• Korean Journal of Microbiology
• /
• v.42 no.4
• /
• pp.313-318
• /
• 2006

A thermophilic bacterium producing the extracellular cellulase was isolated from soybean paste, and the isolate WL-12 has been identified as Bacillus licheniformis on the basis on its 16S rRNA sequence, morphology and biochemical properties. A gene encoding the cellulase of B. licheniformis WL-12 was cloned and its nucleotide sequence was determined. This cellulase gene, designated celA, consisted of 1,551 nucleotides, encoding a polypeptide of 517 amino acid residues. The gene product contained catalytic domain and cellulose binding domain. The deduced amino acid sequence was highly homologous to those of cellulases of B. licheniformis, B. subtilis and B. amytoliquefaciens belonging to the glycosyl hydrolase family 5. When the celA gene was highly expressed using a strong B. subtilis promoter, the extracellular cellulase was produced up to 7.0 units/ml in B. subtilis WB700.

• Journal of Microbiology and Biotechnology
• /
• v.17 no.10
• /
• pp.1688-1694
• /
• 2007

A gene encoding the mannanase of Bacillus subtilis WL-3, which had been isolated from Korean soybean paste, was cloned into Escherichia coli and the nucleotide sequence of a 2.7-kb DNA fragment containing the mannanase gene was subsequently determined. The mannanase gene, designated manA, consisted of 1,080 nucleotides encoding a polypeptide of 360 amino acid residues. The deduced amino acid sequence was highly homologous to those of mannanases belonging to glycosyl hydrolase family 26. The manA gene was strongly expressed in B. subtilis 168 by cloning the gene downstream of a strong B. subtilis promoter of plasmid $pJ27{\Delta}88U$. In flask cultures, the production of mannanase by recombinant B. subtilis 168 reached maximum levels of 300 units/ml and 450 units/ml in LB medium and LB medium containing 0.3% locust bean gum, respectively. Based on the zymogram ofthe mannanase, it was found that the mannanase produced by recombinant B. subtilis could be maintained stably without proteolytic degradation during the culture time.

• Microbiology and Biotechnology Letters
• /
• v.31 no.3
• /
• pp.277-283
• /
• 2003

A bacterium producing the extracellular mannanase was isolated from Korean soybean paste. The isolate WL-7 has been identified as Bacillus subtiis on the basis on its 16S rRNA sequence, fatty acid composition, morphology and biochemical properties. The mannanase of culture supernatant was the most active around $55^{\circ}C$ and pH $6.0^{\circ}C$, and retained 90% of its maximum activity at range of pH 5.0∼7.5 and $50∼60^{\circ}C$. The additional carbohydrates including lactose, $\alpha$-cellulose, avicel, locust bean gum (LBG), wheat bran and konjak increased dramatically the mannanase productivity of strain WL-7. Especially, the maximum mannanase productivity was reached to 224 U/ml in LB medium supplemented with both 0.5% LBG and 0.5% konjak, which was approximately 200-folds more than that in LB medium. It was suggested that the increase of mannanase production was owing to induction of mannanase biosynthesis by both LBG and konjak hydrolysates transported following initial hydrolysis by extracellular mannanase during the cell growth.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.6
• /
• pp.1295-1302
• /
• 2004

A gene encoding the mannanase of Bacillus subtilis WL-7, which had been isolated from Korean soybean paste, was cloned into Escherichia coli, and the gene product was purified from the culture filtrate of the recombinant E. coli. This mannanase gene, designated manA, consisted of 1,086 nucleotides, encoding a polypeptide of 362 amino acid residues. The deduced amino acid sequence was highly homologous to those of mannanases belonging to the glycosyl hydrolase family 26. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. The enzyme had a pH optimum at 6.0 and a temperature optimum at $55^{\circ}C$. The enzyme was active on locust bean gum, konjak, guar gum, and lichenan, while it did not exhibit activity towards yeast mannan, laminarin, carboxymethylcellulose, $\beta$­glucan, xylan, and para-nitrophenyl-$\beta$-mannopyranoside.

• Microbiology and Biotechnology Letters
• /
• v.32 no.3
• /
• pp.212-217
• /
• 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 $^31{\circ}C$.

• Microbiology and Biotechnology Letters
• /
• v.32 no.4
• /
• pp.347-351
• /
• 2004

Hydrolysis of manno-oligosaccharides and galactomannan was studied with the purified Bacillus subtilis WL-7 mannanase from recombinant Eschericoli. The predominant products of hydrolysis were mannose, mannobiose and mannotriose. The enzyme could hydrolyze $\beta$-1 A-linked manno-oligosaccharides larger than mannobiose, but was not active on mannobiose. When the mannanase hydrolyzed manno-oligo saccharides of degree of polymerization(DP) 4-6, it was more active on the substrate of higher DP. Based on analysis of transient reaction products by TLC, the enzyme was found to have a preference for internal $\beta$-IA-mannosidic linkages, which are the central mannosidic bond of mannotetraose and the two middle mannosidic bonds of mannopentaose. The $\beta$-l A-mannosidic bonds situated at the second and fourth positions from the nonreducing end of mannohexaose were preferenhydrolyzed by the mannanase. Locust bean gum(LBG) was enzymatically hydrolyzed with higher efficiency than guar gum, resulting that amount of reducing sugars was liberated more efficiently from LBG than guar gum with same activity of mannanase.

• Microbiology and Biotechnology Letters
• /
• v.33 no.3
• /
• pp.200-206
• /
• 2005

A bacterium with high productivity of poly-$\gamma$-glutamic acid (PGA) was isolated from the traditional Korean seasoning, ChungKookJang. The 16s ribosomal RNA sequence of isolated strain showed 97.6, 98.9 and $90.3{\%}$ of similarity to those of Bacillus sp. WL-3, Bacillus subtilis; ENV1 and B amy-loliquefaciens (T), respectively. Accordingly, this bacterium was identified as a Bacillus sp. However, some biochemical characteristics of this strain were different from those of B. subtilis: D-xylose fermentation and glycogen utility were negative. Maximum production of PGA was achieved when it was grown aerobically in a culture medium containing glutamic acid ($3{\%}$) and fructose ($4{\%}$) as carbon sources. The volumetric yield of PGA reached up to 27 g/l in the optimum culture medium. These results suggest that the present strain can be applicable for industrial purposes such as a prototype strain for food or cosmetics industry.