the optimal condition, the catalytic efficiencies of the mutant toward locust bean gum, konjac powder, and guar gum were improved by 37.4%, 28.9% and 34.4%, respectively. RmMan5AM2 was further expressed in Pichia pastoris for high cell density fermentation. After 156 h, the expression level of the mutant was up to 176 000 U/mL, which is the highest expression level of mannanases as reported so far. The mutant was then used to hydrolyze konjac powder for manno-oligosaccharides production. The products were mainly manno-oligosaccharides with a degree of polymerization of 2~6(relative peak area>85%), and the total yield was 88.5%. RmMan5AM2 showed excellent enzymatic properties, and its hydrolysis efficiency toward konjac powder was significant improved. Thus, the mannanase has great potential in manno- oligosaccharide production.
