• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.257-264
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• 2009

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

• Journal of Microbiology and Biotechnology
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• v.28 no.5
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• pp.776-783
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• 2018

The agarase gene gaa16a was identified from a draft genome sequence of Gilvimarinus agarilyticus JEA5, an agar-utilizing marine bacterium. Recently, three agarase-producing bacteria, G. chinensis, G. polysaccharolyticus, and G. agarilyticus, in the genus Gilvimarinus were reported. However, there have been no reports of the molecular characteristics and biochemical properties of these agarases. In this study, we analyzed the molecular characteristics and biochemical properties of agarases in Gilvimarinus. Gaa16A comprised a 1,323-bp open reading frame encoding 441 amino acids. The predicted molecular mass and isoelectric point were 49 kDa and 4.9, respectively. The amino acid sequence of Gaa16A showed features typical of glycosyl hydrolase family 16 (GH16) ${\beta}$-agarases, including a GH16 domain, carbohydrate-binding region (RICIN domain), and signal peptide. Recombinant Gaa16A (excluding the signal peptide and carbohydrate-binding region, rGaa16A) was expressed as a fused protein with maltose-binding protein at its N-terminus in Escherichia coli. rGaa16A had maximum activity at $55^{\circ}C$ and pH 7.0 and 103 U/mg of specific activity in the presence of 2.5 mM $CaCl_2$. The enzyme hydrolyzed agarose to yield neoagarotetraose as the main product. This enzyme may be useful for industrial production of functional neoagaro-oligosaccharides.

• Korean Journal of Microbiology
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• v.46 no.2
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• pp.207-212
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• 2010

A bacterium producing the extracellular mannanase was isolated from Korean soybean paste. The isolate WL-8 has been identified as Bacillus subtilis on the basis on its 16S rRNA sequence, morphology and biochemical properties. The mannanase productivity of strain WL-8 was increased in LB broth by addition of wheat bran. The maximum mannanase productivity was reached to approximately 20 U/ml in LB medium supplemented with 6% wheat bran. A gene encoding the mannanase of WL-8 was cloned into Escherichia coli and its nucleotide sequence was subsequently determined. The mannanase gene consisted of 1,086 nucleotides encoding a polypeptide of 362 amino acid residues. The deduced amino acid sequence was highly homologous with those of several mannanases from B. subtilis belonging to GH family 26. Reaction temperature and pH profiles were investigated using the culture filtrate and cell-free extract of the recombinant E. coli carrying a WL-8 mannanase gene, respectively. Optimal conditions for the two fractions occurred at pH 5.5 and $60^{\circ}C$. The cell-free extract showed higher mannanase activity than the culture filtrate at above $60^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2036-2045
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel5L, was cloned using the shot-gun method from Bacillus sp.. The gene, which contained a predicted signal peptide, encoded a protein of 496 amino acid residues, and the molecular mass of the mature Cel5L was estimated to be 51.8 kDa. Cel5L contained a catalytic domain of glycoside hydrolase (GH) family 5 and a carbohydrate-binding module family 3 (CBM_3). Chromatography using HiTrap Q and CHT-II resulted in the isolation of two truncated forms corresponding to 50 (Cel5L-p50) and 35 kDa (Cel5L-p35, CBM_3-deleted form). Both enzymes were optimally active at pH 4.5 and $55^{\circ}C$, but had different half-lives of 4.0 and 22.8 min, respectively, at $70^{\circ}C$. The relative activities of Cel5L-p50 and Cel5L-p35 for barley ${\beta}$-glucan were 377.0 and 246.7%, respectively, compared to those for carboxymethyl-cellulose. The affinity and hydrolysis rate of pNPC by Cel5L-p35 were 1.7 and 3.3 times higher, respectively, than those by Cel5L-p50. Additions of each to a commercial enzyme set increased saccharification of pretreated rice straw powder by 17.5 and 21.0%, respectively. These results suggest CBM_3 is significantly contributing to thermostability, and to affinity and substrate specificity for small substrates, and that these two enzymes could be used as additives to enhance enzymatic saccharification.

• Journal of Life Science
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• v.22 no.7
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• pp.912-919
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• 2012

A Bacillus sp. strain producing celluase and xylanase was isolated from environmental soil with LB agar plate containing carboxymethylcellulose (CM-cellulose) and beechwood xylan stained with trypan blue as substrates, respectively. Based on the 16S rRNA gene sequence and API 50 CHL test, the strain was identified as B. subtilis and named B. subtilis NC1. The cellulase and xylanase from B. subtilis NC1 exhibited the highest activities for CM-cellulose and beechwood xylan as substrate, respectively, and both enzymes showed the maximum activity at pH 5.0 and $50^{\circ}C$. We cloned and sequenced the genes for cellulase and xylanase from genomic DNA of the B. subtilis NC1 by the shot-gun cloning method. The cloned cellulase and xylanase genes consisted of a 1,500 bp open reading frame (ORF) encoding a 499 amino acid protein with a calculated molecular mass of 55,251 Da and a 1,269 bp ORF encoding a 422 amino acid protein with a calculated molecular mass of 47,423 Da, respectively. The deduced amino acid sequences from the genes of cellulase and xylanase showed high identity with glycosyl hydrolases family (GH) 5 and 30, respectively.

• Journal of Plant Biotechnology
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• v.45 no.3
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• pp.183-189
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• 2018

The plant hormone auxin regulates the overall metabolic processes essential for plant growth and development. Auxin signaling is mediated by early auxin response genes, which are classified into three major families: AUXIN/INDOLE ACETIC ACID (AUX/IAA), GRETCHEN HAGEN3 (GH3) and SMALL AUIN UP RNA (SAUR). The SAUR gene family is the largest family among early auxin response genes and encodes the small and highly unstable gene products. The functional roles of SAUR genes have remained unclear for many years. The traditional genetic and molecular studies on the SAUR functions have been hampered by their likely genetic redundancy and tandem arrays of highly related genes in the plant genome, together with the molecular characteristics of SAUR. However, recent studies have suggested possible roles of SAUR in a variety of tissues and developmental stages in accordance with the novel approaches such as gain-of-function and RNA silencing techniques. In this review, the recent research progress on the functional roles and regulatory mechanisms of SAUR and a set of possible future works are discussed.

• Journal of Microbiology and Biotechnology
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• v.24 no.11
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• pp.1525-1535
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• 2014

The xynC gene, which encodes high molecular weight xylanase from Paenibacillus sp. DG-22, was cloned and expressed in Escherichia coli, and its nucleotide sequence was determined. The xynC gene comprised a 4,419bp open reading frame encoding 1,472 amino acid residues, including a 27 amino acid signal sequence. Sequence analysis indicated that XynC is a multidomain enzyme composed of two family 4_9 carbohydrate-binding modules (CBMs), a catalytic domain of family 10 glycosyl hydrolases, a family 9 CBM, and three S-layer homologous domains. Recombinant XynC was purified to homogeneity by heat treatment, followed by Avicel affinity chromatography. SDS-PAGE and zymogram analysis of the purified enzyme identified three active truncated xylanase species. Protein sequencing of these truncated proteins showed that all had identical N-terminal sequences. In the protein characterization, recombinant XynC exhibited optimal activity at pH 6.5 and $65^{\circ}C$ and remained stable at neutral to alkaline pH (pH 6.0-10.0). The xylanase activity of recombinant XynC was strongly inhibited by 1 mM $Cu^{2+}$ and $Hg^{2+}$, whereas it was noticeably enhanced by 10 mM dithiothreitol. The enzyme exhibited strong activity towards xylans, including beechwood xylan and arabinoxylan, whereas it showed no cellulase activity. The hydrolyzed product patterns of birchwood xylan and xylooligosaccharides by thin-layer chromatography confirmed XynC as an endoxylanase.

• Journal of Animal Science and Technology
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• v.45 no.1
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• pp.13-22
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• 2003

Growth Hormone (GH) gene is a member of gene family through the evolutionary process from a small common ancestral gene by a series of gene duplications. The role of the GH in growth and performance controls has been extensively studied in human, mice and livestock. Many researchers have considered GH as a strong candidate gene for evaluation of genetic polymorphisms that could be associated with economic traits in cattle. We report here a novel missense mutation within the exon 5 of the bovine Growth Hormone (bGH) gene. We could amplified 522 bp fragments from eight unrelated Hanwoo cattle by PCR, then, subsequently cloned and sequenced. An Msp I RFLP corresponding to a C to T transition was observed at position 2258 nt. From this result, we could predict a missense mutation (Arg to Trp) at codon 166 in a highly conserved region among many mammals. Codominant Mendelian segregation of the two alleles, Msp I (+) and Msp I (－), was observed in two full-sib F2 families (n = 32, African taurine Bos taurus ${\times}$ African zebu Bos indicus) and eight half-sib Hanwoo families. For the availability of genetic marker, we have performed PCR-RFLP with a large number of individual animals from 15 different cattle breeds (European and Asian taurines, and African indicines). Consideration of breed frequencies of Msp I (－) allele in relation to breed type and their geographic origins, shows higher frequencies in humped breeds or Asian cattle breeds than in humpless or European breeds. This result indicates that the missense mutation can be contributed the functional significance such as the signal transduction through the receptor binding, also may be used as a marker for selection of the economic traits in Hanwoo.

• Journal of Preventive Medicine and Public Health
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• v.37 no.2
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• pp.157-165
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• 2004

Objectives : This study aimed to investigate the toxic effects of chromium (VI) on the placental function and reproduction in rats. For the study, the placental prolactin-growth hormone (PRL-GH) gene expression, placental trophoblast cell differentiation and reproductive data were analyzed. Methods : The pregnancies of F344 Fisher rats were checked by the presence of a copulatory plug or sperm in the vaginal smear, which was defined as day 0 of the pregnancy. Pregnant rats were divided into the three groups. The control group was given tap water (chromium level < 0.001 ppm) and the remaining groups were given 250 or 750 ppm of chromium (VI) [as potassium dichromate], from day 7 to 19 of the pregnancy. Rats were sacrificed at days 11 and 20 of pregnancy. The mRNA levels of PRL-GH and Pit-1a and b isotype genes were analyzed by Northern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR). The hormonal concentration was analyzed by radioimmunoassay, and the differentiation of placental trophoblast cells were observed by histochemical studies. Reproductive data, such as placental and fetal weights, pregnancy period, and litter size, were surveyed at day 20 of pregnancy and after birth. A statistical analysis was carried out using the SAS program (version 8.1). Results : The mRNA levels of the prolactin-growth hormone (PRL-GH) family of genes were dose dependently reduced by chromium exposure. The mRNA levels of Pit-1a and b isotype genes that induce the expression of the PRL-GH family of genes were also reduced by chromium exposure. The PRL-GH hormonal concentration in the rat placenta, fetus and maternal blood were decreased by chromium exposure. In the middle stage of pregnancy (day 11), a high dose of chromium suppressed the differentiation of spongiotrophoblast cells that secret the PRLGH hormones. In the last stage of pregnancy (day 20), a high dose of chromium induced apoptosis of placental cells. Reproductive data, such as placental and fetal weights, litter size, were reduced, but the pregnancy period was extended in the group exposed to chromium compared with the controls. Conclusion : Chromium (VI) disrupts the ordered functions of the placenta, which leads to reproductive disorders in rats.

• Korean Journal of Microbiology
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• v.42 no.4
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• pp.313-318
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• 2006

A thermophilic bacterium producing the extracellular cellulase was isolated from soybean paste, and the isolate WL-12 has been identified as Bacillus licheniformis on the basis on its 16S rRNA sequence, morphology and biochemical properties. A gene encoding the cellulase of B. licheniformis WL-12 was cloned and its nucleotide sequence was determined. This cellulase gene, designated celA, consisted of 1,551 nucleotides, encoding a polypeptide of 517 amino acid residues. The gene product contained catalytic domain and cellulose binding domain. The deduced amino acid sequence was highly homologous to those of cellulases of B. licheniformis, B. subtilis and B. amytoliquefaciens belonging to the glycosyl hydrolase family 5. When the celA gene was highly expressed using a strong B. subtilis promoter, the extracellular cellulase was produced up to 7.0 units/ml in B. subtilis WB700.