Characterization, Gene Cloning, and Heterologous Expression of β-Mannanase from a Thermophilic Bacillus subtilis

Pijug Summpunn; Suttidarak Chaijan; Duangnate Isarangkul; Suthep Wiyakrutta; Vithaya Meevootisom

doi:10.1007/s12275-011-0357-1

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Characterization, Gene Cloning, and Heterologous Expression of β-Mannanase from a Thermophilic Bacillus subtilis: Pijug Summpunn ^1,2, Suttidarak Chaijan ², Duangnate Isarangkul ², Suthep Wiyakrutta ², Vithaya Meevootisom ²; Journal of Microbiology 2011;49(1):86-93.
DOI: https://doi.org/10.1007/s12275-011-0357-1
Published online: March 3, 2011

¹Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand, ²Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand¹Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand, ²Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand

Corresponding author: Vithaya Meevootisom , Tel: +66-2201-5671,

Received: 8 September 2010 • Accepted: 3 November 2010

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Abstract

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).

Keywords: β-mannanase;Bacillus subtilis;characterization;gene expression;thermophilic

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Characterization, Gene Cloning, and Heterologous Expression of β-Mannanase from a Thermophilic Bacillus subtilis

J. Microbiol. 2011;49(1):86-93. Published online March 3, 2011

DOI: https://doi.org/10.1007/s12275-011-0357-1

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