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KMID : 0545120120220030331
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Journal of Microbiology and Biotechnology
2012 Volume.22 No. 3 p.331 ~ p.338
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Cloning, High-Level Expression, Purification, and Properties of a Novel Endo-β-1,4-Mannanase from Bacillus subtilis G1 in Pichia pastoris
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Thi Thu Hang Vu
Dinh Thi Quyen
Thi Tuyet Dao
Sy Le Thanh Nguyen
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Abstract
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A novel gene coding for an endo-β-1,4-mannanase (manA) from Bacillus subtilis strain G1 was cloned and overexpressed in P. pastoris GS115, and the enzyme was purified and characterized. The manA gene consisted of an open reading frame of 1,092 nucleotides, encoding a 364-aa protein, with a predicted molecular mass of 41 kDa. The β-mannanase showed an identity of 90.2-92.9% (≤95%) with the corresponding amino acid sequences from B. subtilis strains deposited in GenBank. The purified β- mannanase was a monomeric protein on SDS-PAGE with a specific activity of 2,718 U/mg and identified by MALDITOF mass spectrometry. The recombinant β-mannanase had an optimum temperature of 45oC and optimum pH of 6.5. The enzyme was stable at temperatures up to 50oC (for 8 h) and in the pH range of 5-9. EDTA and most tested metal ions showed a slightly to an obviously inhibitory effect on enzyme activity, whereas metal ions (Hg2+, Pb2+, and Co2+) substantially inhibited the recombinant β-mannanase. The chemical additives including detergents (Triton X- 100, Tween 20, and SDS) and organic solvents (methanol, ethanol, n-butanol, and acetone) decreased the enzyme activity, and especially no enzyme activity was observed by addition of SDS at the concentrations of 0.25-1.0% (w/v) or n-butanol at the concentrations of 20-30% (v/v). These results suggested that the β-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.
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KEYWORD
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Bacillus subtilis G1, Endo- β-1, 4-mannanase gene, Cloning, Over-expression, Pichia pastoris, Properties
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