• Journal of Microbiology and Biotechnology
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• 제24권1호
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• pp.19-25
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• 2014

An extracellular agarase was purified from Bacillus sp. BI-3, a thermophilic agar-degrading bacterium isolated from a hot spring in Indonesia. The purified agarase revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 58 kDa. The optimum pH and temperature of the agarase were 6.4 and $70^{\circ}C$, respectively. The activity of the agarase was stable at high temperatures, and more than 50% activity was retained at $80^{\circ}C$ for 15 min. Furthermore, the enzyme was stable in the pH range of 5.8-8.0, and more than 60% of the residual activity was retained. Significant activation of the agarase was observed in the presence of $K^+$, $Na^+$, $Ca^{2+}$, $Mg^{2+}$, and $Sr^{2+}$; on the other hand, $Ba^{2+}$, $Zn^{2+}$, $Cu^{2+}$, $Mn^{2+}$, $Co^{2+}$, $Fe^{2+}$, and EDTA inhibited or inactivated the enzyme activity. The components of the hydrolytic product analyzed by thin-layer chromatography showed that the agarase mainly produced neoagarobiose. This study is the first to present evidence of agarolytic activity in aerobic thermophilic bacteria.

• 생명과학회지
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• 제31권7호
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• pp.641-653
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• 2021

본 연구에서는 근권토양으로부터 18종의 세균을 순수분리하고, 이들의 식물 병원성 진균 생육억제 활성, 식물생장촉진 활성 및 미네랄 가용화능을 평가하였다. Bacillus 속과 Pseudomonas 속 분리균주의 항진균 활성을 통해 생물학적 방제제로서의 가능성을 확인할 수 있었으며, 이는 식물 병원성 진균의 세포벽에 작용하는 여러 가수분해효소 활성과 siderophore 생성능 등에 기인된 것으로 판단된다. 또한 대부분의 분리균주가 aminocyclopropane-1-carboxylate deaminase 생성능, indole-3-acetic acid 생성능 및 질소 고정능을 갖고 있는 것으로 나타났으며, 이러한 특성은 식물이 흔히 노출될 수 있는 환경 스트레스 조건 하에서 스트레스 에틸렌의 농도를 감소시킴으로써 뿌리 발달 및 성장, 그리고 작물의 생산성에 긍정적인 영향을 줄 것으로 판단된다. 그리고 분리균주의 인산, 규소, 탄산칼슘, 아연 가용화능 등을 확인한 결과, 일부 분리균주들의 미네랄 가용화능을 확인하였으며, 이들 분리균주를 식물 생장 시에 접종한다면 식물 생장에 필요한 영양분을 식물이 흡수할 수 있는 이용가능한 형태로 변환시켜 식물의 생장에 도움을 줄 수 있을 것으로 기대된다. 이러한 항진균 활성, 식물생장촉진 활성 및 미네랄 가용화능의 결과를 통해 분리균주 18종의 biocontrol agent로서의 이용가능성을 제시하고자 한다.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.764-769
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• 2000

The lipase gene(lip) from Bacillus stearothermophilus was recombined in vitro by utilizing the DNA shuffling technique. After four rounds of shuffling, transformation, and screening based on the initial rate of clear zone formation on a tricaprylin plate, a clone (M10) was isolated, the cell extract of which showed about 2.8-fold increased lipase activity. The DNA sequence of the mutant lipase gene (m10) showed 3 base changes, resulting in two cryptic mutations and one amino acid substitution: S113($AGC{\rightarrow}AGT$), L252 ($TTG{\rightarrow}TTA$), and G275E ($GGA{\rightarrow}GAA$). SDS-PAGE analysis revealed that the increased enzyme activity observed in M10 was partly caused by high expression of the m10 lipase gene. The amount of the expressed G275E lipase was estimated to comprise as much as 41% of the total soluble proteins of the cell. The maximum velocity ($V_{max}$) of the purified mutant enzyme for the hydrolysis of olive oil was measured to be 3,200 U/mg, which was 10% higher than that of the parental (WT) lipase (2,900 U/mg). Its optimum temperature for the hydrolysis of olive oil was $68^{\circ}C$ and it showed a typical $Ca^{2+}$-dependent thermostability, properties fo which were the same as those of the WT lipase. However, the mutant enzyme exhibited a high enantiospecificity towards (S)-naproxen compared with the WT lipase. In addition, it showed increased hydrolytic activity towards triolein, tricaprin, tricaprylin, and tricaproin.

• The Plant Pathology Journal
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• 제22권4호
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• pp.323-328
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• 2006

Serratia marcescens W1, isolated from cucumber-cultivated soil in Suwon, Korea, evidenced profound antifungal activity and produced the extracellular hydrolytic enzymes, chitinase and protease. In order to isolate the antifungal genes from S. marcescens W1, a cosmid genomic library was constructed and expressed in Escherichia coli. Transformants exhibiting chitinase and protease expression were selected, as well as those transformants evidencing antifungal effects against the rice blast fungus, Magnaporthe grisea, and the cucumber leaf spot fungus, Cercospora citrullina. Cosmid clones expressing chitinase or protease exerted no inhibitory effects against the growth of fungal pathogens. However, two cosmid clones evidencing profound antifungal activities were selected for further characterization. An 8.2 kb HindIII fragment from these clones conditioned the expression of antagonistic activity, and harbored seven predicted complete open reading frames(ORFs) and two incomplete ORFs. The deduced amino acid sequences indicated that six ORFs were highly homologous with genes from S. marcescens generating pyrroloquinoline quinone(PQQ). Only subclones harboring the full set of pqq genes were shown to solubilize insoluble phosphate and inhibit fungal pathogen growth. The results of this study indicate that the functional expression of the pqq genes of S. marcescens W1 in E. coli may be involved in antifungal activity, via as-yet unknown mechanisms.

• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.771-780
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• 2014

A putative lipolytic enzyme gene, named as est9x, was obtained from a marine microbial metagenome of the South China Sea. Sequence analysis showed that Est9X shares lower than 27% sequence identities with the characterized lipolytic enzymes, but possesses a catalytic triad highly conserved in lipolytic enzymes of the ${\alpha}/{\beta}$ hydrolase superfamily. By phylogenetic tree construction, Est9X was grouped into a new lipase/esterase family. To understand Est9X protein in depth, it was recombinantly expressed, purified, and biochemically characterized. Within potential hydrolytic activities, only lipase/esterase activity was detected for Est9X, confirming its identity as a lipolytic enzyme. When using p-nitrophenol esters with varying lengths of fatty acid as substrates, Est9X exhibited the highest activity to the C2 substrate, indicating it is an esterase. The optimal activity of Est9X occurred at a temperature of $65^{\cric}C$, and Est9X was pretty stable below the optimum temperature. Distinguished from other salt-tolerant esterases, Est9X's activity was tolerant to and even promoted by as high as 4 M NaCl. Our results imply that Est9X is a unique esterase and could be a potential candidate for industrial application under extreme conditions.

• Journal of Ginseng Research
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• 제37권1호
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• pp.80-86
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• 2013

Korean red ginseng has been shown to possess a variety of biological activities. However, little is known about antiviral activity of ginsenosides of Korean red ginseng. Here, we investigated the protective effect by oral administration of various ginsenosides on the lethal infection of haemagglutinating virus of Japan (HVJ) in mice. In a lethal infection model in which almost all mice infected with HVJ died within 15 days, the mice were administered orally (per os) with 1 mg/mouse of dammarane-type (ginsenoside-Rb1, -Rb2, -Rd, -Re, and -Rg2) or oleanolic acid-type (ginsenoside-Ro) ginsenosides 3, 2, and 1 d before virus infection. Ginsenoside-Rb2 showed the highest protective activity, although other dammarane-type and oleanolic acid-type ginsenosides also induced a significant protection against HVJ. However, neither the consecutive administration with a lower dosage (300 ${\mu}g$/mouse) nor the single administration of ginsenoside-Rb2 (1 mg/mouse) was active. In comparison of the protective activity between ginsenoside-Rb2 and its two hydrolytic products [20(S)- and 20(R)-ginsenoside-Rg3], 20(S)-ginsenoside-Rg3, but not 20(R)-ginsenoside-Rg3, elicited a partial protection against HVJ. The protective effect of ginsenoside-Rb2 and 20(S)-ginsenoside-Rg3 on HVJ infection was confirmed by the reduction of virus titers in the lungs of HVJ-infected mice. These results suggest that ginsenoside-Rb2 is the most effective among ginsenosides from red ginseng to prevent the lethal infection of HVJ, so that this ginsenoside is a promising candidate as a mucosal immunoadjuvant to enhance antiviral activity.

• 식물조직배양학회지
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• 제21권6호
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• pp.357-362
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• 1994

키틴 가수분해 효소와 베타-1,3-글루카네이즈는 식물체의 주요 방어효소로 여겨지고 있다. 본 논문에서는 아기장대풀의 잎조직으로부터 고정된 현탁배양주를 만들고 이어서 에틸렌과 유인제 (elicitor) 처리에 의한 키틴 가수분해 효소유도양상을 분석하였다. 키틴 가수분해 효소활성은 $^3$H으로 표지된 키토산을 기질로 한 radiochemical 분석방법이나 담배식물체의 키틴 가수분해 효소를 대상으로 얻어진 항체를 이용하여 Western 분석방법을 사용하였다. 이 결과 에틸렌과 유인제 처리 공히 48시간후에 가장 활성이 높게 나타남을 관찰하였다. 또한 씨앗, 새싹, 식물체의 잎, 뿌리 등에서의 활성을 조사하여 뿌리에서 키틴 가수분해 효소 활성이 높음을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제20권11호
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• pp.1577-1584
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• 2010

In total, 140 halotolerant bacterial strains were isolated from both the soil of barren fields and the rhizosphere of six naturally growing halophytic plants in the vicinity of the Yellow Sea, near the city of Incheon in the Republic of Korea. All of these strains were characterized for multiple plant growth promoting traits, such as the production of indole acetic acid (IAA), nitrogen fixation, phosphorus (P) and zinc (Zn) solubilization, thiosulfate ($S_2O_3$) oxidation, the production of ammonia ($NH_3$), and the production of extracellular hydrolytic enzymes such as protease, chitinase, pectinase, cellulase, and lipase under in vitro conditions. From the original 140 strains tested, on the basis of the latter tests for plant growth promotional activity, 36 were selected for further examination. These 36 halotolerant bacterial strains were then tested for 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. Twenty-five of these were found to be positive, and to be exhibiting significantly varying levels of activity. 16S rRNA gene sequencing analyses of the 36 halotolerant strains showed that they belong to 10 different bacterial genera: Bacillus, Brevibacterium, Planococcus, Zhihengliuella, Halomonas, Exiguobacterium, Oceanimonas, Corynebacterium, Arthrobacter, and Micrococcus. Inoculation of the 14 halotolerant bacterial strains to ameliorate salt stress (150 mM NaCl) in canola plants produced an increase in root length of between 5.2% and 47.8%, and dry weight of between 16.2% and 43%, in comparison with the uninoculated positive controls. In particular, three of the bacteria, Brevibacterium epidermidis RS15, Micrococcus yunnanensis RS222, and Bacillus aryabhattai RS341, all showed more than 40% increase in root elongation and dry weight when compared with uninoculated salt-stressed canola seedlings. These results indicate that certain halotolerant bacteria, isolated from coastal soils, have a real potential to enhance plant growth under saline stress, through the reduction of ethylene production via ACC deaminase activity.

• Journal of Microbiology and Biotechnology
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• 제32권4호
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• pp.447-457
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• 2022

Notoginsenoside R1 and ginsenoside Rg1 are the main active ingredients of Panax notoginseng, exhibiting anti-fatigue, anti-tumor, anti-inflammatory, and other activities. In a previous study, a GH39 β-xylosidase Xln-DT was responsible for the bioconversion of saponin, a natural active substance with a xylose group, with high selectivity for cleaving the outer xylose moiety of notoginsenoside R1 at the C-6 position, producing ginsenoside Rg1 with potent anti-fatigue activity. The optimal bioconversion temperature, pH, and enzyme dosage were obtained by optimizing the transformation conditions. Under optimal conditions (pH 6.0, 75℃, enzyme dosage 1.0 U/ml), 1.0 g/l of notoginsenoside R1 was converted into 0.86 g/l of ginsenoside Rg1 within 30 min, with a molar conversion rate of approximately 100%. Furthermore, the in vivo anti-fatigue activity of notoginsenoside R1 and ginsenoside Rg1 were compared using a suitable rat model. Compared with the control group, the forced swimming time to exhaustion was prolonged in mice by 17.3% in the Rg1 high group (20 mg/kg·d). Additionally, the levels of hepatic glycogen (69.9-83.3% increase) and muscle glycogen (36.9-93.6% increase) were increased. In the Rg1 group, hemoglobin levels were also distinctly increased by treatment concentrations. Our findings indicate that treatment with ginsenoside Rg1 enhances the anti-fatigue effects. In this study, we reveal a GH39 β-xylosidase displaying excellent hydrolytic activity to produce ginsenoside Rg1 in the pharmaceutical and food industries.

• 생명과학회지
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• 제22권2호
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• pp.251-258
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• 2012

본 연구는 동물의 분변으로부터 분리한 유산균의 프로바이오틱 특성을 조사할 목적으로 시행되었다. 생리생화학적 특성과 16S 리보솜 DNA 분석 결과 BCNU 9041, 9042는 Lactobacillus brevis와 99%의 상동성을 나타내었다. 이들 균주를 대상으로 기초적인 안전성 실험을 시행한 결과, 이들 균주는 용혈현상이 나타나지 않으며 ${\beta}$-glucosidase, ${\beta}$-glucuronidase, tryptophanase 및 urease와 같은 유해한 생성물을 생성하지 않는 안전한 생물자 원임이 확인되었다. BCNU 9041 및 9042은 pH 2.5의 산성 조건 및 담즙에서(0.3, 0.6, 1%의 oxgall이 포함된 MRS broth) 높은 생존률을 나타내었다. 뿐만 아니라 식중독 원인 세균에 대하여 항균활성을 가지고 있으며, 특히 Bacillus cereus, Listeria monocytogenes 및 Shigella sonnei 에 대한 항균활성이 뛰어났다. 또한 BCNU 9041, BCNU 9042은 92-95%의 높은 소수성과 BSH (bile salt hydrolytic) 활성 및 cholesterol 흡수력이 우수함이 확인되었다. 이들 결과를 바탕으로 프로바이오틱로서의 우수한 기능성을 가진 BCNU 9041와 BCNU 9042의 기능성 식품 및 건강관련 제품으로의 활용이 기대된다.