• Journal of Plant Biotechnology
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• v.34 no.2
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• pp.111-118
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• 2007

Global warming crisis due primarily to continued green house gas emission requires impending change to renewable alternative energy than continuously depending on exhausting fossil fuels. Bioenergy including biodiesel and bioethanol are considered good alternatives because of their renewable and sustainable nature. Bioethanol is currently being produced by using sucrose from sugar beet, grain starches or lignocellulosic biomass as sources of ethanol fermentation. However, grain production requires significant amount of fossil fuel inputs during agricultural practices, which means less competitive in reducing the level of green house gas emission. By contrast, cellulosic bioethanol can use naturally-growing, not-for-food biomass as a source of ethanol fermentation. In this respect, cellulosic ethanol than grain starch ethanol is considered a more appropriate as a alternative renewable energy. However, commercialization of cellulosic ethanol depends heavily on technology development. Processes such as securing enough biomass optimized for economic processing, pretreatment technology for better access of polymer-hydrolyzing enzymes, saccharification of recalcitrant lignocellulosic materials, and simultaneous fermentation of different sugars including 6-carbon glucose as well as 5-carbon xylose or arabinose waits for greater improvement in technologies. Although it seems to be a long way to go until commercialization, it should broadly benefit farmers with novel source of income, environment with greener and reduced level of global warming, and national economy with increased energy security. Mission-oriented strategies for cellulosic ethanol development participated by government funding agency and different disciplines of sciences and technologies should certainly open up a new era of renewable energy.

• 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 the Korean Wood Science and Technology
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• v.38 no.2
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• pp.140-148
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• 2010

This study was to investigate the enzymatic hydrolysis of cellulose using the cellulase from whole body of the native termite collected in Milyang-si, Kyungsangnamdo, Korea. In the results, optimal temperature and pH for the enzyme of native termites were $45^{\circ}C$ and pH 5.5 for both endo-${\beta}$-1, 4-glucanase and ${\beta}$-glucosidase. Enzyme activity of the termite enzyme was shown $8.8{\times}10^{-2}\;FPU/m{\ell}$. And the highest glucose hydrolysis rate of cellulose by the digestive enzyme from test termites was 24.5% based on the glucan, comparing 59.7% by commercial enzyme (only celluclast 1.5 L) at 1% (w/v) substrate and 36 hours in hydrolysis time. This hydrolysis rate by the digestive enzyme from test termites was comparatively high value in 41% level of the commercial enzyme. When cellulose was hydrolyzed by the digestive enzyme of the native termite, glucose hydrolysis was almost completed in 12 hours which was the considerably reduced time for cellulose hydrolysis. It was suggested that the quiet short reaction time for cellulose hydrolysis by the enzyme from native termite could be a very high advantage for development of hydrolysis cellulase for lignocellulosic biomass.

• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1476-1484
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• 2015

Three endoxylanase-encoding genes from the moderately themophilic chemoorganotrophic bacterium Melioribacter roseus were cloned and expressed in Escherichia coli. Genes xyl2091 (Mros_2091) and xyl2495 (Mros_2495) encode GH10 family hydrolases, whereas xyl2090 (Mros_2090) represents the GH30 family. In addition to catalytic domains, Xyl2090 and Xyl2091 contain carbohydrate-binding modules that could facilitate their binding to xylans and Por sorting domains associated with the sorting of proteins from the periplasm to the outer membrane, where they are covalently attached. Recombinant endoxylanase Xyl2495 exhibited a high specific activity of 1,920 U/mg on birchwood xylan at 40℃. It is active at low temperatures, exhibiting more than 30% of the maximal activity even at 0℃. Endoxylanases Xyl2090 and Xyl2091 have lower specific activities but higher temperature optima at 80℃ and 65℃, respectively. Analysis of xylan hydrolysis products revealed that Xyl2090 generates xylo-oligosaccharides longer than xylopentaose. Xylose and xylobiose are the major products of xylan hydrolysis by the recombinant Xyl2091 and Xyl2495. No activity against cellulose was observed for all enzymes. The presence of three xylanases ensures efficient xylan hydrolysis by M. roseus. The highly processive "free" endoxylanase Xyl2495 could hydrolyze xylan under moderate temperatures. Xylan hydrolysis at elevated temperatures could be accomplished by concerted action of two cell-bound xylanases; Xyl2090 that probably degrades xylans to long xylo-oligosaccharides, and Xyl2091 hydrolyzing them to xylose and xylobiose. The new endoxylanases could be useful for saccharification of lignocellulosic biomass in biofuels production, bleaching of paper pulp, and obtaining low molecular weight xylooligosaccharides.

• Journal of Microbiology and Biotechnology
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• v.20 no.9
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• pp.1351-1358
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• 2010

The demand for ${\beta}$-glucosidases insensitive to product inhibition is increasing in modern biotechnology, for these enzymes would improve the process of saccharification of lignocellulosic materials. In this study, a ${\beta}$-glucosidase gene that encodes a 442-amino-acid protein was isolated from a marine microbial metagenomic library by functional screening and named as bgl1A. The protein was identified to be a member of the glycoside hydrolases 1 family, and was recombinantly expressed, purified, and biochemically characterized. The recombinant ${\beta}$-glucosidase, Bgl1A, exhibited a high level of stability in the presence of various cations and high concentrations of NaCl. Interestingly, it was activated by glucose at concentrations lower than 400 mM. With glucose further increasing, the enzyme activity of Bgl1A was gradually inhibited, but remained 50% of the original value in even as high as 1,000 mM glucose. These findings indicate that Bgl1A might be a potent candidate for industrial applications.

• Journal of Microbiology and Biotechnology
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• v.27 no.9
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• pp.1670-1680
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• 2017

Lignocellulose, composed mostly of cellulose, hemicellulose, and lignin generated through secondary growth of woody plant, is considered as promising resources for biofuel. In order to use lignocellulose as a biofuel, biodegradation besides high-cost chemical treatments were applied, but knowledge on the decomposition of lignocellulose occurring in a natural environment is insufficient. We analyzed the 16S rRNA gene and metagenome to understand how the lignocellulose is decomposed naturally in decayed Torreya nucifera (L) of Bija forest (Bijarim) in Gotjawal, an ecologically distinct environment. A total of 464,360 reads were obtained from 16S rRNA gene sequencing, representing diverse phyla; Proteobacteria (51%), Bacteroidetes (11%) and Actinobacteria (10%). The metagenome analysis using single molecules real-time sequencing revealed that the assembled contigs determined originated from Proteobacteria (58%) and Actinobacteria (10.3%). Carbohydrate Active enZYmes (CAZy)- and Protein families (Pfam)-based analysis showed that Proteobacteria was involved in degrading whole lignocellulose, and Actinobacteria played a role only in a part of hemicellulose degradation. Combining these results, it suggested that Proteobacteria and Actinobacteria had selective biodegradation potential for different lignocellulose substrates. Thus, it is considered that understanding of the systemic microbial degradation pathways may be a useful strategy for recycle of lignocellulosic biomass, and the microbial enzymes in Bija forest can be useful natural resources in industrial processes.

• Mycobiology
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• v.48 no.6
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• pp.501-511
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• 2020

Xylophagous termites are capable of degrading lignocellulose by symbiotic gut microorganisms along with the host's indigenous enzymes. Therefore, the termite gut might be a potential niche to obtain natural yeasts with celluloytic, xylanolytic and ethanologenic traits required for bioethanol production from lignocellulosic biomass. In this study, we cultured 79 yeasts from three different termites viz. Coptotermes heimi, Odontotermes javanicus and Odontotermes obesus. After suitable screening methods, we identified 53 yeasts, which belonged to 10 genera and 16 different species of both ascomycetous and basidiomycetous yeasts. Most yeasts in the present study represent their first-ever isolation from the termite gut. Representative strains of identified yeasts were evaluated for their cellulolytic, xylanolytic, and ethanologenic abilities. None of the isolates showed cellulase activity; 22 showed xylanolytic activity, while six produced substantial quantities of ethanol. Among xylanolytic cultures, Pseudozyma hubeiensis STAG 1.7 and Hannaella pagnoccae STAG 1.14 produced 1.31 and 1.17 IU of xylanase. Among ethanologenic yeasts, the strains belonging to genera Candida and Kodamaea produced high amount of ethanol. Overall, highest ethanol level of 4.42 g/L was produced by Candida tropicalis TS32 using 1% glucose, which increased up to 22.92 g/L at 35 ℃, pH 4.5 with 5% glucose. Fermentation of rice straw hydrolysate gave 8.95 g/l of ethanol with a yield of 0.42 g/g using the strain TS32. Our study highlights the gut of wood-feeding termites as a potential source of diverse yeasts that would be useful in the production of xylanase and bioethanol.

• Journal of Microbiology and Biotechnology
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• v.28 no.4
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• pp.588-596
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel9K, was cloned using the shot-gun method from Paenibacillus sp. X4, which was isolated from alpine soil. The gene was 2,994 bp in length, encoding a protein of 997 amino acid residues with a predicted signal peptide composed of 32 amino acid residues. Cel9K was a multimodular enzyme, and the molecular mass and theoretical pI of the mature Cel9K were 103.5 kDa and 4.81, respectively. Cel9K contains the GGxxDAGD, PHHR, GAxxGG, YxDDI, and EVxxDYN motifs found in most glycoside hydrolase family 9 (GH9) members. The protein sequence showed the highest similarity (88%) with the cellulase of Bacillus sp. BP23 in comparison with the enzymes with reported properties. The enzyme was purified by chromatography using HiTrap Q, CHT-II, and HiTrap Butyl HP. Using SDS-PAGE/activity staining, the molecular mass of Cel9K was estimated to be 93 kDa, which is a truncated form produced by the proteolytic cleavage of its C-terminus. Cel9K was optimally active at pH 5.5 and $50^{\circ}C$ and showed a half-life of 59.2 min at $50^{\circ}C$. The CMCase activity was increased to more than 150% in the presence of 2 mM $Na^+$, $K^+$, and $Ba^{2+}$, but decreased significantly to less than 50% by $Mn^{2+}$ and $Co^{2+}$. The addition of Cel9K to a commercial enzyme set (Celluclast 1.5L + Novozym 188) increased the saccharification of the pretreated reed and rice straw powders by 30.4% and 15.9%, respectively. The results suggest that Cel9K can be used to enhance the enzymatic conversion of lignocellulosic biomass to reducing sugars as an additive.

• Journal of the Korean Wood Science and Technology
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• v.40 no.6
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• pp.424-430
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• 2012

In this study, laccase activity, rate of weight loss and degree of lignin degradation of pine wood chips were determined during the liquid and solid state incubation with Polyporus brumalis. The results showed that laccase enzyme activity at untreated wood chip was gradually decreased after 20 days, but enzyme activity with wood chip treatment showed 10 times higher than untreated ones at 60 incubation days. Rate of weight losses of pine chip and rate of lignin loss were 23.4% and 6.3% by P. brumalis during 80 incubation days. Gene expression of pblac1 from P. brumalis was 3 times increased under pine chip treatment at 40 incubation days. Consequently, laccase activity of white rot fungi, P. brumalis, was increased at incubation with wood chip and pblac1 gene was important factor of lignin degradation. Therefore, to regulate lignin degrading enzyme gene expression by using the tools of biotechnology will be able to develop superior strains and it will be useful for pretreatment of lignocellulosic biomass at bioethanol production.

• Journal of Mushroom
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• v.15 no.3
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• pp.134-138
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• 2017

The white button mushroom, Agaricus bisporus, is commercially the fifth most important edible mushroom, accounting for the production of 9,732 tons of mushrooms in Korea in 2015. The genus Agaricus has been known for its potential to degrade lignocellulosic materials. Chemical analyses carried out during the cultivation of A. bisporus indicated that the cellulose, hemicellulose, and lignin fractions were changed preferentially for both vegetative growth and sexual reproduction. We screened A. bisporus strains for effective biodegradation through extracellular enzyme activity using cellulase, xylanase, and ligninolytic enzymes. The enzyme biodegradations were conducted as follows: mycelia of collected strains were incubated in 0.5% CMC-MMP (malt-mops-peptone), 0.5 Xylan-MMP, and 0.5% lignin-MMP media for 14 days. Incubated mycelia were stained with 0.2% trypan blue. Eighteen strains were divided into 8 groups based on different extracellular enzyme activity in MMP media. These strains were then incubated in sterilized compost and compost media for 20 days to identify correlations between mycelial growth in compost media and extracellular enzyme activity. In this study, the coefficient of determination was the highest between mycelial growth in compost media and ligninolytic enzyme activity. It is suggested that comparison with ligninolytic enzyme activity of the tested strains is a simple method of screening for rapid mycelial growth in compost to select good mother strains for the breeding of A. bisporus.