• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.905-912
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• 2007

A novel ${\beta}-glucosidase$ gene, bglA, was isolated from uncultured soil bacteria and characterized. Using genomic libraries constructed from soil DNA, a gene encoding a protein that hydrolyzes a fluorogenic analog of cellulose, 4-methylumbelliferyl ${\beta}-D-cellobioside$ (MUC), was isolated using a microtiter plate assay. The gene, bglA, was sequenced using a shotgun approach, and expressed in E. coli. The deduced 55-kDa amino acid sequence for bglA showed a 56% identity with the family 1 glycosyl hydrolase Chloroflexus aurantiacus. BglA included two conserved family 1 glycosyl hydrolase regions. When using $p-nitrophenyl-{\beta}-D-glucoside$ (pNPG) as the substrate, the maximum activity of the purified ${\beta}-glucosidase$ exhibited at pH 6.5 and $55^{\circ}C$, and was enhanced in the presence of $Mn^{2+}$. The $K_m\;and\;V_{max}$ values for the purified enzyme with pNPG were 0.16 mM and $19.10{\mu}mol/min$, respectively. The purified BglA enzyme hydrolyzed both pNPG and $p-nitrophenyl-{\beta}-D-fucoside$. The enzyme also exhibited substantial glycosyl hydrolase activities with natural glycosyl substrates, such as sophorose, cellobiose, cellotriose, cellotetraose, and cellopentaose, yet low hydrolytic activities with gentiobiose, salicin, and arbutin. Moreover, BglA was able to convert the major ginsenoside $Rb_1$ into the pharmaceutically active minor ginsenoside Rd within 24 h.

• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.462-469
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• 2012

A metagenomic fosmid library was constructed from genomic DNA isolated from the microbial community residing in hindguts of a wood-feeding higher termite (Microcerotermes sp.) collected in Thailand. The library was screened for clones expressing lignocellulolytic activities. Fourteen independent active clones (2 cellulases and 12 xylanases) were obtained by functional screening at pH 10.0. Analysis of shotgun-cloning and pyrosequencing data revealed six ORFs, which shared less than 59% identity and 73% similarity of their amino acid sequences with known cellulases and xylanases. Conserved domain analysis of these ORFs revealed a cellulase belonging to the glycoside hydrolase family 5, whereas the other five xylanases showed significant identity to diverse families including families 8, 10, and 11. Interestingly, one fosmid clone was isolated carrying three contiguous xylanase genes that may comprise a xylanosome operon. The enzymes with the highest activities at alkaline pH from the initial activity screening were characterized biochemically. These enzymes showed a broad range of enzyme activities from pH 5.0 to 10.0, with pH optimal of 8.0 retaining more than 70% of their respective activities at pH 9.0. The optimal temperatures of these enzymes ranged from $50^{\circ}C$ to $55^{\circ}C$. This study provides evidence for the diversity and function of lignocellulose-degrading enzymes in the termite gut microbial community, which could be of potential use for industrial processes such as pulp biobleaching and denim biostoning.

• Fisheries and Aquatic Sciences
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• v.16 no.4
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• pp.311-318
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• 2013

The gene encoding an esterase from Photobacterium sp. MA1-3 was cloned in Escherichia coli using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (948 bp) corresponded to a protein of 315 amino acid residues with a molecular weight of 35 kDa and a pI of 6.06. The deduced protein showed 74% and 68% amino acid sequence identities with the putative esterases from Photobacterium profundum SS9 and Photobacterium damselae, respectively. Absence of a signal peptide indicated that it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-X-S-X-G included in most serine-esterases and lipases. The MA1-3 esterase was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at $18^{\circ}C$. The enzyme was a serine-esterase and was active against $C_2$, $C_4$, $C_8$ and $C_{10}$ p-nitrophenyl esters. The optimum pH and temperature for enzyme activity were pH 8.0 and $30^{\circ}C$, respectively. Relative activity remained up to 45% even at $5^{\circ}C$ with an activation energy of 7.69 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was inhibited by $Cd^{2+}$, $Cu^{2+}$, $Zn^{2+}$, and $Hg^{2+}$ ions.

• Journal of Plant Biotechnology
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• v.37 no.2
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• pp.153-165
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• 2010

Brassica rapa is considered an ideal candidate to act as a reference species for Brassica genomic studies. Among the three basic Brassica species, B. rapa (AA genome) has the smallest genome (529 Mbp), compared to B. nigra (BB genome, 632 Mbp) and B. oleracea (CC genome, 696 Mbp). There is also a large collection of available cultivars of B. rapa, as well as a broad array of B. rapa genomic resources available. Under international consensus, various genomic studies on B. rapa have been conducted, including the construction of a physical map based on 22.5X genome coverage, end sequencing of 146,000 BACs, sequencing of >150,000 expressed sequence tags, and successful phase 2 shotgun sequencing of 589 euchromatic region-tiling BACs based on comparative positioning with the Arabidopsis genome. These sequenced BACs mapped onto the B. rapa genome provide beginning points for genome sequencing of each chromosome. Applying this strategy, all of the 10 chromosomes of B. rapa have been assigned to the sequencing centers in seven countries, Korea, UK, China, India, Canada, Australia, and Japan. The two longest chromosomes, A3 and A9, have been sequenced except for several gaps, by NAAS in Korea. Meanwhile a China group, including IVF and BGI, performed whole genome sequencing with Illumina system. These Sanger and NGS sequence data will be integrated to assemble a draft sequence of B. rapa. The imminent B. rapa genome sequence offers novel insights into the organization and evolution of the Brassica genome. In parallel, the transfer of knowledge from B. rapa to other Brassica crops would be expected.

• Korean Journal of Soil Science and Fertilizer
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• v.34 no.5
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• pp.333-341
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• 2001

The Compost-decomposing-bacteria was isolated from livestock compost containing sawdust. The isolated bacteria was identified as Bacillus subtilis LYH201 by the method of the composition of the fatty acid with MIDI system and Bergey's manual. Cloning of CMCase encoding gene was accompanied by shotgun method. The pLK100 have yellow activity ring on CMC medium, that was carried 2.2 kb insert DNA in pBluescript II $SK^+$ vector, named BglC gene. The BglC was very similar to Pectobacterium carotovorum Gun_CLOAB(P15704) with score of 57% identity and 71% homology over 508 aa. The BglC was measured molecular weight 56 kDa by CMC-SDS-PAGE. Optimum cellulase activity Bacillus subtilis LYH201 was temperature $50^{\circ}C$ and pH 7.

• Journal of Trauma and Injury
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• v.31 no.2
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• pp.82-86
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• 2018

Craniocerebral gunshot injuries (CGIs) are extremely seldom happened in Korea because possession of individual firearm is illegal. So, CGIs are rarely encountered by Korean neurosurgeons or Korean trauma surgeons, though in other developing countries or Unites states of America their cases are indefatigably increasing. Management goal should focus on early aggressive, vigorous resuscitation. The treatments consist of immediate life salvage through correction of coagulopathy, intracranial decompression, prevention of infection and preservation of nervous tissue. There have been few studies involving penetrating CGIs in Korea. Here we present a case of penetrating gunshot wound in Korea. We present a 58-year-old man who was unintentionally shot by his colleague with a shotgun. The patients underwent computed tomography (CT) for assessment of intracranial injury. The bullet passed through the left parietal bone and right lateral ventricle and exited through the posterior auricular right temporal bone. After CT scan, he arrested and the cardiopulmonary resuscitation was conducted immediately. But we were unable to resuscitate him. This case report underscores the importance of the initial clinical exam and CT studies along with adequate resuscitation to make the appropriate management decision. Physicians should be familiar with the various injury patterns and imaging findings which are poor prognostic indicators.

• Korean Journal of Environmental Biology
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• v.38 no.3
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• pp.424-432
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• 2020

The present study aimed to analyze the metaproteome of the microbial community comprising harmful algal bloom (HAB) in the Daechung reservoir, Korea. HAB samples located at GPS coordinates of 36°29'N latitude and 127°28'E longitude were harvested in October 2013. Microscopic observation of the HAB samples revealed red signals that were presumably caused by the autofluorescence of chlorophyll and phycocyanin in viable cyanobacteria. Metaproteomic analysis was performed by a gelbased shotgun proteomic method. Protein identification was conducted through a two-step analysis including a forward search strategy (FSS) (random search with the National Center for Biotechnology Information (NCBI), Cyanobase, and Phytozome), and a subsequent reverse search strategy (RSS) (additional Cyanobase search with a decoy database). The total number of proteins identified by the two-step analysis (FSS and RSS) was 1.8-fold higher than that by one-step analysis (FSS only). A total of 194 proteins were assigned to 12 cyanobacterial species (99 mol%) and one green algae species (1 mol%). Among the species identified, the toxic microcystin-producing Microcystis aeruginosa NIES-843 (62.3%) species was the most dominant. The largest functional category was proteins belonging to the energy category (39%), followed by metabolism (15%), and translation (12%). This study will be a good reference for monitoring ecological variations at the meta-protein level of aquatic microalgae for understanding HAB.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1993-2005
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• 2016

Bacillus pumilus is one of the most characterized microorganisms that are used for high-level production of select industrial enzymes. A novel B. pumilus SCU11 strain possessing high alkaline protease activity was obtained in our previous work. The culture supernatant of this strain showed efficient dehairing capability with minimal collagen damage, indicating promising potential applications in the leather industry. In this study, the strain's extracellular proteome was identified by LC-MS/MS-based shotgun proteomic analysis, and their related secretory pathways were characterized by BLAST searches. A total of 513 proteins, including 100 actual secreted and 413 intracellular proteins, were detected in the extracellular proteome. The functions of these secreted proteins were elucidated and four complete secretory systems (Sec, Tat, Com, and ABC transporter) were proposed for B. pumilus. These data provide B. pumilus a comprehensive extracellular proteome profile, which is a valuable theoretical and applicative basis for future genetic modifications and development of industrial enzymes.

• Journal of Microbiology and Biotechnology
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• v.27 no.7
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• pp.1345-1358
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• 2017

The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N-acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1714-1723
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• 2012

${\beta}$-Glucosidase is necessary for the bioconversion of glycosidic phytochemicals in food. Two Bifidobacterium strains (Bifidobacterium animalis subsp. lactis SH5 and B. animalis subsp. lactis RD68) with relatively high ${\beta}$-glucosidase activities were selected among 46 lactic acid bacteria. A ${\beta}$-glucosidase gene (bbg572) from B. lactis was shotgun cloned, fully sequenced, and analyzed for its transcription start site, structural gene, and deduced transcriptional terminator. The structural gene of bbg572 was 1,383 bp. Based on amino sequence similarities, bbg572 was assigned to family 1 of the glycosyl hydrolases. To overexpress bbg572 in Bifidobacterium, several bifidobacteria expression vectors were constructed by combining several promoters and a terminator sequence from different bifidobacteria. The maximum activity of recombinant Bbg572 was achieved when it was expressed under its own promoter and terminator. Its enzyme activity increased 31-fold compared with those of its parental strains. The optimal pH for Bbg572 was pH 6.0. Bbg572 was stable at $37-40^{\circ}C$. It hydrolyzed isoflavones, quercetins, and disaccharides with various ${\beta}$-glucoside linkages. Bbg572 also converted the ginsenosides Rb1 and Rb2. These results suggest that this new ${\beta}$-glucosidase-positive Bifidobacterium transformant can be utilized for the production of specific aglycone products.