• The Korean Journal of Mycology
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• v.49 no.2
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• pp.199-209
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• 2021

The present study was performed to improve the technique used for fermenting the mushroom growth medium. Taxonomic analysis of 16S rDNA sequence from the predominant Bacillus strain CY-24 isolated during the fermentation phase of the rice straw medium identified it as Bacillus licheniformis. In addition, the growth environment of B. licheniformis was also examined in this study, which revealed the optimal growth temperature and pH to be 30 ℃ and 6.0, respectively. This study also revealed that carboxymethyl cellulase (CMCase) and polygalacturonase (PGase) enzymes isolated from B. licheniformis achieved their maximal activities at 50 ℃ and 60 ℃ respectively. Furthermore, the study confirmed that the two enzymes, i.e., CMCase and PGase in B. licheniformis are stable at temperatures above 60 ℃. The present study thus demonstrates that B. licheniformis CY-24 possesses excellent enzymatic properties. It also reveals that the action of enzymes during the production of growth mediums used for the cultivation of mushrooms is closely associated with the promotion of fermentation and softening of the rice straw. Overall, this study provides elementary information regarding the role of B. licheniformis enzymes during growth medium fermentation for Agaricus bisporus cultivation.

• Journal of Microbiology and Biotechnology
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• v.5 no.1
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• pp.18-21
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• 1995

Endoglucanase gene of Pseudomonas sp. LBC505 was previously cloned in pUCl9 to yield plasmid pLC1. overproduction of endoglucanase was attempted by following ways. First, the endoglucanase gene of Pseudomonas sp. LBC505 cloned in pUCl9(pLC1) was tandemly inserted, step by step, into a expression vector pKK223-3 in a directly repeated form to enhance productivity of endoglucanase. Escherichia coli containing pKCC30 among the resulting plasm ids showed the higher yield of the endoglucanase. Ecoli harboring pKCC30 which had three inserted endoglucanase genes expressed about 12.3 times as much CMCase activity as Ecoli harboring pLCl. Second, the endoglucanase gene was subcloned into Bacillus subtilis expression vector pgnt41 for both overproduction and extracellular secretion of the endoglucanase. A resulting plasmid pgntc15 in Bacillus subtilis expressed 4.3-fold higher levels of CMCase activity than that of E.coli harboring pLCl and the endoglucanase produced was entirely secreted into the culture medium.

• Journal of Life Science
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• v.23 no.6
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• pp.757-769
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• 2013

A microorganism producing carboxymethylcellulase (CMCase) was isolated from seawater, identified as Bacillus velezensis by analyses of 16S rDNA and partial sequences of the gyrA, and designated as B. velezensis A-68. The optimal conditions for production of CMCase by B. velezensis A-68 were established using response surface methodology (RSM). The optimal concentrations of rice hulls and yeast extract, and initial pH of the medium for cell growth were 60.2 g/l, 7.38 g/l, and 7.18, respectively, whereas those for production of CMCase were 50.0 g/l, 5.00 g/l, and 7.30. The analysis of variance (ANOVA) implied that the most significant factor for cell growth as well as production of CMCase was yeast extract. The optimal concentrations of $K_2HPO_4$, NaCl, $MgSO_4{\cdot}7H_2O$, and $(NH_4)_2SO_4$ in the medium for cell growth were 7.50, 1.00, 0.10, and 0.80 g/l, respectively, which were the same as those for production of CMCase. The optimal temperatures for cell growth and production of CMCase were 30 and $35^{\circ}C$, respectively. The maximal production of CMCase under optimized conditions was 83.8 U/ml, which was 3.3 times higher than that before optimization. In this study, rice hulls, agro-byproduct, were developed as a substrate for production of CMCase and time for production of CMCase was reduced to 3 days using a newly isolated marine bacterium.

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

• Journal of Life Science
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• v.14 no.1
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• pp.193-197
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• 2004

A continuous enrichment culture procedure was used to isolate bacteria from various soil sources capable of suppressing large patch disease of turfgrass. Six isolates consistently suppressed large patch in turfgrass, and ranged in the spectrum of extracellular enzymes that they expressed. The best disease- suppressing isolate, TBM912, expressed protease, CMCase, and pectinase activity and inhibited the growth of Rhizectonin solani and Betrytis cinerea in vitro. Here we show that this strain also produces an antibiotic that was identified by TLC, SDS-PACE and HPLC analysis as lipopeptide.

• Journal of Life Science
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• v.22 no.6
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• pp.799-806
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• 2012

Approximately 80 species of bacteria were isolated from the fermented mushroom first and the HJ-57 antibacterial micro-organism was selected to the final isolation bacteria. It has a high degree of CMCase, amylase, and protease activity as well as high antibacterial activity against mushroom pathogenic bacteria without affecting the growth and development of Flammulina velutipes and Lentinus edodes mushrooms. The finally selected HJ-57 antibacterial micro-organism was identified as Bacillus sp. HJ-57. The initial pH for culture was pH7 and its optimum culture temperature was $35^{\circ}C$. The antibacterial material produced by Bacillus sp. HJ-57 showed a little antibacterial activity even in the 12 hr of culture, but showed the highest antibacterial activity in the 36~48 hr of culture. The HJ-57 antibacterial micro-organism also showed a high antibacterial activity against mushroom pathogenic bacteria and molds in the corn cob contained culture medium is used in Flammulina velutipes cultivators.

• Journal of Animal Science and Technology
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• v.50 no.5
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• pp.713-720
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• 2008

This study was conducted to isolate and identify xylanase- and cellulase-producing thermophilic bacteria from stacked spent mushroom substrates and to determine the optimal medium conditions for their growth. Bacteria with the highest xylanase and CMCase activities were strain 3 and 201-7. Both of them were identified as Bacillus spp. and named Bacillus subtilis KU3 and Bacillus subtilis KU201-7. The optimal medium condition of Bacillus subtilis KU3 was obtained when 3%(w/v) of yeast extract and 1%(w/v) of maltose were used as nitrogen and carbon sources, respectively. That of Bacillus subtilis KU201-7 was obtained when 0.5%(w/v) of yeast extract and 0.5%(w/v) of CMC were used as nitrogen and carbon sources, respectively.

• 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.

• Journal of Microbiology and Biotechnology
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• v.2 no.1
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• pp.7-13
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• 1992

A moderately thermophilic, alkalophlic and powerful crystalline cellulose-digesting bacterium, Bacillus K-12, was isolated from filter paper wastes and found to be similar to Bacillus circulans or Bacillus pumilis, except for its ability to grow at a moderately high pH and temperature. The isolate grew at a pH ranging from 6 to 10 and at a temperature ranging from 35 to $65^{\circ}C$ and produced a large amount of cellulase components containing avicelase, xylanase, CMCase, and FPase when grown in avicel medium for 5 to 7 days at $50^{\circ}C$. The crude enzyme preparation from the culture broth hydrolyzed xylan, raw starch, pullulan and ${\beta}-1,3$ glucan such as laminarin. Furthermore, the enzyme hydrolyzed crystalline cellulose to cellobiose and glucose and had a broad pH activity curve (pH 6~9). The enzyme was stable up to $70^{\circ}C$.

• The Korean Journal of Food And Nutrition
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• v.6 no.4
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• pp.314-321
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• 1993

We have cloned the Bacillus cellulyticus K-12 avicelase (Avi, E.C.3.2.1.4) gene (ace A) In E. coli. This was accompanied by using the vector PT7T3U 19 and Hind W -Hind m libraries of Bacillus cellulyticus K-12 chromosomal inserts created in 5.cofi. The Libraries were screened for the expression of avicelase by monitoring the immunoreaction of the anti-avicelase (immunoscreening). Positive clones (Ac-3, Ac-5, and Ac-7) contained the identical 3.5kb Hind III fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constituvely using its own promoter in the heterologous host. From the immunoblotting analysis, a polypeptide which showed a CMCase activity with an Mr of 54000 was detected.