(593y) 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
3.2.1.78) 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.