• Preventive Nutrition and Food Science
• /
• v.7 no.1
• /
• pp.22-27
• /
• 2002

The cellulase from internal organs of edible snails was purified by fractionation with ammonium sulfate, DEAE-Sephadex chromatography and gel filtration on Sephacryl S-200 and Superose 12 HR 10/30. The specific activity of the purified cellulase was 85.1 units/mg protein with 24.3 purification fold from crude extract. Molecular weight of the enzyme was estimated to be approximately 74,000 dalton by gel filtration chromatography and SDS-PAGE eletrophoresis. T7e isoelectric point of the enzyme was determined to be pH 4.6. The optimum temperature and pH of the enzyme were 5$0^{\circ}C$ and pH 6.0, respectively. The enzyme was stable at 30~5$0^{\circ}C$ and pH 6.0~10.0. It was activates by Mn$^{2+}$, but inhibited by Li$^{2+}$, Zn$^{2+}$, Ag$^{2+}$ and Hg$^{2+}$./TEX> 2+/.

• Journal of Microbiology and Biotechnology
• /
• v.7 no.5
• /
• pp.305-309
• /
• 1997

Bacillus stearothermophilus No. 236, an effective xylanolytic bacterium, produced an extracellular carboxymethyl cellulase when the strain was grown on xylan. The carboxymethyl cellulase was purified to homogeneity as judged by SDS-PAGE and zymogram, The carboxymethyl cellulase had a pI of 4.0, and a molecular mass of 95 kDa. The highest level of enzyme activity was observed at pH 6.5 and $60^{\circ}C$. The $K_m$, and $V_{max}$ values of the enzyme to carboxymethyl cellulose were 20.8 mg/ml and $0.63 {\mu}mole$/min/mg protein, respectively, The enzyme was found to act also on filter paper and xylan as well as carboxymethyl cellulose. Therefore, it is expected that this xylanolytic strain isolated from soil could be efficiently used for xylan biodegradation.

• Journal of Microbiology and Biotechnology
• /
• v.1 no.4
• /
• pp.232-239
• /
• 1991

Caboxymethyl cellulase (CMCase) I was purified from the induced culture filtrate of Penicllium verruculosum F-3 by ammonium sulfate precipitation, DEAE-Sephadex A-50 chromatography and Bio-gel P-150 filtration. The purified enzyme was assumed to be a glycoprotein consisting of 8.5% carbohydrate and having a molecular weight of 70.000 in SDS-polycrylamide gel electrophoresis (SDS-PAGE). The purified enzyme-specific anti-CMCase I IgG was obtained by rabbit immunization and protein A-sepharose CL-4B chromatography. The fungal poly($A^+$) RNA was isolated from the total RNA of the mycelium grown under cellulase induction conditions by oligo(dT)-cellulosse chromatography. The translation products in vitro were prepared by translating the isolated poly ($A^+$) RNA in rabbit reticulocyte lysate and analyzed by SDS-PAGE and fluorography. Of the translation products, CMCase I was identified by the immunoprecipitation against anti-CMCase I IgG.

• The Korean Journal of Mycology
• /
• v.21 no.1
• /
• pp.28-37
• /
• 1993

An endo-type cellulase, carboxymethyl cellulase(CMCase) IV, was purified from culture filtrate of cellulolytic fungus Penicillium verruculosum. The CMCase IV was acidic glycoprotein having carbohydrate of 13% as glucose and pI value of 4.0. The CMCase IV was 52 KDa of molecular weight in SDS-polyacrylamide gel electrophoresis and have optimum temperature and pH of $50^{\circ}C$ and 5.0 for enzyme activity. The CMCase IV liberated glucose and cellobiose as major products of the enzyme against carboxymethyl cellulose(CMC) and seemed to has transglycosylation activity simultaneously. Cellulase activity staining(zymogram) showed that the cellulase components of P. verruculosum were not aggregated in the medium. P. vrttuculosum mRNA was translated in vitro and translation product by the mRNA coding for CMCase IV was identified by immunoprecipitation.

• Applied Biological Chemistry
• /
• v.12
• /
• pp.99-105
• /
• 1969

The yield of cellulase derived from Trichoderma $(O_2-1)$ was remarkably varied with various concentration of ethanol and acetone in purification of the enzyme. In the purification with ethanol of ${\beta}-glucosidase$, the best result was obtained in the concentration of 60% and, of CMCase and of filter paper disintegrating enzyme 80%. And in the purification with acetone of ${\beta}-glucosidase$, filter paper disintegrating enzyme, and CMCase, in the concentration of 60%, 80%, and 90% respectively, was shown the best yield. The activities of crude Cellulase preparation could be seperated into few of fractions by column chromatography with Silica gel, Cellulose powder, and gauze. Most of CMCase, avicelase, and ${\beta}-glucosidase$ were eluted, but most of filter paper disintegrating enzyme and the rest of enzymes mentioned the above were absorbed, and were eluted with water. Therefore, it was considered that CMCase is different from filter paper disintegrating enzyme in properties. The relative activity of CMCase was different from that of avicelase in the peak of elusion part. And it was considered that filter paper disintegrating enzyme and cellulose powder saccharifying enzyme was seperated respectively as absorption part and non absorption part. The auther came to the conclusion that at least there were more than three sorts of cellulase in Trichoderma $(O_2-1)$ cellulase preparation.

• Mycobiology
• /
• v.37 no.2
• /
• pp.121-127
• /
• 2009

Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was $45^{\circ}C$ and the highest activity was exhibited in 35 to $45^{\circ}C$. The enzyme lost their activities almost completely (95${\sim}$100%) at $80^{\circ}C$ or above and as well as bellow $25^{\circ}C$.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.11
• /
• pp.1501-1509
• /
• 2012

A novel bacterial strain, MG7, with high cellulase activity was isolated and identified by morphological characteristics and molecular phylogeny analysis as Paenibacillus barcinonensis. Maximum production of cellulase by MG7 was observed at pH 7.0 and $35^{\circ}C$. The enzyme was purified with a specific activity of 16.88 U/mg, the cellulase activity was observed in a zymogram, and its molecular mass (58.6 kDa) was confirmed by SDS-PAGE. The purified enzyme showed maximum activity at pH 6.0 and $65^{\circ}C$ and degraded cellulosic substrates such as carboxy methyl cellulose (CMC), Avicel, filter paper, and ${\beta}$-glucan. The enzyme showed stability with 0.5% concentration of various surfactants. The $K_m$ and $V_{max}$ of cellulase for CMC and Avicel were found to be 0.459mg/ml and 10.46mg/ml/h, and 1.01 mg/ml and 10.0 mg/ml/h, respectively. The high catalytic activity and its stability to temperature, pH, surfactants, and metal ions indicated that the cellulase enzyme by MG7 is a good candidate for biotechnological applications.

• Korean Journal of Microbiology
• /
• v.45 no.3
• /
• pp.257-262
• /
• 2009

Carboxymethyl celluase (cellulase) was purified from cell-free extract of the recombinant Escherichia coli carrying a Bacillus licheniformis WL-12 cellulase gene by DEAE-Sepharose and phenyl-Sepharose column chromatography with specific activity of 163 U/mg protein. The molecular mass of the purified enzyme was estimated to be approximately 49.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had a pH optimum at 5.5 and a temperature optimum at $55^{\circ}C$. The activity of the enzyme was completely inhibited by SDS (5 mM), and slightly enhanced by $Cu^{2+}$ (5 mM). The cellulase was active on CMC, konjac, barely glucan and lichenan, while it did not exhibit activity towards xylan, locust bean gum, and p-nitrophenyl-$\beta$-glucopyranoside. The predominant products resulting from the cellulase hydrolysis were cellobiose and cellotriose for cellooligosaccharides including cellotriose, cellotetraose and cellopentaose. The enzyme could hydrolyze cellooligosaccharides larger than cellobiose.

• Journal of Microbiology
• /
• v.34 no.1
• /
• pp.90-94
• /
• 1996

The carboxymethyl cellulase (CMCase) was purified from the induced culture filtrate of hybrid TAPW15703 between Aspergillus niger and penicillium verruculosum made by nuclear transfer. The enzyme was purified 80 fold with an overall yield 17% from the culture medium by ammonium sulfate fractionation, Sephadex G-75 gel permeation chromatography, and DEAE-ion exchange column chromatography. The molecular weight of the CMCase has estimated to be 32,000 daltons on SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel permeation chromatography. The purified enzyme functions optimally at pH 4.0 and 4$0^{\circ}C$ The Km value for carbosymethyl cellulose was 68 mM. The enzyme activity was increased by the presence of $Mg^{2+}$and Mn$^{2+}$.

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