An endophyte with high yield of β-mannanase, designated as Paenibacillus sp. CH-3, was isolated from black locust (Robinia pseudoacacia) seeds. The β-mannanase gene (manB) has been cloned, expressed, purified, and characterized. With an open reading frame of 984 bp, the manB gene encodes 327 amino acid residues, which belongs to GH family 5. The recombinant β-mannanase (ManB) has an apparent molecular weight of 50.4 kDa with an optimal activity level at pH 7.0 and 45 °C, and it is stable in the range of pH 4-9. By optimizing, the temperature, isopropyl β-d-thiogalactoside concentration, and induction time were 28 °C, 0.05 mM, and 12 H, respectively. The highest activity reached at 1,054.17 U/mL. In addition, ManB had Km values of 7.30, 8.69, and 27.17 mg/mL for locust bean gum (LBG), guar gum, and konjac glucomannan (KGM), respectively. Its catalytic efficiency (kcat /Km ) was 176.31 mL/(mg·Sec) for LBG, 116.69 mL/(mg·Sec) for guar gum, and 115.49 mL/(mg·Sec) for KGM. The products of glucomannans, which were hydrolyzed by ManB, mainly contain mannobiose, mannotetrose, and a higher manno-oligosaccharide. The characteristics of ManB revealed that it could be exploited as an effective additive in the animal feed industry.
Keywords: TAIL-PCR; endophytes; manno-oligosaccharide; β-mannanase.
© 2016 International Union of Biochemistry and Molecular Biology, Inc.