• Applied Biological Chemistry
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• v.51 no.4
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• pp.299-304
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• 2008

A ${\beta}$-1,4-endoglucanase gene from Bacillus subtilis H12 was cloned into Escherichia coli JM109 (pBC8) and sequenced. The endoglucanase gene with an insert DNA of 2.5 kb possessed an open reading frame of 1,500 bp encoding a mature protein of 499 amino acids with a calculated molecular mass of 55 kDa. The deduced amino acid sequence showed similarity to those of the known neutral cellulase genes of B. subtilis PAP115 (99.2%) and BSE616 (97.8%), as well as the alkaline gene of Bacillus sp. N4 (55.1%). The endoglucanase activity expressed by E. coli (pBC8) was localized in the periplasmic fraction (80%) and the cytoplasmic fraction (20%). An endoglucanase was purified from the periplasmic fraction by performing gel filtration and anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 31 kDa by SDS-PAGE, and the maximum activity occurred at pH 7 and $40^{\circ}C$. The enzyme easily hydrolyzed soluble substrates such as carboxymethyl cellulose and barely ${\beta}$-glucan, whereas the sigmacell and xylan, the known insoluble substrates, were not entirely hydrolyzed.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.505-510
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• 1992

An endo-$\beta$-1,4-glucanase gene of Clostridium thermocellum was cloned in Escherichia coli and was considered as a novel gene by comparison with the restriction patterns of the C. thermocellum cellulase genes so far reported. The endoglucanase from recombinant E. coli was purified by column chromatography after heat treatment. The purified enzyme was a monomer having molecular weight of 40,000. The enzyme hydrolyzed CMC to glucose and cello-oligosaccharides at :naximum activities at pH 5.0 and $65^{\circ}C$. One of the endproducts, glucose, showed no inhibitory effect on the enzyme activity, while the other endproduct, cellobiose, inhibited slightly. The values of $K_{m}$ and $V_{max}$ of the enzyme for CMC were 0.39% (w/v) and 268 Ulmg protein, respectively.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.800-805
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• 2007

Two endoglucanases with processive cellulase activities, produced from Fomitopsis palustris grown on 2% microcrystalline cellulose(Avicel), were purified to homogeneity by anion-exchange and gel filtration column chromatography systems. SDS-PAGE analysis indicated that the molecular masses of the purified enzymes were 47 kDa and 35 kDa, respectively. The amino acid sequence analysis of the 47-kDa protein(EG47) showed a sequence similarity with fungal glycoside hydrolase family 5 endoglucanase from the white-rot fungus Phanerochaete chrysosporium. N-terminal and internal amino acid sequences of the 35-kDa protein(EG35), however, had no homology with any other glycosylhydrolases, although the enzyme had high specific activity against carboxymethyl cellulose, which is a typical substrate for endoglucanases. The initial rate of Avicel hydrolysis by EG35 was relatively fast for 48 h, and the amount of soluble reducing sugar released after 96 h was $100{\mu}g/ml$. Although EG47 also hydrolyzed Avicel, the hydrolysis rate was lower than that of EG35. Thin layer chromatography analysis of the hydrolysis products released from Avicel indicated that the main product was cellobiose, suggesting that the brown-rot fungus possesses processive EGs capable of degrading crystalline cellulose.

• Microbiology and Biotechnology Letters
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• v.35 no.2
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• pp.128-134
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• 2007

Previously, we isolated plant growth promoting rhizobacterium (PGPR) Bacillus licheniformis K11 which could produce auxin, cellulase and siderophore. The siderophore of B. licheniformis K11 $(siderophore_{K11})$ was determined to be a catechol type siderophore which is produced generally by Bacillus spp. B. licheniformis K11 could produce the siderophore most highly after 96 h of incubation under nutrient broth at $20^{\circ}C$ with initial pH 9.0. For the production of the $siderophore_{K11}$, trehalose and $NH_4Cl$ were the best carbon and nitrogen sources in Davis minimal medium, respectively. The $siderophore_{K11}$ was Produced in M9 medium (pH 9.0) after 4 days at $20^{\circ}C$, and purified from culture broth of B. licheniformis K11 by using Amberlite XAD-2, Sephadex LH-20 column chromatography, and reversed-phase HPLC. The $siderophore_{K11}$ had the biocontrol activity against spore germination of P. capsici and F. oxysporum on potato dextrose agar (PDA). The results indicate that the $siderophore_{K11}$ is an antifungal mechanism of B. licheniformis K11 against phytopathogenic fungi.

• Journal of Korean Society of Forest Science
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• v.71 no.1
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• pp.66-73
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• 1985

Factors affecting isolation and fusion of protoplasts of three Quercus species were investigated and procedures for isolation, purification and fusion of protoplasts of the three species were also established. Unhardened leaves and rapidly growing callus cultures were good source of viable protoplasts. The optimum composition of enzyme mixture for rapid isolation of protoplasts from leaf mesophyll tissues and calli was Cellulase Onozuka R-10 (20g/l, Macerozyme R-10(10g/l), Pectinase(250 units/l, $CaCl_2$, $2H_2O$(14mM), $MgSO_4{\cdot}7H_2O$(1.8mM), $KNO_3$(1.0mM), $H_3BO_3$(1.0mM), $KH_2PO_4$(0.2mM), KI($1.0{\mu}M$), 1,4-dithiothreitol (0.1mM) and mannitol (0.6M). Optimum density of protoplasts for maximum fusion was $2{\times}10^5/ml$ which was the highest protoplast density given in this study. Optimum concentration and duration of PEG 1450 treatment for inducing fusion appeared to be 29%(W/V) final PEG 1450 concentration and 5-10 minutes, respectively.

• Current Research on Agriculture and Life Sciences
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• v.2
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• pp.15-22
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• 1984

This study was conducted to identify the enzyme treatment time, enzyme concentration and plant growth condition for isolation of potato mesopyll, it was also performed to determine the adquate sucrose molarity on purification of protoplasts and to investigate the incubation time, PEG concentration and DMSO effect for potato-petunia protoplast fusion. The results were summarized as follows: The optimal time of incubation in enzyme solution was 3 - 4 hours and high humidity and low light intensity made plants more effective to protoplast releasing enzymes. Our experimental results showed that the pectolyase Y-23 was an ideal agent for isolation from mesophyll cultured in vitro compared with macerozyme. The enzyme solution with 0.5 % macerozyme and 2 % cellulase was very effective and the purity of healthy protoplast was better in 0.4 and 0.5 M sucrose than in others. It was revealed that the rate of potato-petunia fusion according to the incubation time with PEG was effective at 30 min incubation and percentage of protoplast aggregation was increased by high molecular weight and concentration of PEG. Percentage of potato-petunia protoplast heteroplasmic aggregation was increased by 4 to 16 % in PEG 6000 compared with PEG 4000 and PEG 1500. Addition of 5 to 10 % DMSO to the PEG solution increased to the heteroplasmic aggregation of potato-petunia from 2 to 4 %.

• Journal of Korean Society of Forest Science
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• v.73 no.1
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• pp.33-42
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• 1986

This study was carried out to investigate the optimum conditions for isolation and culture of mesophyll protoplasts from Populus alba ${\times}$ P. glandulosa. The results obtained from the experiments are as follows; 1) The suitable concentration of BAP for shoot multiplication was 0.4 mg/l. 2) High yield and viability of isolated protoplasts were obtained by our high enzyme-short time incubation method. 3) Optimum enzyme concentrations for mesophyll protoplast isolation were Cellulase 2%, Macerozyme 0.8%, Hemicellulase 1.2%, Driselase 2%, and Pectolyase Y-23 0.05%. 4) 0.6M mannitol in enzyme solution was the most effective for protoplast isolation and viability. 5) The most adequate pH level of enzyme solution was pH 5.6. 6) The effect of DTT and MES buffer was significant. 7) For protoplast purification, 0.6M sucrose was the most proper concentration. 8) The adding effect of Dextran T40 in floating solution was important. 9) The mesophyll protoplasts isolated through our high enzyme-short time incubation method revealed successful response to culture condition over 3 weeks of culture.

• Journal of Microbiology and Biotechnology
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• v.21 no.3
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• pp.284-292
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• 2011

An endocellulase-free multienzyme complex was produced by a thermophilic anaerobic bacterium, Thermoanaerobacterium thermosaccharolyticum strain NOI-1, when grown on xylan. The temperature and pH optima for growth were $60^{\circ}C$ and 6.0, respectively. The bacterial cells were found to adhere to insoluble xylan and Avicel. A scanning electron microscopy analysis showed the adhesion of xylan to the cells. An endocellulase-free multienzyme complex was isolated from the crude enzyme of strain NOI-1 by affinity purification on cellulose and Sephacryl S-300 gel filtration. The molecular mass of the multienzyme complex was estimated to be about 1,200 kDa. The multienzyme complex showed one protein on native PAGE, one xylanase on a native zymogram, 21 proteins on SDS-PAGE, and 5 xylanases on a SDS zymogram. The multienzyme complex consisted of xylanase, ${\beta}$-xylosidase, ${\alpha}$-L-arabinofuranosidase, ${\beta}$-glucosidase, and cellobiohydrolase. The multienzyme complex was effective in hydrolyzing xylan and corn hulls. This is the first report of an endocellulase-free multienzyme complex produced by a thermophilic anaerobic bacterium, T. thermosaccharolyticum strain NOI-1.

• Current Research on Agriculture and Life Sciences
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• v.5
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• pp.19-26
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• 1987

The experiments were conducted to identify several factors affecting isolation and culture of mesophyll protoplasts in Solanum melongena var. fructualbo. Higher viable plotoplasts were obtained, when isolated in 1.5% macerozyme, 0.2% macerozyme, 0.6M mannitol, 0.01M MES, 0.2% BSA containing solution adjusted to pH 6.3 for 4 hours. One hour plasmolysis of the material before digestion of leaf tissue was effective for protoplast yield and viability. The method of washing and purification of crude protoplasts, ESS process with 0.7M mannitol and 0.6M sucrose solution. was the best way to get purified protoplasts with viability. As isolated protoplasts were cultured in 8P-KM medium at a density of $2.5{\times}10^4/ml$, the cells were enlarged after 3 to 5 days from culture, subsequently the cells were divided and resulted in colonies.

• Microbiology and Biotechnology Letters
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• v.31 no.3
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• pp.235-241
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• 2003

For the biological control of Phytophthora blight of red-pepper caused by Phytophthora capsici, an antibiotic-producing plant growth promoting rhizobacteria (PGPR) Bacillus sp. KL 39 was selected from a local soil of Kyongbuk, Korea. The strain KL 39 was identified as Bacillus megaterium by various cultural, biochemical test and API and Microlog system. B. megaterium KL 39 could produce the highest antifungal antibiotic after 40 h of incubation under the optimal medium which was 0.4% fructose, 0.3% yeast extract, and 5 mM KCl at 30 C with initial pH 8.0. The antifungal antibiotic KL 39 was purified by Diaion HP-20 column, silica gel column, Sephadex LH-20 column, and HPLC. Its RF value was confirmed 0.32 by thin-layer chromatography with Ethanol:Ammonia:Water = 8:1:1. The crude antibiotic KL39 was active against a broad range of plant pathogenic fungi, Rhizoctonia solani, Pyricularia oryzae, Monilinia fructicola, Botrytis cinenea, Alteranria kikuchiana, Fusarium oxysporum and Fusarium solani. The purified antifungal antibiotic KL39 had a powerful biocontrol activity against red-pepper phytophthora blight disease with in vivo pot test as well as the strain B. megaterium KL 39.