Biochemical analyses of a novel thermostable GH5 endo β-1,4-mannanase with minor β-1,4-glucosidic cleavage activity from Bacillus sp. KW1 and its synergism with a commercial α-galactosidase on galactomannan hydrolysis.

A novel GH5 endo-1,4-β-mannanase (BaMan5A) was identified from Bacillus sp. KW1, it shares the highest sequence identity (86%) with another characterized Bacillus endo-1,4-β-mannanase. The recombinant BaMan5A displayed maximum activity at pH 7.0 and 70 °C, it was stable at a broad pH range (pH 3.5-11.0) after 12-h incubation at 25 °C, and exhibited good thermostability, retaining about 100% and 85% activity after incubating at 60 °C for 12 h and 65 °C for 8 h, respectively. The results of polysaccharide hydrolysis revealed that the enzyme can only hydrolyze mannan substrates, including carob galactomannan, konjac glucomannan, 1,4-β-D-mannan, locust bean gum, and guar gum, yielding mannose, mannobiose, mannotriose, and some other oligosaccharides. The best substrate was carob galactomannan, the corresponding specific activity and K value were 10,886 μmol/min/μmol and 3.31 mg/mL, respectively. Interestingly, BaMan5A was capable to hydrolyze both manno-oligosaccharides and cello-oligosaccharides, including mannotetraose, mannopentaose, mannohexaose, cellopentaose and cellohexaose. Furthermore, BaMan5A acted synergistically with a commercial α-galactosidase (CbAgal) on galactomannan depolymerization, a best synergy degree of 1.58 was achieved after optimizing enzyme ratios. This study not only expands the diversity of Bacillus GH5 β-mannanase, but also discloses the potential of BaMan5A in industrial application.