• Journal of Applied Biological Chemistry
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• v.60 no.1
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• pp.79-86
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• 2017

Microbes in forest are very important due to not only to enhance soil fertility but also maintain a healthy ecosystem by supplying the energy available to living organisms by producing various kinds of enzymes related to degradation of lignocellulosic biomass. In order to isolate a lignocellulosic biomass degrading bacterial strain from the Jurassic park located in Gyeongnam National University of Science and Technology, We used the Luria-Bertani-Carboxymethyl cellulose (CMC) agar trypan blue method containing 0.4 % carboxymethyl cellulose and 0.01 % trypan blue. As a result, we isolated a bacterial strain showing both activity on the CMC and xylan. To identify the isolated strain, 16S rRNA sequencing and API kit analysis were used. The isolated strain turned out to belong to Bacillus species and then named Bacillus sp. GJY. In the CMC zymogram analysis, it showed that one active band of about 28kDa in size is present. Xylan zymogram analysis also showed to have one active band of about 25kDa in size. The optimal growth temperature of Bacillus sp. GJY was $37^{\circ}C$. The maximal activities of CMCase and xylanase were 12 hour after incubation. The optimal pH and temperature for CMCase were 5.0 and $40^{\circ}C$, respectively, whereas the optimal pH and temperature for xylanase was 4.0 and $40^{\circ}C$. Both activities for CMCase and xylanase showed to be thermally stable at 40and $50^{\circ}C$, while both activities rapidly decreased at over $60^{\circ}C$.

• Journal of Applied Biological Chemistry
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• v.61 no.1
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• pp.59-67
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• 2018

This study was conducted to isolate the cellulolytic bacteria able to grow on LB- Carboxymethyl cellulose (CMC) agar trypan blue medium from the mixed forest and Larix leptolepis stands. Three bacterial strains with high activity against both CMC and xylan were isolated. Both API kit test and 16S rRNA gene sequence analysis revealed that the three different isolates belong to the gene Bacillus. Therefore, the isolates named as Bacillus sp. EFL1, Bacillus sp. EFL2, and Bacillus sp. EFP3. The optimum growth temperature of Bacillus sp. EFL1, EFL2, and EFP3 were $37^{\circ}C$. The optimum temperature for CMCase and xylanase from Bacillus sp. EFL1 were $50^{\circ}C$. The optimum pH of Bacillus sp. EFL1 xylanase was pH 5.0 but the optimum pH of CMCase from Bacillus sp. EFL1 was pH 6.0. The optimum temperature of CMCase and xylanase from Bacillus sp. EFL2 was $60^{\circ}C$, respectively. The optimum pH of CMCase of Bacillus sp. EFL2 was 5.0, whereas xylanase showed high activity at pH 3.0-9.0. The optimum temperature for CMCase and xylanase of Bacillus sp. EFP3 was $50^{\circ}C$. The optimum pH for CMCase and xylanse was 5.0 and 4.0, respectively. CMCases from Bacillus sp. EFL1, EFL2, and EFP3 were thermally unstable. Although xylanase from Bacillus sp. EFL1 and EFP3 showed to be thermally unstable, xylanase from Bacillus sp. EFL2 showed to be thermally stable. Therefore, Bacillus sp. EFL2 has great potential for animal feed, biofuels, and food industry applications.

• Journal of Animal Science and Technology
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• v.48 no.3
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• pp.415-424
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• 2006

The purpose of this study was to investigate the fungal growth and enzyme production under different carbohydrate substrate conditions. The anaerobic fungus Neocallimastix sp. NLRI-3 isolated from the rumen of Korean native goat was incubated with different carbohydrate media containing 0.2% of glucose, starch, rice straw, filter paper, carboxymethyl cellulose(CMC), Sigmacell cellulose, xylan or xylose, respectively. The culture head gas production was the highest in the culture of filter paper medium, and the lowest in the culture of CMC medium at 96h incubation (P<0.05). The fungal zoospore production reached peak at 72h incubation, and its number was the highest in rice straw medium among the treatments (P<0.05). At 96h incubation, carboxymethyl cellulase(CMCase) activity was the highest in the culture of filter paper medium and the lowest in the culture of starch medium (P<0.05). While xylanase activity was the highest in the culture of rice straw medium and the lowest in the culture of xylose medium(P<0.05) at 72h incubation. There were no differences in culture supernatant protein expression among the treatments. However, the patterns of enzyme expression were different among the treatments with zymogram analysis. Six CMCases and 4 xylanase were detected from the results of zymogram analysis. Therefore the present study indicating that the fungal enzyme expression could be stimulated with insoluble substrates in the culture medium.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.11
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• pp.1610-1613
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• 2003

The aim of the experiment was to study the changes in the activities of various rumen fibre degrading enzymes due to the feeding of chemically treated mustard (Brassica campestris) straw in sheep. Mustard straw (MS) (<5 cm particle size) was treated either with urea (4% (w/w), or with 2% sodium hydroxide (NaOH), or with alkaline hydrogen peroxide (2% NaOH and 1.5% hydrogen peroxide ($H_2O_2$)) and/or supplemented with 2% (w/w) urea. Seven maintenance type rations were prepared using MS (70 parts) with molasses (5 parts) and concentrate (25 parts). They were untreated MS (CMS), urea treated MS (UMS), urea supplemented MS (MSUS), alkali treated MS (AMS), alkali treated and urea supplemented MS (AMS-US), alkali $H_2O_2$ treated MS (AHMS) and alkali $H_2O_2$ treated and urea supplemented MS (AHMS-US). They were then compressed into a complete feed block with the help of block making machine. Forty two male hoggets of Malpura breed sheep were equally distributed into each treatment group and (were) offered feed and water ad libitum. At the end of 21 days of feeding trial, rumen liquor was collected through stomach tube from three animals in each group at 0 h, 4 h, 8 h, 12 h of post feeding. Results showed that the level of enzyme varied from 8.52 to 11.12, 40.85 to 50.37, 3.22 to 3.78, 2.09 to 2.77 and 31.44 to 44.24 units/100 ml SRL respectively for carboxymethyl cellulase (CMCase), $\alpha$-amylase, microcrystalline cellulase (MCCase), filter paper (FP) degrading enzyme and $\alpha$-glucosidase. Processing of MS affected the enzyme activities, in a way, that NaOH and AHP treatment significantly reduced CMCase and FP degrading enzyme. The effect of urea treatment showed an increase in the activity of MCCase and $\alpha$-glucosidase. But the supplementation of urea increased the activity of CMCase, FP degrading enzyme and $\alpha$-glucosidase. The CMCase, $\alpha$-amylase, $\alpha$-glucosidase activities were highest at 4hr whereas MCCase and FP degrading enzyme had maximum activities at 12 h post feeding Results suggested that MS might need longer time in the rumen for its effective degradation.

• 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.

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.404-409
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• 2008

The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and ${\beta}$-glucosidases. A gene encoding endoglucanase, designated as cel12, was cloned from total RNA prepared from F. palustris grown at the expense of Avicel. The gene encoding Cel12 has an open reading frame of 732 bp, encoding a putative protein of 244 amino acid residues with a putative signal peptide residing at the first 18 amino acid residues of the N-terminus of the protein. Sequence analysis of Cel12 identified three consensus regions, which are highly conserved among fungal cellulases belonging to GH family 12. However, a cellulose-binding domain was not found in Cel12, like other GH family 12 fungal cellulases. Northern blot analysis showed a dramatic increase of cel12 mRNA levels in F. palustris cells cultivated on Avicel from the early to late stages of growth and the maintenance of a high level of expression in the late stage, suggesting that Cel12 takes a significant part in endoglucanase activity throughout the growth of F. palustris. Adventitious expression of cel12 in the yeast Pichia pastoris successfully produced the recombinant protein that exhibited endoglucanase activity with carboxymethyl cellulose, but not with crystalline cellulose, suggesting that the enzyme is not a processive endoglucanase unlike two other endoglucanases previously identified in F. palustris.

• BMB Reports
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• v.39 no.1
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• pp.105-110
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• 2006

We have screened 766 strains of fungi from the BIOTEC Culture Collection (BCC) for xylanases working in extreme pH and/or high temperature conditions, the so-called extreme xylanases. From a total number of 32 strains producing extreme xylanases, the strain BCC7928, identified by using the internal transcribed spacer (ITS) sequence of rRNA to be a Marasmius sp., was chosen for further characterization because of its high xylanolytic activity at temperature as high as $90^{\circ}C$. The crude enzyme possessed high thermostability and pH stability. Purification of this xylanase was carried out using an anion exchanger followed by hydrophobic interaction chromatography, yielding the enzyme with >90% homogeneity. The molecular mass of the enzyme was approximately 40 kDa. The purified enzyme retained broad working pH range of 4-8 and optimal temperature of $90^{\circ}C$. When using xylan from birchwood as substrate, it exhibits $K_m$ and $V_{max}$ values of $2.6{\pm}0.6\;mg/ml$ and $428{\pm}26\;U/mg$, respectively. The enzyme rapidly hydrolysed xylans from birchwood, beechwood, and exhibited lower activity on xylan from wheatbran, or celluloses from carboxymethylcellulose and Avicel. The purified enzyme was highly stable at temperature ranges from 50 to $70^{\circ}C$. It retained 84% of its maximal activity after incubation in standard buffer containing 1% xylan substrate at $70^{\circ}C$ for 3 h. This thermostable xylanase should therefore be useful for several industrial applications, such as agricultural, food and biofuel.

• Journal of Microbiology and Biotechnology
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• v.24 no.5
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• pp.585-591
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• 2014

To evaluate the potential utility of pretreatment of raw biomass with a complex microbial system, we investigated the degradation of rice straw by BMC-9, a lignocellulose decomposition strain obtained from a biogas slurry compost environment. The degradation characteristics and corresponding changes in the bacterial community were assessed. The results showed that rapid degradation occurred from day 0 to day 9, with a peak total biomass bacterium concentration of $3.3{\times}10^8$ copies/ml on day 1. The pH of the fermentation broth declined initially and then increased, and the mass of rice straw decreased steadily. The highest concentrations of volatile fatty acid contents (0.291 mg/l lactic acid, 0.31 mg/l formic acid, 1.93 mg/l acetic acid, and 0.73 mg/l propionic acid) as well as the highest xylanse activity (1.79 U/ml) and carboxymethyl cellulase activity (0.37 U/ml) occurred on day 9. The greatest diversity among the microbial community also occurred on day 9, with the presence of bacteria belonging to Clostridium sp., Bacillus sp., and Geobacillus sp. Together, our results indicate that BMC-9 has a strong ability to rapidly degrade the lignocelluloses of rice straw under relatively inexpensive conditions, and the optimum fermentation time is 9 days.

• Proceedings of the Korea Technical Association of the Pulp and Paper Industry Conference
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• 2011.04a
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• pp.81-91
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• 2011

Current fuel ethanol research and development deals with process engineering trends for improving biotechnological production of ethanol. Recently, a large amount of studies regarding the utilization of lignocellulosic biomass as a good feedstock for producing fuel ethanol is being carried out worldwide. The plant biomass is mainly composed of cellulose, hemicellulose and lignin. The main challenge in the conversion of biomass into ethanol is the complex, rigid and harsh structures which require efficient process and cost effective to break down. The isolation of microorganisms is one of the means for obtaining enzymes with properties suitable for industrial applications. For these reasons, crude cultures containing cellulosic biomass degrading microorganisms were isolated from rice field soil, cow farm soil and rotten rice straw from cow farm. Carboxymethyl cellulose (CMC), xylan and Avicel (microcrystalline cellulose) degradation zone of clearance on agar platefrom rice field soil resulted approximately at 25 mm, 24 mm and 22 mm respectively. As for cow farm soil, CMC, xylan and Avicel degradation clearancezone on agar plate resulted around at 24mm, 23mm and 21 mm respectively. Rotten rice straw from cow farm also resulted for CMC, xylan and Avicel degradation zone almost at 24 mm, 23 mm and 22 mm respectively. The objective of this study is to isolatebiomass degrading microbial strains having good efficiency in cellulose hydrolysis and observed the effects of different substrates (CMC, xylan and Avicel) on the production of cellulase enzymes (endo-glucanase, exo-glucanase, cellobiase, xylanase and avicelase) for producing low cost biofuel from cellulosic materials.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.5
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• pp.382-390
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• 2006

Various microorganisms were isolated from the surface waters and sediments of eutrophic lakes and reservoirs in Korea to enable an investigation of bacteria having algal lytic activities against Anabaena flos-aquae when water blooming occurs and to study enzyme profiles of algal lytic bacteria. Two bacterial strains, AFK-07 and AFK-13, were cultured, characterized and identified as Acinetobacter johnsonii and Sinorhizobium sp., respectively. The A. johnsonii AFK-07 exhibited a high level of degradatory activities against A. flos-aquae, and produced alginase, caseinase, lipase, fucodian hydrolase, and laminarinase. Moreover, many kinds of glycosidase, such as ${\beta}-galactosidase,\;{\beta}-glucosidase,\;{\beta}-glucosaminidase,\;and\; {\beta}-xylosidase$, which hydrolyzed ${\beta}-O-glycosidic$ bonds, were found in cell-free extracts of A. johnsonii AFK-07. Other glycosidases such as ${\alpha}-galactosidase,\;{\alpha}-N-Ac-galactosidase,\;{\alpha}-mannosidase,\; and\;{\alpha}-L-fucosidase$, which cleave ${\alpha}-O-glycosidic$ bonds, were not identified in AFK-07. In the Sinorhizobium sp. AFK-13, the enzymes alginase, amylase, proteinase (caseinase and gelatinase), carboxymethyl-cellulase (CMCase), laminarinase, and lipase were notable. No glycosidase was produced in the AFK-13 strain. Therefore, the enzyme system of A. johnsonii AFK-07 had a more complex mechanism in place to degrade the cyanobacteria cell walls than did the enzyme system of Sinorhizobium sp. AFK-13. The polysaccharides or the peptidoglycans of A. flos-aquae may be hydrolyzed and metabolized to a range of easily utilized monosaccharides or other low molecular weight organic substances by strain AFK-07 of. A. johnsonii, while the products of polysaccharide degradation or peptidoglycans were more likely to be utilized by Sinorhizobium sp. AFK-13. These bacterial interactions may offer an alternative effective approach to controlling the water choking effects of summer blooms affecting our lakes and reservoirs.