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.
