Cloning and characterization of bacterial mannanase isolated from dynastes hercules larvae

Abstract

Bacterial isolate HM7 was isolated from Dynastes hercules larvae excrement. It was identified as Bacillus sp. by 16S rRNA gene analysis. When the bacteria were cultured for β-mannanase production in medium containing konjac flour it had the highest specific activity of 138 U/mg at 36 h, and when cultured in medium containing coconut meal it had the highest specific of 114 U/mg at 48 h. The 1089 base pairs long of a β-mannanase encoding gene (Man26HM7) was successfully cloned and expressed in Escherichia coli BL21 (DE3), using pET21-b expression system. The enzyme was purified and characterized. The optimal condition MAN26HM7catalysis was 50 o C and pH 6.0. MAN26HM7 showed surfactant-tolerant ability when compared to Bacillus subtilis CAe24 mannanase (MAN26CAe24). The residual activity of MAN26HM7 and MAN26CAe24 was 90% and 60% after incubated with 1.0 % (w/v) SDS at 37 °C for 180 min, and retaining 60 % and 40 % of MAN26HM7 and MAN26CAe24 activity at 2.0% (w/v) SDS at 50 and 60 °C, respectively. Powder detergent at 0.5 % (w/v) decreased β-mannanase activity, retaining more than 55 % and 51 % of MAN26HM7 and MAN26CAe24) activity, respectively, more than 5 % (v/v) liquid detergent, retaining more than 95 % and 90 % of MAN26HM7 and MAN26CAe2424) activity. Amino acid sequence alignment of MAN26HM7 and MAN26CAe24 showed amino acid changes at 7 positions, one of which was found in the interior of the protein, leucine 238. Leucine at this position in MAN26HM7 was replaced with Isoleucine in MAN26CAe24. Sited-directed mutagenesis of amino acid at this position in MAN26CAe24 from I to L improved SDS-tolerant of the enzyme. Surfactant-tolerant β -mannanase can be applied in detergent industry.