• Food Science and Preservation
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• v.21 no.3
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• pp.442-450
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• 2014

In this study, we isolated a cellulase-producing bacterium isolated from traditional Korean fermented soybean paste and investigated the effect of culture conditions on the production of cellulase. This bacterium, which was identified as Bacillus subtilis 4-1 through 16S rRNA gene sequence analysis, showed the highest cellulase activity when the cells were grown at $45^{\circ}C$ for 24 hours in the CMC medium supplemented with 1.0% of soluble starch and 0.1% yeast extract. The initial optimum pH of the medium was observed in the range of 5.0~9.0. The optimal pH and temperature for the production of cellulase from B. subtilis 4-1 were pH 9.0 and $60^{\circ}C$ respectively. In addition, the enzyme showed significant activity in the temperature range of $20{\sim}90^{\circ}C$, which indicates that B. subtilis 4-1 cellulase is an alkaline-resistance and thermo-stable enzyme. This enzyme showed higher activity with CMC as the substrate for endo-type cellulase than avicel or pNPG as the exo-type substrates for exo-type cellulase and ${\beta}$-glucosidase. These results suggest that the cellulase produced from B. subtilis 4-1 is a complex enzyme rather than a mono-enzyme.

• Journal of Chitin and Chitosan
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• v.23 no.4
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• pp.228-233
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• 2018

We isolated microorganisms from soil, which is sampled at forest, Kyeonbuk, Korea, as cellulolytic microorganisms. The isolated strains were identified by analysis of 16S rRNA gene from the starins. The result, four kinds of Bacillus subtilis, one kind of Bacillus amyloliquefaciens, and one kind of Bacillus cereus were identified. Among these strains, Bacillus subtilis was selected due to its high cellulase activity and this strain was named as Bacillus subtilis CNS. The optimum pH and temperature of the cellulase from Bacillus subtilis CNS was pH 5.0 and $40^{\circ}C$, respectively. In the investigation of pH and temperature stability, the cellulase from Bacillus subtilis NSC stabled pH 4.0~6.0 range and until $40^{\circ}C$ for 30 min perfectly. In the enzyme activity for various cellulosic substrate, cellulase from Bacillus subtilis CNS showed the highest activity for CM-cellulose. And, the enzyme activities for alkali swollen cellulose, Alpha-cellulose, Sigmacell-cellulose, and Avicel were approximately 31%, 8%, 8% and 4% of activity for CM-cellulose, respectively. In the degradation of CM-cellulose, the 0.26 U/ml and 0.52 U/ml of cellulase showed 0.43 and 0.76 U/ml activity for CM-cellulose after the reaction of 120 min, respectively.

• Journal of Microbiology and Biotechnology
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• v.28 no.9
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• pp.1473-1481
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• 2018

A cellulase hyperproducing mutant strain, JNDY-13, was obtained using the ARTP mutation system and with Trichoderma reesei RUT-C30 as the parent strain. Whole-genome sequencing of JNDY-13 confirmed that 105 of the 653 SNPs were point mutations, 336 mutations were deletions and 165 were insertions. Moreover, 99 mutations were insertions and duplications. Among all the mutations, the one that occurred in the galactokinase gene might be related to the production of cellulases in T. reesei JNDY-13. Moreover, the up-regulation of cellulase and hemicellulase genes in JNDY-13 might contribute to higher cellulases production. Under optimal conditions, the highest cellulase activity by batch fermentation reached 4.35 U/ml, and the highest activity of fed-batch fermentation achieved was 5.40 U/ml.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.2
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• pp.108-112
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• 2007

A cellulase (endo-${\beta}$-1,4-D-glucanase(E.C.3.2.1.4)) was isolated from the hepatopancreas of abalone Haliotis discus hannai by EST analysis. The abalone cellulase named HdEG compassed 1977 bp, including 195 bp in the 5'untranslated region, 1680 bp in the open reading frame which encodes 560 amino acid residues, and 92 bp in the 3'-untranslated region. The C-terminal region of the HdEG showed 44-52% identity to the catalytic domains of glycoside hydrolase family 9 (GHF9)-cellulases from arthropods and bacteria. The recombinant cellulase, pEHdEG was produced in E. coli with being fused with C-terminal His-tag. The expressed protein showed a single band (~62 kDa) on Western blotting which was consistent with the value (61,878 Da) calculated from the DNA sequence.

• Journal of Animal Science and Technology
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• v.50 no.3
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• pp.429-436
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• 2008

A bacterium producing cold-active cellulase and xylanase was isolated from pig feces. The isolate, DK42 strain, was found to be the Gram-positive, non-motile, catalase-positive, and spore-forming stain. Under an electron microscope, the cells were observed to be rod-shaped. The isolate was identified as Bacillus licheniformis DK42 on the basis of morphological and biochemical properties as well as 16S rRNA gene sequences. The characterization of crude cellulase and xylanase from B. licheniformis DK42 was investigated. Cellulase exhibited an optimum temperature and pH at 45℃ and 6.0, whereas xylanase exhibited an optimum temperature and pH at 55℃ and 6.0. Especially cellulase maintained approx. 50% of its maximum activity even at 10℃, indicating that it is cold-active. Both cellulase and xylanase were stable after 2hr at 35℃, whereas they lost their activities after 30min at 65℃.

• Journal of Life Science
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• v.22 no.7
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• pp.912-919
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• 2012

A Bacillus sp. strain producing celluase and xylanase was isolated from environmental soil with LB agar plate containing carboxymethylcellulose (CM-cellulose) and beechwood xylan stained with trypan blue as substrates, respectively. Based on the 16S rRNA gene sequence and API 50 CHL test, the strain was identified as B. subtilis and named B. subtilis NC1. The cellulase and xylanase from B. subtilis NC1 exhibited the highest activities for CM-cellulose and beechwood xylan as substrate, respectively, and both enzymes showed the maximum activity at pH 5.0 and $50^{\circ}C$. We cloned and sequenced the genes for cellulase and xylanase from genomic DNA of the B. subtilis NC1 by the shot-gun cloning method. The cloned cellulase and xylanase genes consisted of a 1,500 bp open reading frame (ORF) encoding a 499 amino acid protein with a calculated molecular mass of 55,251 Da and a 1,269 bp ORF encoding a 422 amino acid protein with a calculated molecular mass of 47,423 Da, respectively. The deduced amino acid sequences from the genes of cellulase and xylanase showed high identity with glycosyl hydrolases family (GH) 5 and 30, respectively.

• Journal of Mushroom
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• v.15 no.4
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• pp.249-253
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• 2017

Twelve strains of bacteria with cellulase and xylanase activities were isolated from spent mushroom substrates collected from button mushroom cultivation farm, Buye, Chungcheongnam-do in Korea. Among them, one strain, designated NO12, with higher cellulase and xylanase activities was selected by agar diffusion method. The strain NO12 was identified to be a Bacillus sp. by biochemical characteristics using Bacillus ID kit and MicroLog system. Comparative 16S rDNA gene sequence analysis showed that strain NO12 formed a distinct phylogenetic tree within the genus Bacillus and was most closely related to Bacillus subtilis with 16S rDNA gene sequence similarity of 99.2%. Based on its physiological properties, biochemical characteristics, and phylogenetic distinctiveness, strain NO12 was classified within the genus Bacillus, for which the name Bacillus subtilis NO12 was proposed. The cellulase and xylanase activities of B. subtilis NO12 were slightly increased according to bacterial population from exponential phase to stationary phase in the growth curve for B. subtilis NO12. The xylanase activity continuously increased from the beginning of the exponential phase and exhibited maximum activity in the middle of the exponential phase.

• Proceedings of the Korean Society of Life Science Conference
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• 2006.09a
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• pp.50.1-50.1
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• 2006

• Korean Journal of Microbiology
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• v.26 no.1
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• pp.6-12
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• 1988

Cellobiase ($\beta$-glucosidase) is an enzyme of the cellulase system in cellulolytic microor-ganisms. The chromosomal DNA fragment which include cellobiase gene of Cellulomonas biazotea was cloned in Eschericia coli via plasmid pBR 322 vector. Restriction enzyme Sal I was used to obtain adequate size of fragments from C. biazotea. chromosomal DNA. The transformant of E. coli HB101 with recombinant plasmid pBG101 showed cellobiase activity, which is not ordinary in E. coli HB101. The enzyme activity of the transformant was as of 20% lower than that of C. biazotea.

• Food Science and Biotechnology
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• v.17 no.6
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• pp.1221-1227
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• 2008

The application of the cellulase gene (celA) as a selection marker of food-grade integration system was investigated in Lactobacillus (Lb.) casei, Lactococcus lactis, and Leuconostoc (Leu.) mesenteroides. The 6.0-kb vector pOC13 containing celA from Clostridium thermocellum with an integrase gene and a phage attachment site originating from bacteriophage A2 was used for site-specific recombination into chromosomal DNA of lactic acid bacteria (LAB). pOC13 was also equipped with a broad host range plus replication origin from the lactococcal plasmid pWV01, and a controllable promoter of nisA ($P_{nisA}$) for the production of foreign proteins. pOC13 was integrated successfully into Lb. casei EM116, and pOC13 integrants were easily detectable by the formation of halo zone on plates containing cellulose. Recombinant Lb. casei EM 116::pOC13 maintained these traits in the absence of selection pressure during 100 generations. pOC13 was integrated into the chromosome of L. lactis and Leu. mesenteroides, and celA acted as an efficient selection marker. These results show that celA can be used as a food-grade selection marker, and that the new integrative vector could be used for the production of foreign proteins in LAB.