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Kim M.K.,Environment friendly Research Division | Seo W.T.,Gyeongnam National University of Science and Technology | Lee Y.B.,Gyeongsang National University | Cho K.M.,Gyeongnam National University of Science and Technology
Food Science and Biotechnology | Year: 2013

The archaeal diversity in the soybean fermented food was analysed by culture-independent methods based on the 16S rRNA sequences. The 21 doenjang archaea - clones from the doenjang library were grouped into 5 groups: uncultured archaeon clone AE34 (42. 8%), uncultured archaeon clone AS17 (28. 6%), uncultured compost archaeon clone 4A29 (4. 8%), uncultured compost archaeon clone 0A10 (19. 0%), and uncultured compost archaeon clone 5A16 (4. 8%). The 21 ganjang archaea - clones from the ganjang library were grouped into 5 groups: Halophilic archaeon MH1-34-1 (19. 0%), H. archaeon MH1-16-3 (61. 9%), Halococcus thailandensis (4. 8%), Haloplanus sp. RO5-8 (9. 5%), and H. archaeon MH1-136-2 (4. 8%). © 2013 The Korean Society of Food Science and Technology and Springer Science+Business Media Dordrecht.


Kang Y.M.,Korea Institute of Oriental Medicine | Kim M.G.,Environment friendly Research Division | Yun H.D.,Gyeongsang National University | Cho K.M.,Gyeongnam National University of Science and Technology
Journal of the Korean Society for Applied Biological Chemistry | Year: 2013

A shotgun method was adopted to clone the β-xylanase and lichenase genes from a genomic library of a Paenibacillus polymyxa GS01 genome library. Also, a fusion enzyme, Xyn3A-Lin16A, was designed by overlap extension polymerase chain reaction (PCR). The cloned Xyn3A and Lin16A proteins were successfully expressed and exhibited both xylanase and lichenase activities. The xyn43A and lin16A gene amplicons were 1,917 bp and 714 bp in size and encoded proteins of 635 and 238 amino acids, respectively. The Xyn43A and Lin16A gene products showed predicted molecular masses of 65 and 24 kDa with respective calculated pIs of 5.97 and 5.77, respectively. Furthermore, the fusion enzyme gene, Xyn43A-Lin16A, was 4,466 bp in length and encoded a protein of 847 amino acids, with apparent molecular mass of 89 kDa and a calculated pI of 5.93. This fusion enzyme showed optimum activity at pH 6.0-7.0 and 50°C. Thus, the xyn43A and lin16A genes from P. polymyxa GS01were able to exist in tandem, and recombinant DNA technologies can be used to improve enzyme productivity. Therefore, the development of functional fusion enzymes (xylanase-lichenase) using recombinant DNA technologies may lead to further improvements and their successful enzyme engineering in industrial application. © 2013 The Korean Society for Applied Biological Chemistry.


Kang Y.M.,Korea Institute of Oriental Medicine | Kim M.K.,Environment Friendly Research Division | An J.M.,Gyeongsang National University | Haque M.A.,Gyeongnam National University of Science and Technology | Cho K.M.,Gyeongnam National University of Science and Technology
Journal of Molecular Catalysis B: Enzymatic | Year: 2015

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. Genetic engineering facilitates the artificial fusion of genes that encode functional proteins into a single open reading frame. The cloning of the cellulase and xylanase genes from the cow rumen metagenome resulted in the construction of a bifunctional fusion gene via site-directed mutagenesis for further specific industrial processes. The metagenome of cow rumen bacteria was the source of a gene that encodes an extracellular β-glucanase for cloning and expression in Escherichia coli DH5α. The cellulase (cel9E) gene of un-culturable rumen bacteria existed in tandem with the xylanase (xyn10A) gene. The genes were 2268 bp and 1.578 bp and encoded 756- and 526-aa proteins, respectively. BLAST analyses and domain predictions assigned Cel9E and Xyn10A to glycosyl hydrolase families 9 and 10. The molecular weight of the individual proteins Cel9E and Xyn10A were estimated to be approximately 76.0 kDa and 56.0 kDa by CMC-SDS-PAGE and OSX-SDS-PAGE, respectively. The 3909 bp cel9E-xyn10A fusion gene encoded a 1303-amino acid residue protein with a molecular weight of approximately 137.0 kDa according to CMC/OSX-SDS-PAGE. The maximum cellulase and xylanase activities from the fusion protein Cel9E-Xyn10A were observed at pH 6.0 and pH 8.0, respectively. The optimal temperature for the bifunctional enzyme was found to be 50 °C. The improved catalytic efficiency of the Cel9E-Xyn10A for the cellulase and xylanase activity was equivalent to 1.47- and 2.21-fold of the parental efficiency. We report the presence of the cel9E gene in tandem with the xyn10A gene in the metagenome of un-culturable cow rumen bacteria. The construction, expression and characterization of the cel9E-xyn10A bifunctional gene fusion obtained by site-directed mutagenesis are also reported. © 2015 Published by Elsevier B.V.


Kim B.,Gyeongnam National University of Science and Technology | Seo W.T.,Gyeongnam National University of Science and Technology | Kim M.G.,Environment friendly Research Division | Yun H.D.,Gyeongsang National University | Cho K.M.,Gyeongnam National University of Science and Technology
Journal of the Korean Society for Applied Biological Chemistry | Year: 2012

Lactic acid bacterial diversity and the composition of individual bacterial communities during the fermentation of mulkimchi were examined using a polymerase chain recation (PCR)-based approach. Based on 16S rRNA sequence similarity values, a total of fifteen different lactic acid bacterial species were found in eight sampling sites, including Lactobacillus alimentarius, Lactobacillus brevis, Lactobacillus farciminis, Lactobacillus fabifermentans, Lactobacillus nantensis, Lactobacillus parabrevis, Lactobacillus plantarum, Lactobacillus versnoldensis, Lactobacillus zymae, and Lactobacillus sp., Leuconostoc pseudomesenteroides, Weissella cibaria, Weissella confusa, and Weissella sp. The prevalence of We. cibaria, belonging to the Weissella genus, was the highest (86. 7%) at 0 h (initial stages) and gradually decreased at 72 h (rancid stage). In contrast, La. plantarum was observed at 36 h (16. 7%, over-ripening stage) and gradually increased up to 84 h (70. 0%, rancid stage) during mulkimchi fermentation. We. cibaria was found to be associated with the microorganisms that were present during the initial stage of fermentation, whereas La. plantarum was associated with the production of lactic acid in the over-ripening and rancid stages during fermentation at 30°C±2. © 2012 The Korean Society for Applied Biological Chemistry.


Lee J.H.,NAKDONG River Basin Environmental Office | Nam S.H.,Gyeongnam National University of Science and Technology | Seo W.T.,Gyeongnam National University of Science and Technology | Yun H.D.,Gyeongsang National University | And 3 more authors.
Food Chemistry | Year: 2012

Bacillus subtilis CSY191, the potential probiotics and surfactin-like compound producer, was isolated from doenjang (Korean traditional fermented soybean paste).The survival rate of this strain appeared to be the 58.3% under artificial gastric conditions after 3 h at pH 3.0. Surfactin was purified from the strain CSY191. Three potential surfactin isoforms were detected, with protonated masses of m/z 1030.7, 1044.7, and 1058.71. These different structures were detected in combination with Na+, K+ and Ca 2+ ions by MALDI-TOF mass spectrometry. Upon 500 MHz 1H NMR analysis, the surfactin isoforms had identical amino acids (GLLVDLL) and hydroxy fatty acids (of 13-15 carbons in length). The MTT assay showed that surfactin inhibited growth of MCF-7 human breast cancer cells in a dose-dependent manner, with an IC50 of approximately 10 μg/ml at 24 h. Additionally, the surfactin contents, during cheonggukjang fermentation with strain CSY191, increased from 0.3 to 48.2 mg/kg over 48 h of fermentation, while the level of anticancer activity increased from 2.6- to 5.1-fold. © 2011 Elsevier Ltd. All rights reserved.


PubMed | Gyeongnam National University of Science and Technology, Korea Research Institute of Bioscience and Biotechnology and Environment Friendly Research Division
Type: Journal Article | Journal: The plant pathology journal | Year: 2014

In 2009-2010, unusual symptoms were observed on Pleurotus eryngii grown in mushroom farms in Gyeongnam Province, Republic of Korea. One of the main symptoms was a cobweb-like growth of fungal mycelia over the surface of the mushroom. The colonies on the surface rapidly overwhelmed the mushrooms and developed several spores within 3-4 days. The colonized surface turned pale brown or yellow. The fruit body eventually turned dark brown and became rancid. Kochs postulates were completed by spraying and spotting using isolated strains. The phylogenetic tree obtained from the internal transcribed spacer sequence analysis showed that the isolated fungal pathogen corresponded to Cladobotryum mycophilum (99.5%). In the fungicide sensitivity tests, the ED50 values for the isolate with respect to benomyl and carbendazim were from 0.29 to 0.31 ppm. Benzimidazole fungicides were most effective against C. mycophilum, a causal agent of cobweb disease in P. eryngii.


Kim M.K.,Environment Friendly Research Division | Lee Y.H.,Environment Friendly Research Division | Kim H.,Korea Research Institute of Bioscience and Biotechnology | Lee J.,Korea Research Institute of Bioscience and Biotechnology | Ryu J.S.,Environment Friendly Research Division
Microbiological research | Year: 2015

To characterize of the pathogenicity gene from the soft rot pathogen Pantoea sp. PPE7 in Pleurotus eryngii, we constructed over 10,000 kanamycin-resistant transposon mutants of Pantoea sp. strain PPE7 by transposon mutagenesis. One mutant, Pantoea sp. NPPE9535, did not cause a soft rot disease on Pleurotus eryngii was confirmed by the pathogenicity test. The transposon was inserted into the wzc gene and the disruption of the wzc gene resulted in the reduction of polysaccharide production and abolished the virulence of Pantoea sp. strain PPE7 in P. eryngii. Analysis of the hydropathic profile of this protein indicated that it is composed of two main domains: an N-terminal domain including two transmembrane α-helices and a C-terminal cytoplasmic domain consisting of a tyrosine-rich region. Comparative analysis indicated that the amino acid sequence of Wzc is similar to that of a number of proteins involved in the synthesis or export of polysaccharides in other bacterial species. Purified GST-Wzc was found to affect the phosphorylation of tyrosine residue in vivo. These results showed that the wzc gene might play an important role in the virulence of Pantoea sp. strain PPE7 in P. eryngii. Copyright © 2014 Elsevier GmbH. All rights reserved.


Im C.H.,Environment friendly Research Division | Park Y.-H.,Pusan National University | Hammel K.E.,U.S. Department of Agriculture | Park B.,Environment friendly Research Division | And 3 more authors.
Fungal Genetics and Biology | Year: 2016

Breeding new strains with improved traits is a long-standing goal of mushroom breeders that can be expedited by marker-assisted selection (MAS). We constructed a genetic linkage map of Pleurotus eryngii based on segregation analysis of markers in postmeiotic monokaryons from KNR2312. In total, 256 loci comprising 226 simple sequence-repeat (SSR) markers, 2 mating-type factors, and 28 insertion/deletion (InDel) markers were mapped. The map consisted of 12 linkage groups (LGs) spanning 1047.8 cM, with an average interval length of 4.09 cM. Four independent populations (Pd3, Pd8, Pd14, and Pd15) derived from crossing between four monokaryons from KNR2532 as a tester strain and 98 monokaryons from KNR2312 were used to characterize quantitative trait loci (QTL) for nine traits such as yield, quality, cap color, and earliness. Using composite interval mapping (CIM), 71 QTLs explaining between 5.82% and 33.17% of the phenotypic variations were identified. Clusters of more than five QTLs for various traits were identified in three genomic regions, on LGs 1, 7 and 9. Regardless of the population, 6 of the 9 traits studied and 18 of the 71 QTLs found in this study were identified in the largest cluster, LG1, in the range from 65.4 to 110.4 cM. The candidate genes for yield encoding transcription factor, signal transduction, mycelial growth and hydrolase are suggested by using manual and computational analysis of genome sequence corresponding to QTL region with the highest likelihood odds (LOD) for yield. The genetic map and the QTLs established in this study will help breeders and geneticists to develop selection markers for agronomically important characteristics of mushrooms and to identify the corresponding genes. © 2016 Elsevier Inc.


Ryu J.-S.,Environment Friendly Research Division | Kim M.K.,Environment Friendly Research Division | Im C.H.,Environment Friendly Research Division | Shin P.-G.,Mushroom
Scientia Horticulturae | Year: 2015

Pleurotus eryngii has recently become the most commonly cultivated mushroom in East Asia. However, a long shelf life is required for export to distant regions. To obtain a long shelf life for mushrooms, different ratios of raw materials were mixed to develop new media based on crude protein and nitrogen free extract contents and thus 7 media designated A-G were prepared. Fruiting bodies from the "D" and "G" media containing 4.5 and 4.7% crude protein (with nitrogen free extract and crude protein ratios of 2.9 and 3.3), respectively, had the longest shelf life period (35.5 and 35.3 days) as a B grade, representing intact mushrooms with water and spores. Moreover, G medium showed significantly better mushrooms in yield (113.7g/bottle), quality (7.3) and required days to harvest (15.4 days) compared to mushrooms produced in control medium (A), it was statistically proved by analysis of variance and Duncan's multiple-range test (P<0.05). The shelf life as a B grade was significantly extended with increased the crude protein (R2=0.774) and CaO contents (R2=0.608) in the media and regression analyses indicated that crude protein and CaO presented synergistic effects on shelf life (R2=0.728-0.819, P<0.05). These results might be useful for mushroom farmers to produce more exportable mushrooms with a long shelf life. © 2015 Elsevier B.V.


PubMed | Korea Research Institute of Bioscience and Biotechnology and Environment Friendly Research Division
Type: | Journal: Microbiological research | Year: 2014

To characterize of the pathogenicity gene from the soft rot pathogen Pantoea sp. PPE7 in Pleurotus eryngii, we constructed over 10,000 kanamycin-resistant transposon mutants of Pantoea sp. strain PPE7 by transposon mutagenesis. One mutant, Pantoea sp. NPPE9535, did not cause a soft rot disease on Pleurotus eryngii was confirmed by the pathogenicity test. The transposon was inserted into the wzc gene and the disruption of the wzc gene resulted in the reduction of polysaccharide production and abolished the virulence of Pantoea sp. strain PPE7 in P. eryngii. Analysis of the hydropathic profile of this protein indicated that it is composed of two main domains: an N-terminal domain including two transmembrane -helices and a C-terminal cytoplasmic domain consisting of a tyrosine-rich region. Comparative analysis indicated that the amino acid sequence of Wzc is similar to that of a number of proteins involved in the synthesis or export of polysaccharides in other bacterial species. Purified GST-Wzc was found to affect the phosphorylation of tyrosine residue in vivo. These results showed that the wzc gene might play an important role in the virulence of Pantoea sp. strain PPE7 in P. eryngii.

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