Cloning of the gene encoding endo-1, 4-β-mannosidase from Bacillus subtilis HB002 and expression in Pichia pastoris

• 1、College of Life Sciences,Hubei University, Wuhan,430062

Abstract：Endo-1, 4-β-mannosidase (E.C. 3.2.1.78), which is also called mannanase, catalyzes the random hydrolysis of β-1, 4-mannosidic linkages in the main chain of mannan and its heteropolysaccharides. Therefore, mannanases are widely applied not only in researches, but also in industries. Mannanases can be produced by various organisms, including bacteria, fungi, plants, and as well as animals. Bacillus subtilis is a major source of this enzyme. A gene from Bacillus subtilis HB002 coding an endo-1, 4-β-mannosidase belonging to glycoside hydrolyase family 26 (GH26), was first cloned from the genome with shot-gun method and the enzyme was heterogenously expressed in Pichia pastoris GS115, driven by a mutant of AOX1 promoter, the d1+2x201 AOX1 and the MF4I signal peptide. The expression of this enzyme was confirmed by SDS-PAGE and plate assay. The molecular weight of the recombinant protein is about 42 kDa. The expression of target protein is up to 0.25mg/mL and the enzyme activity is about 222 U/mL. According to the analysis of enzyme characteristics, the optimal pH is between pH5.6-7.0 and the optimal temperature is 55 C when locust bean gum is used as substrate. This enzyme is fairly stable at various pH and temperature. Accordingly, it is still stable at pH5.0 to 7.0 after incubation at room temperature for 5h. The enzyme remains about 95% activity after 10h incubated at 50 C. In addition, it shows strong substrate specificity to galactomannans and konjac powder. The enzyme activity is strongly inhibited by Co2+ and Mn2+, but it is not sensitive to other ions. Analysis of hydrolytic products by thin layer chromatography (TLC) and MALDI-MS revealed that the degrees of polymerization of products are mostly less than 13, which indicated it's suitable for animal feed and industry. This study is the first report on the cloning, and heterogenous expression of an endo-1, 4-β-mannosidase from Bacillus subtilis HB002 in Pichia pastoris. In addition, the expression level of this endo-1, 4-β-mannosidase increases dramatically when the AOX1 promoter and α-mating factor signal of Sacchromyces cerevisiae in the Pichia pastoris expression system are replaced with the combination of d1+2x201 AOX1 promoter and the MF4I signal peptide coding sequence, respectively. This improvement implies a potential of its application in large-scale.

keywords： Endo-1, 4-β-mannosidase, Bacillus subtilis HB002, d1+2x201 AOX1 promoter, MF4I signal peptide, Pichia pastoris

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