• Microbiology and Biotechnology Letters
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• v.28 no.4
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• pp.195-201
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• 2000

Glucoamylase of Saccharomyces diastaticus is produced as a large precursor composed of signal peptide (21 amino acid residues), Thr and Ser-rich region and functional glucoamylase. To evaluate the utility of the glucoamylase signal peptide (GSP) for the secretion of foreign proteins, four types of GSP mutants (ml : Pro-18 longrightarrowLeu-18, m2 : Tyr-13 longrightarrowLeu, m3 : Ser-9longrightarrowLeu-9, m4 : Asn-5 longrightarrowPro-5) were constructed and secretion efficiency of each mutant was compared with that of native GSP by the expression and secretion of Bacillus subtilis CMCase under the control of GAP in N-terminal domain and hydrophobic domain. n mutant 4, a polar amino acid was replaced by a helix - breaking Pro residue. CMCase activity assay and Western blot analysis revealed that CMCase secretion by GSP mutants replaced by Leu were increased compared with native GSP. In the case of m2 and m3, the substitution of Leu for Tyr-13 and Ser-9 in the hydrophobic region resulted in a twofold increase in the extracellular CMCase activity.

• Journal of Applied Biological Chemistry
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• v.42 no.3
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• pp.107-112
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• 1999

A carboxymethyl-cellulase (CMCase) was purified from the culture supernatant of Bacillus sp. KD1014 by ultrafiltration, ammonium sulfate precipitation, and a series of chromatography on QAE-Sephadex A-50, hydroxylapatite and Sephadex G-75. The purified CMCase was a single protein of 32 kDa, showed an optimum activity at $60^{\circ}C$ and pH 6.0, and had a half-life of 23 min at $70^{\circ}C$. The enzyme activity was not influenced by metal ions such as $Mg^{2+},\;Fe^{3+},\;K^+,\;Zn^{2+}$, and $Cu^{2+}$ at a concentration of 1.0 mM, partially inhibited by $Mn^{2+}$ and $Ag^+$, and significantly inhibited by pentachlorophenol (PCP). The purified enzyme showed a 3.9-times higher activity on lichenan than on CMC, but hardly cleaved xylan, starch, avicel, laminarin, filter paper and levan. The results of activity staining of the purified enzyme separated by native and denaturing gel electrophoresis suggested that the CMCase might exist in dimeric, oligomeric or aggregated form as well as in monomeric form. The enzymatic cleavage products from cellotetraose indicated that the CMCase possessed transglycosylation activity.

• Journal of Applied Biological Chemistry
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• v.60 no.1
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• pp.79-86
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• 2017

Microbes in forest are very important due to not only to enhance soil fertility but also maintain a healthy ecosystem by supplying the energy available to living organisms by producing various kinds of enzymes related to degradation of lignocellulosic biomass. In order to isolate a lignocellulosic biomass degrading bacterial strain from the Jurassic park located in Gyeongnam National University of Science and Technology, We used the Luria-Bertani-Carboxymethyl cellulose (CMC) agar trypan blue method containing 0.4 % carboxymethyl cellulose and 0.01 % trypan blue. As a result, we isolated a bacterial strain showing both activity on the CMC and xylan. To identify the isolated strain, 16S rRNA sequencing and API kit analysis were used. The isolated strain turned out to belong to Bacillus species and then named Bacillus sp. GJY. In the CMC zymogram analysis, it showed that one active band of about 28kDa in size is present. Xylan zymogram analysis also showed to have one active band of about 25kDa in size. The optimal growth temperature of Bacillus sp. GJY was $37^{\circ}C$. The maximal activities of CMCase and xylanase were 12 hour after incubation. The optimal pH and temperature for CMCase were 5.0 and $40^{\circ}C$, respectively, whereas the optimal pH and temperature for xylanase was 4.0 and $40^{\circ}C$. Both activities for CMCase and xylanase showed to be thermally stable at 40and $50^{\circ}C$, while both activities rapidly decreased at over $60^{\circ}C$.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.307-311
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• 2005

Marine microorganisms to produce functional biopolymers were isolated from the seashore of the Kyungsang province. Microorganisms to hydrolyze carboxy-methyl cellulose(CMC) were cultured in marin broth and the other liquid medium that contained 2.0% (w/v) glucose, 0.25% yeast extract, 0.5% $K_2HPO_4$, 1% NaCl, 0.02% $MgSO_4{\cdot}7H_2O$ and 0.06% $(NH_4)_2SO_4$ to investigate the ability to produce carboxymethyl cellululase (CMCase) under aerobic conditions. Twelve microorganisms among them showed higher activities of CMCase than B. amyloliquefaciens DL-3, which was known as a cellulase-producing strain. The microorganism showing highest activity of CMCase in this study was identified as Bacillus subtilis subsp. subtilis with 16S rDNA partial sequencing and gyrase A partial sequencing and named as B. subtilis subsp. subtilis A-53.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.528.1-528
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• 1986

Cellulose utilising hybrids between Cellulomonas fimi and Bacillus pumilus were isolated after PEG mediated protoplast fusion. 33% (w/v) PEG #6, 000 and 50mM $Ca^{++}$were optimum concentration. The intergeneric fusion frequency was 3.2$\times$10$^{-7}$ xtracellular CMCase and $\beta$-glucosidase activities were detected from one hydrid unlike only CMCase was detected from Cellulomonas fimi.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.248-254
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• 2005

One hundred thirty nine bacteria were isolated from rotten onions collected from main producing districts, Chang-Nyung, Eui-Ryung, and Ham-Yang in Korea. The $18\%$ (25 strains) of bacterial isolates have carboxymethylcellulase (CMCase) activity and the $53\%$ (74 strains) have polygalacturonase (PGase) activity. Thirty one among randomly selected 45 strains of PGase producing bacteria have pathogenicity to onions. The isolates were classified into Pseudomonas sp. (18 strains), Bacillus sp. (11 strains), Yers-inia sp. (7 strains), and others (9 strains) on the basis of FAMEs patterns. Eighteen strains of Pseudomonas sp. were mainly divided into three cluster in the dendrogram and only the two clusters of them showed pathogenicity to onions. CMCase and PGase activities of Pseudomonas sp. weaker than those of Bacillus sp.. However, the pathogenicity of pseudomonas sp. to soften onions was stronger than that of Bacillus sp. Inoculation of $10^{2}$ cfu of Pseudomonas sp. gives rise to softening of onions. Pseudomonas sp. was identified as Pseudomonas gladioli by biochemical and physiological characteristics. P. gladioli is the first reported bacterium as a pathogen of onion in Korea. In low temperature, P. gladioli showed better growth and higher PGase activity than those of Bacillus sp. identified as Bacillus subtilis. And pH 9.0 is optimal pH for PGase activity of B. subtilis while that of P. gladioli is pH $5.0\∼6.0$ which is the acidity of onions. Taken together, P. gladioli may be a main pathogene of onion rot during the cold storage condition.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.529.1-529
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• 1986

For isolation of the CMCase gene of the alkalophilic Bacillus sp. strain N-4 to analyze their genetic information for the multicomponents of the cellulase, Bscherichia coli K12 and plasmid DNA pBR322 was used as host-vector system. After the digestion of purified chromosomal DNA and plasmid DNA pBR322 with HindIII, these were ligated. The ligated DND were transformed into Escherichia coli, and recombinant plasmid 107 carried the gene coding for CMCase was constructed. The CMCase produced by Escherichia coli cells containing plasmid DNA pYBC107 was found in the cells as intracellular enzyme and nearly 60% of the total CMCase activity was localized in cellular fraction. Also, the optimum pH for the reaction of CMCase produced by Escherichia coli was appeared at pH .8.0 and the enzyme was stable between pH 7.0 and pH 8.0.

• Korean Journal of Microbiology
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• v.35 no.4
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• pp.277-282
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• 1999

A bacterium producing the extracellular cellulase was isolated from cattle feces and screened as cellulase activity was excellent upon congo red straining method and activity measurements. Isolate was identified as Bacillus subtilis CH-10 on the basis of morphological and biochemical properties as well as cellular fatty acids composition. The enzyme which the isolate secretes had the optimum initial pH and temperature for its induction was 7.5 and 50${\circ}C$, respectively. The maximum CMCase activity in crude enzyme solution was observed at pH 7.5 and 75${\circ}C$ and was stable for pH 7.5 to 9.0 to maintain 70% activity. When the isolate was cultured in CMC media at 37${\circ}C$ for 24 hrs, CMCase and FPase activity was 1.13 U/㎖and 0.16U/㎖, respectively whereas Avicelase and ${\beta}$-glucosidase activity was not detected. When crude supernatant was used for zymogram, three major bands, cel 1, cel 2 and cel 3, were detected approximately 39, 41 and 57 KDa, respectively on CMC-SDS-PAGE.

• KSBB Journal
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• v.28 no.1
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• pp.42-47
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• 2013

This study was performed to find cellulolytic strain of enzymatic saccharification for bioethanol production. Cellulolytic strains were isolated from 59 different feces of herbivores from Seoul Grand Park located in Gwacheon Gyeonggi-Do. The celluloytic strain was selected by congo red staining and DNS method. Among the isolated strains, H9-1 strain isolated from the feces of rabbit has the highest CMCase activity. H9-1 strain was identified as Bacillus sp. based on 16S rDNA gene sequencing. The optimal conditions for CMCase activity by Bacillus sp. H9-1 were at $40^{\circ}C$ and at initial pH 8.

• Journal of Life Science
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• v.22 no.10
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• pp.1295-1306
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• 2012

A microorganism producing carboxymethylcellulase (CMCase) was isolated from seawater and identified as Bacillus atrophaeus. This species was designated as B. atrophaeus LBH-18 based on its evolutionary distance and the phylogenetic tree resulting from 16S rDNA sequencing and the neighbor-joining method. The optimal conditions for rice bran (68.1 g/l), peptone (9.1 g/l), and initial pH (7.0) of the medium for cell growth was determined by Design Expert Software based on the response surface method; conditions for production of CMCase were 55.2 g/l, 6.6 g/l, and 7.1, respectively. The optimal temperature for cell growth and the production of CMCase by B. atrophaeus LBH-18 was $30^{\circ}C$. The optimal conditions of agitation speed and aeration rate for cell growth in a 7-l bioreactor were 324 rpm and 0.9 vvm, respectively, whereas those for production of CMCase were 343 rpm and 0.6 vvm, respectively. The optimal inner pressure for cell growth and production of CMCase in a 100-l bioreactor was 0.06 MPa. Maximal production of CMCase under optimal conditions in a 100-l bioreactor was 127.5 U/ml, which was 1.32 times higher than that without an inner pressure. In this study, rice bran was developed as a carbon source for industrial scale production of CMCase by B. atrophaeus LBH-18. Reduced time for the production of CMCase from 7 to 10 days to 3 days by using a bacterial strain with submerged fermentation also resulted in increased productivity of CMCase and a decrease in its production cost.