• Journal of Microbiology and Biotechnology
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• 제19권3호
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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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• 제28권5호
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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.

• 미생물학회지
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• 제46권2호
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• pp.207-212
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• 2010

전통 발효식품인 된장으로부터 mannanase의 생산균으로 분리된 WL-8 균주는 형태적 특성, 생화학적 성질 및 16S rRNA의 염기서열에 근거하여 Bacillus subtilis로 동정되었다. B. subtilis WL-8은 locust bean gum 보다는 밀기울이 첨가된 LB 배지에서 mannanase 생산성이 높았으며, 24시간 배양하였을 때 약 20 U/ml에 이르렀다. 분리균 WL-8의 mannanase 유전자를 클로닝하여 그 염기서열을 결정한 결과 mannanase 유전자는 362 아미노산으로 구성된 단백질을 코드하며 1,086 뉴클레오티드로 이루어졌다. 아미노산 잔기배열을 분석한 결과 WL-8의 mannanase는 GH family 26에 속하며 B. subtilis의 mannanases와 매우 상동성이 높았다. B. subtilis WL-8의 mannanase 유전자를 함유한 재조합 대장균의 배양상등액과 균체파쇄상등액을 사용하여 반응특성을 조사한 결과 pH 5.5와 $60^{\circ}C$에서 최대 반응활성을 보였으며, $60^{\circ}C$보다 높은 온도에서 배양상등액보다는 균체파쇄상등액에 존재하는 mannanase가 더 높은 활성을 보였다.

• Journal of Microbiology and Biotechnology
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• 제28권12호
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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.

• 생명과학회지
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• 제22권7호
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• pp.912-919
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• 2012

Carboxymethylcellulose (CM-cellulose)와 Beechwood xylan을 각각 기질로 사용하여 trypan blue를 첨가하여 제작한 Agar-LB 배지 상에서 명확한 활성환을 형성하는 균주를 cellulase와 xylanase 생산 균주로 단리하였다. 단리한 균주 유래의 16S rRNA 유전자 및 API 50 kit를 분석한 결과 Bacillus subtilis와 약 99.5%의 높은 상동성을 보였기에 본 균주를 Bacillus subtilis로 동정하여 B. subtilis NC1로 명명하였다. B. subtilis NC1 유래 cellulase와 xylanase는 CM-cellulose와 Beechwood xylan에 대하여 각각 높은 효소 활성을 보였으며, 두 효소 모두 pH 5.0과 $50^{\circ}C$의 조건하에서 가장 높은 효소 활성을 보였다. B. subtilis NC1 균주 유래 cellulase와 xylanase 유전자를 cloning하기 위하여 shot-gun cloning 방법을 이용하여 B. subtilis NC1 염색체 DNA로부터 효소 유전자를 cloning하여 유전자 배열을 규명한 결과 cellulase 유전자는 아미노산 499개를 암호화하는 1,500 bp의 open reading frame (ORF)으로 이루어져 있었으며, 아미노산 배열로부터 추정되는 분자량은 55,251 Da 이었다. 그리고, xylanase에 대한 유전자는 아미노산 422개를 암호화하는 1,269 bp의 ORF로 이루어져 있었으며 유전자 유래 아미노산 배열로부터 추정되는 단백질 분자량은 47,423 Da 이었다. 두 효소의 아미노산 배열을 이용하여 상동성을 검토한 결과 cellulase는 glycoside hydrolase family (GH) 5에 속하는 cellulase와 xylanase는 GH30에 속하는 xylanase와 높은 상동성을 나타내었다.

• Journal of Plant Biotechnology
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• 제45권3호
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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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• 제24권11호
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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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• 제45권1호
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• pp.13-22
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• 2003

성장호르몬 유전자는 하나의 작은 공통 선조 유전자로부터 아주 오랜 기간동안 유전자 중복에 의해 진화되어 온 그룹들 중의 하나이다. 이들에 속하는 유전자들은 동물 종간에 구조적인 상동성과 기능적 공통성 등 유사성이 비교적 높게 나타난다. 이런 연구결과들을 근거로 하여 소 성장호르몬 유전자에서 아미노산을 암호화하는 영역으로부터 새로운 아미노산의 변이(missense mutation)를 검출하였고, 이 변이의 대립유전자 빈도는 소(cattle)의 종(species) 및 품종의 지리적 분포에 따라 일정한 경향 치를 보여 주었다. 한편 변경되어진 아미노산은 Tryptophan으로 이는 생물체에 존재하는 많은 단백질들을 구성하는 아미노산중에서도 그 출현빈도가 가장 낮은 것이다. 또한 검출된 변이는 성장호르몬이 그의 수용체와 강하게 결합하는 부위로서, 성장호르몬의 구조적 변이를 초래하여 수용체와의 결합이 비정상적으로 이루어져, 이후 성장호르몬이 표적세포로의 신호전달과 같은 역할을 제대로 수행치 못하게 되고, 이로 인하여 가축의 표현되어지는 경제형질에 영향을 미칠 것으로 추정된다. 그러므로 이러한 대립유전자를 보유하는 개체는 집단에서 제거하는 방법에 의한 개량이 가능할 것으로 사료된다.

• Journal of Preventive Medicine and Public Health
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• 제37권2호
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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.

• 미생물학회지
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• 제42권4호
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• pp.313-318
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• 2006

가정에서 제조된 된장으로부터 cellulase 생산균으로 분리된 고온성 WL-12는 형태적 특성, 생화학적 성질 및 16S rRNA의 염기서열에 근거하여 Bacillus licheniformis로 동정되었다. B. licheniformis WL-12의 cellulase 유전자를 클로닝하여 그 염기서열을 결정한 결과 cellulase 유전자(celA)는 517 아미노산으로 구성된 단백질을 코드하며 1,551 뉴클레오티드로 이루어졌다. 아미노산 잔기배열을 분석한 결과 WL-12의 cellulase는 활성영역과 cellulose 결합영역으로 구성되어 있었으며, glycosyl hydrolase (GH) family 5에 속하는 B. licheniformis, B. subtilis와 B. amyloliquefaciens의 cellulase와 높은 상동성을 보였다. 클론된 celA를 발현용 vector에 도입하여 B. subtilis에서 발현시켜 cellulase 최대생산성이 7.0 units/ml에 이르렀다.