Heterologous expression of α-1,3-glucanase Agn1p from Schizosaccharomyces pombe, and efficient production of nigero-oligosaccharides by enzymatic hydrolysis from solubilized α-1,3;1,6-glucan

Abstract

ABSTRACT The glycoside hydrolase family 71 α-1,3-glucanase (Agn1p) of Schizosaccharomyces pombe was expressed in Escherichia coli Rosetta-gami B (DE3). Agn1p (0.5 nmol/mL) hydrolyzed insoluble α-1,3-glucan (1%), and about 3.3 mm reducing sugars were released after 1440 min of reaction. The analysis of reaction products by high-performance liquid chromatography revealed that pentasaccharides accumulated in the reaction mixture as the main products, along with a small amount of mono-, di-, tri-, tetra-, and hexasaccharides. Soluble glucan was prepared from insoluble α-1,3;1,6-glucan by alkaline and sonication treatment to improve the hydrolytic efficiency. As a result, this solubilized α-1,3;1,6-glucan maintained a solubilized state for at least 6 h. Agn1p (0.5 nmol/mL) hydrolyzed the solubilized α-1,3;1,6-glucan (1%), and about 8.2 mm reducing sugars were released after 240 min of reaction. Moreover, Agn1p released about 12.3 mm reducing sugars from 2% of the solubilized α-1,3;1,6-glucan.