• Microbiology and Biotechnology Letters
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• v.29 no.4
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• pp.201-205
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• 2001

A bacteria strain producing extracellular $\beta$-mannanase was isolated from soil and was identified as Aeromonas sp. A genomic DNA library constructed from Aeromonas, sp that secrets a $\beta$-mannanase was screened for mannan hydrolytic acticity. Recombinant $\beta$-mannanase activity was detercted on the basis of the clear zones around Escherichia coli colonies grown on a LB medium supplemented locust bean gum, EcoRI restriction analysis of plasmid prepared from recombinant E. coli which showed a $\beta$-mannanase activity revealed 10 kb DNA insert, The optimum pH and temperature for the activity of reconmbinant $\beta$-mannanase were 6.0 and $50^{\circ}C$ respectively and were identical to those of the native enzyme.

• Microbiology and Biotechnology Letters
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• v.32 no.3
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• pp.212-217
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• 2004

The gene coding for mannanase from Bacillus subtilis WL-7, a number of glycosyl hydrolase family 26, was hyperexpressed in Escherichia coli. Two recombinant plasmids, pE7MAN and pENS7, were constructed by introducing the complete mannanase gene and the mature mannanase gene lacking N-terminal signal peptide region into a expression vector pET24a(＋), respectively. The level of mannanase produced by E. coli BL21 (DE3) carrying pENS7, which included the mature mannanase gene, was considerably higher than that by E. coli BL21 (DE3)/pE7MAN. Almost mannanase produced by the recombinant E. coli carrying pENS7 at growth temperature of $37^{\circ}C$ existed as inactive enzyme of insoluble form. Growth at temperature below $31^{\circ}C$ increased the soluble fraction of mannanase having catalytic activity in the recombinant E. coli cells. The highest productivity of active mannanase was observed in cell-free extract of the recombinant E. coli grown at growth temperature ranging from $25^{\circ}C$ to $28^{\circ}C$, while mannanase activity per soluble protein of the cell-free extract was highest in the cells grown at $^31{\circ}C$.

• KSBB Journal
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• v.11 no.5
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• pp.565-571
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• 1996

Medium optimization for ${\beta}$-mannanase production by Aspergillus oryzae ATCC 2114 was performed. Effect of carbon source (locust bean gum) concentration on ${\beta}$-mannanase production was investigated. Above 20 g/L locust bean gum, a lag time for ${\beta}$-mannanase production was appeared because high concentration of locust bean gum caused high viscosity which made the mixing of medium poor. As the locust bean gum concentration in the medium increased, ${\beta}$-mannanase activity and cell growth increased proportionally. Effect of various nitrogen sources on ${\beta}$-mannanase production was also studied. (NH4)2SO4 and malt extract were the most effective for ${\beta}$-mannanase production among the inorganic nitrogenous compounds and organic nitrogen nutrients. Inorganic compounds such as KH2SO4, NaCl, Na2CO3, and MgSO4, on ${\beta}$-mannanase production were optimized for ${\beta}$-mannanase production. Locust bean gum of 10 g/L, malt extract of 3 g/L, (NH4)2SO4 of 2 g/L, KH2SO4, of 10 g/L were selected as the optimal medium. Culture in a fermentor by using the optimal medium was carried out. Lag time of ${\beta}$-mannanase production was shorter due to the better mixing of the fermentor. The maximum ${\beta}$- mannanase activity of 9.7 unit/mL and specific ${\beta}$-mannanase activity of 1.9 unit/mg-cell could be obtained at 27 hours and the productivity of ${\beta}$-mannanase was 0.36 unit/mL$.$h.

• Microbiology and Biotechnology Letters
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• v.25 no.2
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• pp.212-217
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• 1997

A strain of Bacillus sp. WS-14 was isolated from soil. Medium optimization for ${\beta}-mannanase$ production by Bacillus sp. WS-14 was performed. Effect of various carbon sources on ${\beta}-mannanase$ production was investigated and locust bean gum was the most effective for ${\beta}-mannanase$ production. ${\beta}-mannanase$ activity and cell growth increased with increasing the concentration of locust bean gum, however, the amounts were not significant. Among nitrogen sources, soytone was the most effective for ${\beta}-mannanase$ production. Inorganic compounds such as $KH_2PO_4,\;NaCl\;Na_2CO_3\;and\;MgSO_4{\cdot}7H_2O\;on\;{\beta}-mannanase$ production were optimized for ${\beta}-mannanase$ production. Locust bean gum of 10.0 g/l, soytone of 5.0 g/l, $KH_2PO_4$ of 2.0 g/l, NaCl of 10.0 g/l, $MgSO_4{\cdot}7H_2O\;of\;0.2\;g/l,\;Na_2CO_3$, of 2.0 g/l were selected as optimum content. Production of ${\beta}-mannanase$ by using the optimum medium was carried out. The maximum ${\beta}-mannanase$ activity of 20.8 unit/ml could be obtained after 14 h fermentation which corresponed to the productivity of ${\beta}-mannanase$ of 1.48 unit/ml-h.

• Microbiology and Biotechnology Letters
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• v.31 no.1
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• pp.57-62
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• 2003

A bacteria strain producing extracellular $\beta$-mannanase was isolated from soil and was identified as Bacillus subtilis by 16S rRNA sequence comparison and biochemical determinations. The optimum pH and temperature for the $\beta$-mannanase activity were 5.0 and 5.5$^{\circ}C$, respectively. The zymogram technique revealed a single protein band exhibiting $\beta$-mannanase activity from the culture supernatant. The molecular mass of the enzyme was estimated at approximately 130 kDa. The addition of 0.5% lactose or 0.5% locust bean gum to the LB medium caused to Increase significantly the $\beta$-mannanase productivity from Bacillus subtilis JS-1. The cells grown on LB medium supplemented with lactose produced maximal enzyme activity at the stationary phase. In contrast to this, the $\beta$-mannanase was induced at the logarithmic phase from the cells grown on LB medium supplemented with locust bean gum. The discrepancy in induction times suggests that $\beta$-mannanase was induced by different induction mechanisms depending on the carbon sources in Bacillus subtilis JS-1 .

• Microbiology and Biotechnology Letters
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• v.37 no.4
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• pp.405-407
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• 2009

A Bacillus subtilis mannanase gene was subcloned into an Escherichia coli- Corynebacterium lactofermentum shuttle vector pHE83, and the resultant plasmid pHE83M was transferred into an endogenous plasmid-free strain of C. lactofermentum. Mannanase produced by the recombinant C. lactofermentum (pHE83M) was secreted extracellulary at the level of 86%, and the remaining activity of mannanase was detected in the cell-free extract. The maximum mannanase productivity of the recombinant strain reached 8.1 unit/mL in the culture filtrate of LB medium. According to the zymogram of mannanase on SDS-PAGE, it was found that the mannanase produced by the recombinant C. lactofermentum could be maintained stably with a migration identical to the mannanase produced by the parental strain, B. subtilis WL-3.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.331-338
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• 2012

A novel gene coding for an endo-${\beta}$-1,4-mannanase (manA) from Bacillus subtilis strain G1 was cloned and overexpressed in P. pastoris GS115, and the enzyme was purified and characterized. The manA gene consisted of an open reading frame of 1,092 nucleotides, encoding a 364-aa protein, with a predicted molecular mass of 41 kDa. The ${\beta}$-mannanase showed an identity of 90.2-92.9% ${\leq}95%$) with the corresponding amino acid sequences from B. subtilis strains deposited in GenBank. The purified ${\beta}$-mannanase was a monomeric protein on SDS-PAGE with a specific activity of 2,718 U/mg and identified by MALDI-TOF mass spectrometry. The recombinant ${\beta}$-mannanase had an optimum temperature of $45^{\circ}C$ and optimum pH of 6.5. The enzyme was stable at temperatures up to $50^{\circ}C$ (for 8 h) and in the pH range of 5-9. EDTA and most tested metal ions showed a slightly to an obviously inhibitory effect on enzyme activity, whereas metal ions ($Hg^{2+}$, $Pb^{2+}$, and $Co^{2+}$) substantially inhibited the recombinant ${\beta}$-mannanase. The chemical additives including detergents (Triton X-100, Tween 20, and SDS) and organic solvents (methanol, ethanol, n-butanol, and acetone) decreased the enzyme activity, and especially no enzyme activity was observed by addition of SDS at the concentrations of 0.25-1.0% (w/v) or n-butanol at the concentrations of 20-30% (v/v). These results suggested that the ${\beta}$-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.

• Korean Journal of Microbiology
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• v.46 no.2
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• pp.207-212
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• 2010

A bacterium producing the extracellular mannanase was isolated from Korean soybean paste. The isolate WL-8 has been identified as Bacillus subtilis on the basis on its 16S rRNA sequence, morphology and biochemical properties. The mannanase productivity of strain WL-8 was increased in LB broth by addition of wheat bran. The maximum mannanase productivity was reached to approximately 20 U/ml in LB medium supplemented with 6% wheat bran. A gene encoding the mannanase of WL-8 was cloned into Escherichia coli and its nucleotide sequence was subsequently determined. The mannanase gene consisted of 1,086 nucleotides encoding a polypeptide of 362 amino acid residues. The deduced amino acid sequence was highly homologous with those of several mannanases from B. subtilis belonging to GH family 26. Reaction temperature and pH profiles were investigated using the culture filtrate and cell-free extract of the recombinant E. coli carrying a WL-8 mannanase gene, respectively. Optimal conditions for the two fractions occurred at pH 5.5 and $60^{\circ}C$. The cell-free extract showed higher mannanase activity than the culture filtrate at above $60^{\circ}C$.

• Journal of the Korean Society of Food Science and Nutrition
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• v.23 no.3
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• pp.509-514
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• 1994

Penicillium purpurogenum , which produces a copra galactomannan degrading enzyme extracellularyl, was isolated from soil , and its properties and formation condition of mannooligosaccharides were investigated. The optimum ph and temperature for the activity of the mannanase were 5.5 and 55$^{\circ}C$, respectively. The mannanase was stable in between pH 3.5 and 7.0 after 2 hr incubation at 3$0^{\circ}C$ lost 90% of the original activity after incubation at 55$\AA$ and pH 5.5 for 2 hr. With two different substrate concentration, hydrolysis of white coprameal proceeded rapidly at the early stage of the reaction, but gradually solwed thereafter especially at a higher concentration of copra meal (20 %). The enzyme hydrolyzed white copra meal to monosaccharides, mannobiose and mannotriose at the final stage of the reaction.

• Journal of Applied Biological Chemistry
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• v.53 no.2
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• pp.71-76
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• 2010

A gene encoding the mannanase of Bacillus licheniformis WL-12, which had been isolated from Korean soybean paste, was cloned into Escherichia coli and nucleotide sequence of the mannanase gene was subsequently determined. The mannanase gene consisted of 1,080 nucleotides encoding a polypeptide of 360 amino acid residues. The deduced amino acid sequence was identical to that of putative mannanase from B. liceniformis DSM13 belonging to GH family 26. The mannanase was partially purified from cell-free extract of the recombinant Escherichia coli carrying a WL-12 mannanase gene by ammonium sulfate fractionation and DEAE-Sepharose column chromatography. Optimal conditions for the partially purified enzyme occurred at pH 6.0 and $65^{\circ}C$. The enzyme showed higher activity on locust bean gum (LBG) galactomannan and konjac glucomannan than on guar gum galactomannan. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.