• The Korean Journal of Mycology
• /
• v.14 no.3
• /
• pp.225-229
• /
• 1986

Cellulosic substance which plays an important role in carbon cycle is most abundant in the nature world. Some higher fungi are able to digest cellulose directly to satisfy their carbohydrate requirement. Then, in order to investigate the enzymatic components of Lenzites betulina (Fr.) being wood rot, that fungus was collected in Kwangneung area. The carpophore of the fungus was smashed with cool distilled water, extracted and salted out by ammonium sulfate. And then the precipitate was purified by dialysing with visking tube and dissolved with pH 7.8 ammonia water, and the extract was filtrated. The fraction of filtrate was obtained as light brown powder after lyophilization, and determined cellulolytic activity. Cellulolytic potency of Lenzites betulina (Fr.) was 1. 65 unit/ml. The cellulase of Lenzites betulina (Fr.) was stable at below $45^{\circ}C$ and range of pH $4.5{\sim}6.0$ and is completely inactivated at $60^{\circ}C$ for 15 minutes. The optimum condition for the enzymatic reaction was $40^{\circ}C$ and pH 4.0. The enzyme activity was not influenced by the presence of $Ca^{2+}$ and $Fe^{2+}$.

• Korean Journal of Food Science and Technology
• /
• v.27 no.3
• /
• pp.370-374
• /
• 1995

By exposing the complex enzyme solution to alkaline condition, it was possible to remove the protease activity selectively without inactivation of soybean cell wall degrading activity of the crude enzyme complex produced by Aspergillus niger CF-34. Optimum reaction conditions were as follow. pH was $9.0{\pm}0.1$, temperature was $20^{\circ}C$ and reaction time was 30 min with gentle stirring. Over 90% of protease activity could be eliminated while the activities of pectinase, polygalacturonase, xylanase, carboxymethyl cellulase and soybean cell wall degrading enzyme were maintained to $80{\sim}100%$. Through alkali treatment, it was discovered that the quality and organoleptic properties of soy protein produced by this enzymes were improved because the hydrolysis of protein and formation of bitter peptide were decreased.

• The Korean Journal of Food And Nutrition
• /
• v.3 no.1
• /
• pp.57-68
• /
• 1990

In order to obtain the fundamental informations on cellulase of Trichoderma viride QM 9414 for its production and utilization, some physico-chemical properties of the enzyme were reviewed. When T. viride QM 9414 was cultured on wheat bran medium, filter paper-disintegrating and carboxymethyl cellulose-saccharifying activity were increased with the cell growth, and thereafter CMC-saccharifying activity was kept on almost the same leved while filter-paper disintegrating activity was decreased sharply. And B-glucosidase was formed maximally on the late stationary phase of growth. The crude cellulase of cell-free extracts was purified by (NH4)2SO4 fractionation, Sephadex-G 200 column chromatography and DEAE Sephadex A-50 column chromatography. Filter paper-disintegrating, CMC-saccharifying and B-glucosidase activity were purified 10-fold, 47-fold and 38-fold, respectively. The crude enzyme was proved to be a complex of three different enzyme proteins which were showing filter paper-disintegrating, CMC-saccharifying and B-glucosidase activity. The optimal pH of the three enzyme components was alike pH 4.0, and the optimal temperature for CMC-saccharifying, filter paper-disintegrating and B-glucosidase activity were 4$0^{\circ}C$, 45$^{\circ}C$ and 5$0^{\circ}C$ respectively. The Km and Vmax values of CMC saccharifying activity for CMC were 0.485% and 3.10, and the Km and Vmax vallues of B-glucosidase for PNPG were 0.944$\times$10-3M and 0.097, respectively. The Km and Vmax values of filter paper-disintegrating activity for Avicel were determined to be 0.09% and 0.178, respectively. B-Glucosidase activity was competitively inhibited by glucose, and the Ki value for this enzyme was 3.54$\times$10-3M, CMC saccharifying activity was found to be greatly inhibited by cellobiose.

• Microbiology and Biotechnology Letters
• /
• v.20 no.2
• /
• pp.164-168
• /
• 1992

Enzymatic properties of avicelase, carboxymethyl cellulase (CMCase) and P-glucosidase produced by Cellulomonas sp. YE-5 were studied. Optimal temperature and pH of avicelase were 40t and 6.0, and those of CMCase and P-glucosidase were $45^{\circ}C$ and 6.5. Avicelase and CMCase were stable between pH 5.0 and 9.5, and &glucosidase was stable between pH 5.5 and 8.0. Avicelase and P-glucosidase were inactivated when incubated at $35^{\circ}C$ for 6 hrs, and CMCase was at $40^{\circ}C$ for 6 hrs. All cellulases were strongly inhibited by $Cu^{2+} \; and \; Zn^{2+}. K_m$ values of avicelase for avicel, CMCase I and CMCase II for CM-cellulose, and ($\beta$-glucosidase for p-nitrophenyl-$\beta$-D-glucoside (PNPG) were 4.76, 16.4, 16.4 $\mu g$/ml and 3.51 mM, respectively.

• Korean Journal of Food Science and Technology
• /
• v.23 no.1
• /
• pp.31-36
• /
• 1991

Cellulase genes from thermophilic alkalophilic Bacillus sp. F204 a potent cellulase complex-producing bacterium, were cloned in Escherichia coli with pUC 19. Plasmids pBC191 and pBC192, isolated from transformants forming yellow zone around colony on the LB agar plate containing 0.5% carboxymethyl cellulose and ampicillin, contained 4.6 Kb and 5.8 Kb HindIII fragments, respectively. The 4.6 Kb insert of pBC191 had single sites for BamHI EcoRI, KpnI and pvuII. DNA hybridization and immunodiffusion studies showed that pBC191-encoded cellulase gene was homologous with that of host strain. pKC231, constructed by inserting 4.6 Kb insert of pBC191 at the HindIII site of pKK223-3, E. coli expression vector, and pGC711, constructed by inserting 4.6 Kb insert of pBC191 at the HindIII site of pGR71, E. coli and B. subtilis shuttle vector, had 3.2 times and 2.8 times as much cellulase activity as pBC191, respectively. Substrate specificity analysis showed that cellulases cloned were CMCase.

• Microbiology and Biotechnology Letters
• /
• v.31 no.3
• /
• pp.216-223
• /
• 2003

A 1.3-kb of chitosanase gene (choA) encoding 45-kDa polypeptide was cloned, expressed, and characterized from a newly isolated Bacillus cereus H-1. The chitosanase protein (ChoA) of B. cereus H-l was purified to homogeneity by ammonium sulfate precipitation and CM-sephadex column chromatography. Optimum pH was around 7, and stable pH range in the incubation at 50 C was 4-11. Optimum temperature was around 50 C, and enzyme activity was relatively stable below 45 C. ChoA showed the activities toward carboxymethyl cellulose (CMC) in addition to soluble or glycol chitosan. Based on MALDI-TOF MS analysis of purified ChoA, the entire amino acid sequence of ChoA was interpreted by database searching of previously known Bacillus chitosanases. A 1.6 kb of PCR product of corresponding chitosanase gene was obtained and its DNA sequence was determined. The deduced amino acid of choA revealed that ChoA have a 98% homology with those of Bacillus sp. No.7-M strain and Bacillus sp. KCTC0377BP. The recombinant ChoA protein was expressed in E. coli DH5$\alpha$. Deduced amino acid comparison of choA with other chitosanases suggested that it belongs to family 8 microbial endo-chitosanase with chitosanase-cellulase activity.

• Applied Biological Chemistry
• /
• v.28 no.3
• /
• pp.162-166
• /
• 1985

Three isozymes of Carboxymethyl Cellulase $(FI^*,\;FII^*,\;FIII)$ and two fractious of ${\beta}-1,4-D-Cellobiohydrolase$(CI, CIl) from Aspergillus niger were purified by Sephadex G-150, DEAE-Sephadex and Sephadex G-75 column chromatography. From the results of enzymatic hydrolysis and X-ray diffraction, ${\beta}-1,4-D-Cellobiohyarolase$ has a high activity toward highly crystalline cellulose such as filter paper and acts synergistically with Cx enzyme.

• KSBB Journal
• /
• v.30 no.6
• /
• pp.283-290
• /
• 2015

In this study was selected the cellulolytic microorganism and investigated optimum condition of cellulase production for the cellulosic bioethanol production. A bacterial strain Paenibacillus jamilae BRC15-1, was isolated from soil of domestic reclaimed land. For optimizing cellulase production from the selected strain, various culture parameters were investigated such as culture medium, pH (pH 4~10), temperature ($25{\sim}50^{\circ}C$) and culture time (2~72 h). As a result, P. jamilae BRC15-1 efficiently produced cellulase from cellulosic biomass under following conditions: 24 h of culture time (pH 7, $40^{\circ}C$) in manufactured media of CMC (carboxymethyl cellulose) with peptone. Optimum saccharifying condition of crude enzyme produced from P. jamilae BRC15-1 was identified on pH 6 and $40^{\circ}C$ of reaction temperature, respectively. This crude enzyme from P. jamilae BRC15-1 was used for saccharification of pretreated sweet sorghum (Sorghum bicolor var. dulciusculum Ohwi) bagasse under the optimal condition. Finally, pretreated sweet sorghum bagasse including 0.1 g of glucan was saccharified by crude enzyme of P. jamilae BRC15-1 into 2.75 mg glucose, 0.79 mg xylose and 1.12 mg arabinose.

• Korean Journal of Agricultural Science
• /
• v.21 no.2
• /
• pp.142-147
• /
• 1994

Characteristics of TNP-cellulose which prepared from carboxymethyl cellulose powder, CM32, as substrate for cellulase activity assay were investigated. Enzymatic hydrolysis of TNP-cellulose occured on the cellulose moiety but not on amide bonds, following Michaelis-Menten kinetics. Three cellulase preparations from Trichoderma viride, Aspergillus niger, and Cellulomonas sp. were tested for their pH and temperature dependences and compared with the method determining the increase in reducing power. The enzyme activity was found to have the same temperature range in both methods, however the pH range was broadened in the case of using TNP-cellulose as substrate. The colorimetric method for cellulase assay using TNP-cellulose as substrate was compared with the other methods: one based on determination of the increase in reducing power; and the other based on determining the decrease in viscosity of Na-CM-cellulose solution. The activities measured by the colorimetric method showed a linear correlation with the enzyme concentration of certain range in all three enzymes tested, and the activity values were proportional to those obtained from the other methods. Depending on the enzyme, however, the activity values from this method were not always in proportion to those from the viscometric method. suggesting that this method was not specific for determination of the endo-type cellulase.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.11
• /
• pp.1835-1841
• /
• 2015

A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.