• Journal of Korea Technical Association of The Pulp and Paper Industry
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• v.29 no.1
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• pp.43-51
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• 1997

The various culture conditions of Trichoderma viride(ATCC 3454) and Phanerochaete chrysosporium(ATCC 26921) with glucose-pepton medium, Mandels medium, YMG medium for wood degradable enzyme were examined. Mycellium of the two species grew profusely on glucose-pepton medium. Maximum fungal growth was observed about 10days. But CMCase, Fpase, laccase activity in the culture medium with glucose-pepton was not detected. When grown in fermenter culture using Mandels medium, Trichoderma viride produced CMCase and Fpase. Its CMCase activity was 0.15 lU/ml and Fpase activity was 0.3 IU/ml within about 4-6days. Phanerochaete chrysosporium grown in a YMG medium gave the best enzyme activity when they were grown under stationary culture with an atmosphere of 100% oxygen. Levels of laccase activity of 3.0 mull were achieved in stationary culture under 100% oxygen. The enzyme condensation by ultrafiltration method caused a 2-fold(cellulase) and 6-fold(laccase) as compared to control activity.

• 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.

• Applied Biological Chemistry
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• v.42 no.3
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• pp.199-204
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• 1999

Soft-rot bacterial pathogen, Erwinia sp., was isolated from chinese cabbage tissue showing soft-rot symptom. This bacterial strain caused soft-rot to chinese cabbage and potato, and identified as Erwinia chrysanthemi PY35(Ech PY35). Ech PY35 have extracellular CMCase, pectinase, pectate lyase, and protease activity, but not hemicellulase activity. The results of the microscopy showed that chinese cabbage tissue and potato tissue were macerated by infection of Ech PY35. In analysis of the CMCases activity in the total protein of Ech PY35, three CMCases were detected as intracellular protein while two CMCases were as extracellular protein by CMC-SDS-PAGE direct stain method.

• Journal of Life Science
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• v.20 no.4
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• pp.487-495
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• 2010

A microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Psychrobacter aquimaris by analysis of 16S rDNA sequences, and named P. aquimari LBH-10. This strain produced an acidic carboxymethylcellulase (CMCase), which hydrolyzed carboxymethylcellulose (CMC), cellobiose, curdlan, filter paper, p-nitrophenyl-$\beta$-D-glucopyranoside (pNPG), pullulan, and xylan, but there was no detectable activity on avicel and cellulose. The optimal temperature for CMCase produced by P. aquimari LBH-10 was $50^{\circ}C$ and more than 90% of its original activity was maintained at broad temperatures ranging from 20 to $50^{\circ}C$ after 24 hr. The optimal pH of the CMCase was 3.5, and more than 70% of its original activity was maintained under acidic conditions between pH 2.5 and 7.0 at $50^{\circ}C$ after 24 hr. The optimal pH of CMCase produced by P. aquimaris LBH-10 seems to be lower than those produced by any other bacterial and fungal strain. $CoCl_2$, EDTA, and $PbCl_2$ at a concentration of 0.1 M enhanced CMCase-produced P. aquimaris LBH-10, whereas $HgCl_2$, KCl, $MnCl_2$, $NiCl_2$, and $SrCl_2$ inhibited it.

• 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.

• Microbiology and Biotechnology Letters
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• v.25 no.6
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• pp.546-551
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• 1997

A bacterium producing the extracellular cellulases was isolated from soil and has been identified as Bacillus sp. The isolate, named Bacillus sp. 79-23, was shown to be very similar to B. subtilis on the basis of its biochemical properties. The carboxymethyl cellulase (CMCase) of culture supernatant was most active at 60$\circ$C and pH 6.0, and retained 90% of its maximum activity at pH 7.0. The additional carbon sources affected the CMCase productivity than nitrogen sources in the culture medium. The carbon sources including wheat bran, rice straw, maltose and glucose increased the enzyme productivty. Especially, the maximum CMCase production was 5.2 units/ml in LB medium supplemented with 3% (w/v) wheat bran, which was 13-folds more than that in LB medium. It was found that the enzyme production was in association with the growth of Bacillius sp. 79-23. But, whean bran did not affect the growth of isolate, suggesting that increasement of CMCase production was owing to the induction of CMCase biosynthesis by wheat bran. In addition, both water-soluble and insoluble components of wheat bran was involved in induction of CMCase biosynthesis.

• Journal of Korea Technical Association of The Pulp and Paper Industry
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• v.35 no.1
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• pp.34-40
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• 2003

This study was carried out to select the useful bacteria which secret extracellula enzymes for enzymatic deinking agent of old newspaper. CMCase, FPase and xylanase activities of the bacteria liquid culture were measured at optimal growth conditions. Clear zone test was checked on the solid culture. The results of this study were as follow: Eight strains of 28 bacteria isolated from a paper mill soil ground were shown strong CMCase and xylanase activity with the clear zone test. The optimal pH and temperature for culture growth were 6~8 and 26~$34^{\circ}C$, respectively and optimal culture period were less than 60 hours. Based on CMCase, FPase and xylanase activity, strain No. 18, 21, 22 and 28 which were relatively higher than the other strains, were selected for further enzymatic deinking research.

• Journal of Life Science
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• v.22 no.2
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• pp.142-147
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• 2012

The sporulation-specific glucoamylase (SGA) of Saccharomyces diastaticus is known to be produced in the cytoplasm during sporulation. For the purpose of proving that SGA has secretory potential, we constructed a hybrid plasmid, pYESC25, containing the promoter and the putative signal sequence of the SGA fused in frame to the endo-1,4-${\beta}$-D-glucanase (CMCase) gene of Bacillus subtilis without its own signal sequence. The recipient yeast strain of S. diastaticus YIY345 was transformed with the hybrid plasmid. CMCase secretion from S. diastaticus harboring pYESC25 into culture medium was confirmed by the formation of yellowish halos around transformants after staining with Congo red on a CMC agar plate. The transformant culture was fractionated to the extracellular, periplasmic, and intracellular fraction, followed by the measurement of CMCase activity. About 63% and 13% enzyme activity were detected in the culture supernatant (extracellular fraction) and periplasmic fraction, respectively. Furthermore, ConA-Sepharose chromatography, native gel electrophoresis, and activity staining revealed that CMCase produced in yeast was glycosylated and its molecular weight was larger than that of the unglycosylated form from B. subtilis. Taking these findings together, SGA has the potential of secretion to culture medium, and the putative signal sequence of SGA can efficiently direct bacterial CMCase to the yeast secretion pathway.

• Microbiology and Biotechnology Letters
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• v.23 no.3
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• pp.275-281
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• 1995

From the plasmid pYA300 carring a CMCase of Rhizobium fredii USDA193 plasmid was subcloned into pBluescript II KS(+)/pBluescript II SK(+) vectors and designated pYA500 and pYA600, respectively. Escherchia coli cells transformed with pYA500 porduced the CMCase more than with pYA600. The orientation of the cloned fragment in pBluescript vector had the effect on gene expression in E. coli background. When the 1.7 kb CMCase gene fragment of R. fredii USDA193 was hybridized to EcoRI-digested total DNA from R. meliloti and R. fredii USDA 191 the unique bands hybridized respectively, indicating that some genetic diversity exists in the EcoRI restriction enzyme site for CMCase gene in Rhizobium strains. The optimum pH of enzyme activity was 7 and the optimum temperature of that was nearly 37$\circ$C. The cellulase-minus derivatives of pYA500 were constructed by Tn5 insertional mutation. Among 6000 transconjugants, two mutant plasmids (designated pYA500::Tn5a and pYA500::Tn5b) were detected from the cellulase- negative transconjugants. The product of CMCase gene was analyzed by one dimensional SDS- PAGE of the cell extracts. About 45 kDa protein was considered to be a product of CMCase gene.