Research Support, Non-U.S. Gov't
- Characterization, Gene Cloning, and Heterologous Expression of β-Mannanase from a Thermophilic Bacillus subtilis
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Pijug Summpunn , Suttidarak Chaijan , Duangnate Isarangkul , Suthep Wiyakrutta , Vithaya Meevootisom
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J. Microbiol. 2011;49(1):86-93. Published online March 3, 2011
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DOI: https://doi.org/10.1007/s12275-011-0357-1
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Abstract
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Bacillus subtilis BCC41051 producing a thermostable β-mannanase was isolated from soybean meal-enriched soil and was unexpectedly found to be thermophilic in nature. The extracellular β-mannanase (ManA) produced was hydrophilic, as it was not precipitated even with ammonium sulfate at 80% saturation. The estimated molecular weight of ManA was 38.0 kDa by SDS-PAGE with a pI value of 5.3. Optimal pH and temperature for mannan-hydrolyzing activity was 7.0 and 60°C, respectively. The enzyme was stable over a pH range of 5.0-11.5, and at temperatures of up to 60°C for 30 min, with more than 80% of its activity retained.
ManA was strongly inhibited by Hg2+ (1 mM), but was sensitive to other divalent ions to a lesser degree. The gene of ManA encoded a protein of 362 amino acid residues, with the first 26 residues identified as a signal peptide. High expression of recombinant ManA was achieved in both Escherichia coli BL21 (DE3) (415.18 U/ml) and B. megaterium UNcat (359 U/ml).
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Journal Article
- Characterization of the Bacillus subtilis WL-3 Mannanase from a Recombinant Escherichia coli
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Ki-Hong Yoon , Seesub Chung , Byung-Lak Lim
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J. Microbiol. 2008;46(3):344-349. Published online July 5, 2008
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DOI: https://doi.org/10.1007/s12275-008-0045-y
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Abstract
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A mannanase was purified from a cell-free extract of the recombinant Escherichia coli carrying a Bacillus subtilis WL-3 mannanase gene. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. Optimal conditions for the purified enzyme occurred at pH 6.0 and 60°C. The specific activity of the purified mannanase was 5,900 U/mg on locust bean gum (LBG) galactomannan at pH 6.0 and 50°C. The activity of the enzyme was slightly inhibited by Mg2+, Ca2+, EDTA and SDS, and noticeably enhanced by Fe2+. When the enzyme was incubated at 4°C for one day in the presence of 3 mM Fe2+, no residual activity of the mannanase was observed. The enzyme showed higher activity on LBG and konjac glucomannan than on guar gum galactomannan. Furthermore, it could hydrolyze xylans such as arabinoxylan, birchwood xylan and oat spelt xylan, while it did not exhibit any activities towards carboxymethylcellulose and para-nitrophenyl-β-mannopyranoside. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides including mannotriose, mannotetraose, mannopentaose and mannohexaose. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.