287090 Production, Purification and Characterization of b-Mannanase From Bacillus Subtilis TJ-101 and Its Application in the Preparation of Gluco-Mannooligosaccharides
b-mannanase (b-1,4-D-mannanmannohydrolase, EC 18.104.22.168) is an important hemicellulase, which can catalyze the hydrolysis of internal b-1,4-D-mannopyranosyl linkages in various b-mannan such as galactomannan, glucomannan, galactoglucomannan. b-mannanase has been widely used in the food, feed, paper and oil industries. In the food industry, b-mannanase is an excellent biocatalyst for the production of gluco-mannooligosaccharides (GMOS). Several studies have verified that GMOS with the degree of polymerization (DP) of 2~6 have the outstanding bioactivities on human being. For example, GMOS could increase the growth of intestinal microorganisms, decrease pathogenic bacteria and improve the integrity of intestinal mucosa [Zhang et al., 2009]. Among various microorganisms being reported as b-mannanase producers, Bacillus subtilis is recommended because of its safety, fast growth and the high secretion ability of b-mannanase [Jiang et al., 2006]. b-mannanase of Bacillus subtilis is an inducible and extracellular enzyme whose activity and production is greatly influenced by media components, such as carbon sources, nitrogen sources and metal ions.
In this work, strain TJ-101 was firstly identified by 16S rDNA as Bacillus subtilis (Fig. A). The response surface method was applied to improving and enhancing the enzyme production. The optimized media components were obtained (g/L): konjac 34.68, (NH4)2SO4 4.69, Na2HPO4 12H2O 4.00, KH2PO4 0.30, CaCO3 2.69, CaCl2 1.00, NaCO3 1.00, MgCl2 6H2O 0.60 (Fig. B). Under these conditions, the maximum enzyme activity increased 27.68% from 120.42U/mL to 153.75U/mL. Then, b-mannanase was produced in a 7-L fermentor and 7.39-fold purified through the salting out, ultrafiltration, anion-exchange and size-exclusion preparative chromatography with a recovery of 21.41% and a specificity of 125.36U/mg proteins. b-mannanase was optimally active at pH 5.0-8.0 and 60°C (Fig. C) and its molecular weight was 38kDa determined by SDS-PAGE (Fig. D). The enzyme activity was slightly stimulated by some metal ions, such as Fe3+, Cu2+ and Cr2+. The purified b-mannanase showed excellent catalysis efficiency in hydrolysis of konjac flour to prepare GMOS. The GMOS yield of 57.76% has been achieved with 8.71% of mannose and 14.49% of glucose (Fig. E), demonstrating the potential use of b-mannanase in food industry.
(1) Jiang, Z., Wei, Y., Li, D., et al. High-level production, purification and characterization of a thermostable beta-mannanase from the newly isolated Bacillus subtilis WY34, Carbohydrate Polymers, 66, 88-96, 2006.
(2) Zhang, Y.Z., Zhang, M., Chen, X.L., et al. Purification and functional characterization of endo-beta-mannanase MAN5 and its application in oligosaccharide production from konjac flour, Applied Microbiology and Biotechnology, 83, 865-873, 2009.
This work was supported by the Program for New Century Excellent Talents in Chinese University (NCET-08-0386), the 863 Program of China (2008AA10Z318), the Natural Science Foundation of China (20976125; 31071509; 51173128) and Tianjin (10JCYBJC05100), and the Program of Introducing Talents of Discipline to Universities of China (No. B06006).
Figure (A) Phylogenetic tree of Bacillus subtilis TJ-101, (B) 3D surface plots of RSM, (C) Optimum pH and temperature of b-mannanase, (D) SDS¨CPAGE of b-mannanase, (E) HPLC of gluco-mannooligosaccharides.
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