• Microbiology and Biotechnology Letters
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• v.35 no.3
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• pp.255-260
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• 2007

This study was conducted to isolate and identify bacteria producing xylanase and cellulase from spent mushroom substrates and to determine the optimal medium conditions for their growth. Bacteria showing high xylanase and carboxymethyl cellulase activities and low protease and amylase activities were strain 201-3 and strain 206-3. Strain 201-3 was identified as Enterobacter ludwigii and named Ent. ludwigii KU201-3. 206-3 was identified as Bacillus cereus and named B. cereus KU206-3. The optimal medium condition of Ent. ludwigii KU201-3 was obtained when 1%(w/v) of soybean meal and 3%(w/v) of sucrose were used as nitrogen and carbon source, respectively. That of B. cereus KU206-3 was obtained when 3%(w/v) of soybean meal and 1%(w/v) of molasses were used as nitrogen and carbon sources, respectively.

• Korean Journal of Soil Science and Fertilizer
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• v.22 no.4
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• pp.323-328
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• 1989

This study was conducted to find out the behavior of anaerobic microorganisms and anaerobic decomposition products of rice straw in the strict anaerobic condition. The number of methanogenic bacteria were more isolated than cellulolytic bacteria from the digester decaying rice straw during the entire incubation time. The activity of anaerobic microorganisms, such as methanogens and cellulolytics, were high the early incubation time in the treatment of rice straw with urea, but without urea was low at that time and increased moderately after 10 days incubation. Volatile fatty acid as intermediate anaerobic decomposition products had a longer retention time and higher accumulation rate in the treatment of rice straw without urea than with urea, and predominant fatty acid was propionic acid. Gas generation rate as final products were very intimate relationship with the activity of methanogenic bacteria. Average Eh value was -250mV during the incubation time and $CH_4$ : $CO_2$ percent ratio was about 60~65 : 35~40 in this Eh value. Decomposition rate of rice straw calculated from $CH_4$ and $CO_2$ gas wars 45.6% for 50 days in the treatment of rice straw with urea, and 36.8% without urea.

• Journal of Animal Science and Technology
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• v.47 no.5
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• pp.789-804
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• 2005

This study was conducted to determine effects of supplementation levels of aqueous direct-fed microbials (DFM; Bacillus spp.) to TMR(exp. 1.) and aqueous DFM addition under the various ratios of starch and cellulose(exp. 2.) on ruminal fermentation and fibrolytic enzyme activity. In experiment 1, ruminal fluids taken from rumen-cannulated Holstein cows were incubated during 24 hr by using TMR as substrates. Aqueous DFM was applied at a rate of 0, 0.025 and 0.05%, respectively. The pH of 0.025% treatment was not significantly different from that of control at 6 and 9 hr, but it was significantly lower (P<0.05) than 0.05% treatment. Concentrations of ammonia-N and VFAs were not affected by supplementing aqueous DFM. The A:P ratio of 0.05% treatment was significantly increased(P<0.05) by supplementation of aqueous DFM as compared with that of control at 24 hr. Although overall fibrolytic enzyme activities were not significantly affected by supplementing aqueous DFM, CMCase(carboxymethylcellulase) activity showed significant increase(P<0.05) compared to control at 6hr. However, the xylanase activity of 0.05% treatment significantly decreased(P<0.05) at 12 hr due to the application of aqueous DFM. There was no significant difference for in vitro dry matter disappearance among treatments. In experiment 2, ruminal fluids were incubated under the condition of various ratios of starch to cellulose(90:10, 70:30, 50:50, 30:70 and 10:90) with or without aqueous DFM(0.025%). Ruminal pH was unaffected by the addition of aqueous DFM, however, as increased level of starch, ruminal pH partially showed significant decrease(P<0.05). Ammonia-N concentration was not affected by aqueous DFM and ratio of starch and cellulose. On 9 hr incubation, DFM addition at a ratio of 70:30 showed significantly (P<0.05) lower value of ammonia-N(35.65 mg/dL) than that(65.05 mg/dL) of control. Concentrations of VFAs were significantly increased(P<0.05) by aqueous DFM addition compared with control at the same ratio on 6 hr incubation. The overall CMCase activity was not affected by aqueous DFM addition. However, the xylanase activity by aqueous DFM partially showed significant differences at the ratios of 90:10, 30:70 and 10:90. Our results indicated that supplementation of aqueous DFM did not significantly improve in vitro fermentation and fibrolytic enzyme activity. In addition, the DFM utilized in this study did not show consistent results by having various effects on ruminal fermentation under different feeding regimens.

• Journal of the Korea Organic Resources Recycling Association
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• v.14 no.4
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• pp.141-150
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• 2006

To develop the microbial agents for the environmentally-friendly treatment and recycling of food waste, useful microorganisms, which showed higher degradation activities to various organic compounds and possessed flocculating activities, were isolated from the earthworm. One of the isolated strains, named JS-01, was further selected due to its higher flocculating activity against 0.5% Kaolin clay. JS-01 was identified as Bacillus pumilus sp. by 16s rDNA analysis. The optimal temperature and pH for the growth of JS-01 to express the flocculating activity was found to be at $37^{\circ}C$ in pH 7.0 of EPS broth medium. JS-01 also expressed good degrading activity against cellulose, which is one of the representative organic materials in food waste. We propose that JS-01 will be a good candidate for the efficient treatment of food waste and leachate due to the property to degrade cellulose and flocculating activity.

• Applied Biological Chemistry
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• v.35 no.3
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• pp.173-178
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• 1992

A bacterium having CMCase activity was isolated form soil and identifed as a Pseudomonas sp YD-15. The optimum conditions for the production of CMCase were avicel 1.2%, yeast extract 0.5%, $KNO_3$ 0.06%, $K_2HPO_4$ 0.2%, $MgSO_4$ $7H_2O$ 0.15%, pH 8.0, $30^{\circ}C$ and 60 hours cultivation. The CMCase was purified 15.2 folds with 14ft yield through ammonium sulfate precipitation, DEAE-sepharose column chromatography and sephadex G-100 gel filtration chromatography. The optimum pH and temperature for the enzyme activity were 6.0 and $50^{\circ}C$, respectively. The enzyme was stable between pH 5.0 and 8.0, below $50^{\circ}C$. The molecular weight was calculated about 100,000 by SDS-polyacrylamide gel electrophoresis. $K_m$ value for CMC used as a substrate was 40 mg/ml.

• Journal of Life Science
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• v.22 no.4
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• pp.437-446
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• 2012

A metagenomic library of cow rumen in the pCC1FOS phage vector was screened in $E.$ $coli$ EPI300 for cellulase activity on carboxymethyl cellulose agar plates. One clone was partially digested with $Sau$3AI, ligated into the $Bam$HI site of the pBluescript II SK+ vector, and transformed into $E.$ $coli$ $DH5{\alpha}$. We obtained a 1.5 kb insert DNA, designated $cel$5C, which hydrolyzes carboxymethyl cellulose. The cel5C gene has an open reading frame (ORF) of 1,125 bp encoding 374 amino acids. It belongs to the glycosyl hydrolase family 5 with the conserved domain LIMEGFNEIN. The molecular mass of the Cel5C protein induced from $E.$ $coli$ $DH5{\alpha}$, as analyzed by CMC SDS-PAGE, appeared to be approximately 42 kDa. The enzyme showed optimum cellulase activity at pH 4.0, and $50^{\circ}C$. We examined whether the $cel$5C gene comes from the 49 identified cow rumen bacteria using PCR. No PCR bands were identified, suggesting that the $cel$5C gene came from the unidentified cow rumen bacteria.

• Korean Journal of Microbiology
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• v.14 no.3
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• pp.117-127
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• 1976

A cellulase fraction (F IV-1) purified to about 8-folds was obtained from crude cellulase prepared from the wheat bran culture of S.atra. The partial purification of the enzyme was made by DEAE Sephadex and Sephdex cloumn chromatography in conjuction with ammonium sulfate precipitation. After stading at various pH's for 22 hours at $20^{\circ}C$, F IV-1 was most stable at pH 5.0 but when the enzyme fraction was stood for 74 hours, the point of pH stability was raised to around pH 6.0-7.0. After heating at various temperatures for 1 hour, F IV-1 was most stable at $20^{\circ}C$. The optimal enzyme activities of F IV-1 were seen at pH 6.0 and $50^{\circ}C$. The optimal concentrations of $Zn^{++}\;and\;Ca^{++}$ for the activities of crude cellulase were 6 and 4 mM respectively, but $Ca^{++}$ inhibited the enzyme activity at concentrations below 2 mM and above 6mM. Both $Cu^{++}\;and\;Mn^{++}$ ions inhibited cellulase activities and a ocmplete inactivation of crude cellulase was achieved at concentratioins of 5 and 2 mM of ions respectively. When Na-CMC was used as substrate, the Km values of crude cellulase and F IV-1 were calculated to be $5{\times}10^{-4}\;and\;2{\times}10^{-5}mM$, and V values 32 and 1.35 mmoles/hour, respectively. The Ki values of $Mn^{++}$ for crude cellulase and F IV-1 were found to be $8{\times}10^{-2}\;and\;3{\times}10^{-2}\;mM\;while\;those\;of\;Cu^{++}\;were\;at\;2{\times}10^{-1}\;and\;1{\times}10^{-1}\;mM\;respectively.\;Both\;Mn^{++}\;and\;Cu^{++}$ showed competitive inhibition with substrate.

• Microbiology and Biotechnology Letters
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• v.34 no.4
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• pp.311-317
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• 2006

Using PCR amplification, we cloned a cellulase gene (ce/H) from the Bacillus subtilis AH18 which has plant growth-promoting activity and antagonistic ability against pepper blight caused by Phytophthora capsici. The 1.6 kb PCR fragment contained the full sequence of the cellulase gene and the 1,582 bp gene deduced a 508 amino acid sequence. Similarity search in protein database revealed that the cellulase of B. subtilis AH18 was more than 98% homologous in the amino acid sequence to those of several major Bacillus spp. The ce/H was expressed in E. coli under an IPTG inducible lac promoter on the vector, had apparent molecular weight of about 55 kDa upon CMC-SDS-PAGE analysis. Partially purified cellulase had not only cellulolytic activity toward carboxymethyl-cellulose (CMC) but also insoluble cellulose, such as Avicel and filter paper (Whatman No. 1). In addition, the cellulase could degrade a fungal cell wall of Phytophthora capsici. The optimum pH and temperature of the ce/H coded cellulase were determined to be pH 5.0 and $50^{\circ}C$. The enzyme activity was activated by $AgNO_3$ or $CoCl_2$. However its activity was Inhibited by $HgC1_2$. The enzyme activity was activated by hydroxy urea or sodium azide and inhibited by CDTA or EDTA. The results indicate that the cellulase gene, ce/H is an antifungal mechanism of B. subtilis AH18 against phytophthora blight disease in red-pepper.

• Applied Biological Chemistry
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• v.50 no.1
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• pp.23-28
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• 2007

Auxin-producing antagonistic bacterium K11, which can inhibit Phytophtora capsici, was isolated from a local red-pepper field soil in Gyeong-buk. In order to check for additional PGPR(plant growth promoting rhizobacterium) functions of the strain K11, we confirmed siderophore and cellulase productions by CAS (chrome azurol S) blue agar and CMC plate with congo red, respectively. The strain K11 was identified as Bacillus licheniformis with 98% similarity on 16s rDNA comparison and Biolog analyses. B. licheniformis K11 promoted mung bean adventitious root induction and enhanced root growth of mung bean (160%), pea (150%), and Chinese cabbage (130%), Also, B. licheniformis K11 was able to effectively suppress (63%) P. capsici causing red-pepper blight in the pot in vivo test. Therefore, we could select a triple-functional PGPR which has auxin, siderophore, and cellulase producing ability for effective crops production in organic farming.

• 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.