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.

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.

Nigero-oligosaccharide production by enzymatic hydrolysis from alkaline-pretreated α-1,3-glucan.

Nigero-oligosaccharides are α-1,3-linked oligomers of glucose. Glycoside hydrolase 87 type α-1,3-glucanase Agl-KA from Bacillus circulans KA304 is an endo-lytic enzyme that releases nigero-oligosaccharides (tetra-, tri-, and di-saccharide) from α-1,3-glucan. α-1,3-Glucan is insoluble under natural conditions, thus the efficiency of enzymatic hydrolysis is low and only 5 mM of reducing sugars were released from 1% glucan by Agl-KA. To improve hydrolytic efficiency, α-1,3-glucan was solubilized by 1 M NaOH and alkaline-solubilized glucan was adjusted to approximately pH 8. As a result, glucan maintained a solubilized state. This alkaline-pretreated α-1,3-glucan (1%) was hydrolyzed by Agl-KA (0.64 nmol/mL) and approximately 11.6 mM of reducing sugars were released at 240 min of reaction. When 0.016, 0.032, and 0.13 nmol/mL enzyme were added, reducing sugar reached approximately 5.1, 7.5, and 9.8 mM, respectively, and reaction mixtures containing 0.016 and 0.032 nmol/mL enzyme gradually became cloudy. Our findings suggest α-1,3-glucan cannot maintain its solubilized state and gradually becomes insoluble. For deletion enzyme of α-1,3-glucan binding domains from Agl-KA (AglΔDCD-UCD) on glucan hydrolysis (2%), reducing sugar concentrations released by AglΔDCD-UCD were almost the same as Agl-KA. These findings suggest that alkaline-pretreated α-1,3-glucan maintains a soluble state during a short time period and that glucan is efficiently hydrolyzed even by α-1,3-glucanase without α-1,3-glucan binding domains.