| β-Mannanases(EC3.2.1.100) are endo-cleaving enzyme that can hydrolyze both the polysaccharide mannan and manno-oligosaccharides linked by a β-1, 4 glycosidic bond(including mannan, glucomannan, galactomannan, and galactoglucomannan), and it belong to the hemicellulose. β-Mannanases have significant potential in the paper, food, feed, pharmaceutical and oil development industries. In recent years, it has attracted wide attention because of its producing functional oligosaccharides. This study describes the β-mannanase of Bacillus subtilis YH12, strain identification, fermentation optimization, purification and enzymatic characterization. And, the β-mannanase gene is cloned and expressed in B. subtilis WB600 system. The main results of this paper are listed as follows: 1. The strain was identified to be Bacillus subtilis by 16 Sr DNA sequencing, and named as Bacillus subtilis YH12. The optimized fermentation conditions were 4% konjac flour as carbon source, 2% tryptone as nitrogen source, initial p H 7.5, 30 oC and 33 h of culture conditions. The mannanase activity was increased from initial 55 U/m L to 280 U/m L. 2. To obtain a purified β-mannanase though 40%-90% ammonium sulfate precipitation, dialysis, desalting, ion exchange DEAE Sepharose column, and the G-75 chromatography separation and purification steps. The activity recovery was 27.3%, and the specific activity was 7302.4 U / mg with 7.1 times of purification factor. SDS-PAGE analysis showed that the molecular weight of the enzyme was about 40 k Da. 3. The β-mannanase of B. subtilis YH12 was purified to homogeneity and its biochemical characterization was performed. The optimal reaction conditions for the purified enzyme were p H 6.5 and 55°C, and it remained stable at temperature up to 55°C and p H of 4.0-8.0. The activity of the enzyme was activated by Na+, K+, Ca2+, Mg2+ and Ba2+, while inhibited by Ag+ and EDTA. And it showed high activity on many polysaccharides. The mannanase exhibited higher activity on galactomannan branched with(1â†'6)-linked α-D-galactose than glucomannan. And the predominant products resulting from mannanase hydrolysis were 1-7 units of manno-oligosaccharides from locust bean gum, 2-7 units of manno-oligosaccharides from konjac powder, and 1-2 units of manno-oligosaccharides from xanthan gum. 4. The mannanase gene was cloned and the ORF was 1083 bp, encoding 360 amino acid and the theoretical molecular weight was 40 k Da. To construct p MA5-man WH(without the signal peptide) and p MA5-man YH(with the signal peptide) recombinant plasmid, and transport to B. subtilis WB600 host cells. Then the recomninant engineering bacteria B. subtilis p MA5-man WH/WB600 and B. subtilis p MA5-man YH/WB600 were successful, the mannanase activity were 13.04 U/m Land 34.01 U/m L. It showed that the signal peptide could improve the production of the mannanase. And this study successfully constructed β-mannanase mutants A56 M and P128 L, activity were 113.6 U/m L and 37.1 U/m L... |
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