• Journal of Microbiology and Biotechnology
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• 제25권5호
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• pp.620-628
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• 2015

A fungal strain, R9, was isolated from the South Atlantic sediment sample and identified as Aspergillus fumigatus. An antifungal protein, AfAFP_R9, was purified from the culture supernatant of Aspergillus fumigatus R9. AfAFP_R9 was identified to be restrictocin, which is a member of the ribosome-inactivating proteins (RIPs), by MALDI-TOF-TOF-MS. AfAFP_R9 displayed antifungal activity against plant pathogenic Fusarium oxysporum, Alternaria longipes, Colletotrichum gloeosporioides, Paecilomyces variotii, and Trichoderma viride at minimum inhibitory concentrations of 0.6, 0.6, 1.2, 1.2, and 2.4 μg/disc, respectively. Moreover, AfAFP_R9 exhibited a certain extent of thermostability, and metal ion and denaturant tolerance. The iodoacetamide assay showed that the disulfide bridge in AfAFP_R9 was indispensable for its antifungal action. The cDNA encoding for AfAFP_R9 was cloned from A. fumigatus R9 by RT-PCR and heterologously expressed in E. coli. The recombinant AfAFP_R9 protein exhibited obvious antifungal activity against C. gloeosporioides, T. viride, and A. longipes. These results reveal the antifungal properties of a RIP member (AfAFP_R9) from marine-derived Aspergillus fumigatus and indicated its potential application in controlling plant pathogenic fungi.

• 한국축산식품학회지
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• 제35권6호
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• pp.867-873
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• 2015

1,4-Dihydroxy-2-naphthoic acid (DHNA), a precursor of menaquinone (vitamin K2), has an effect on growth stimulation of bifidobacteria and prevention of osteoporosis, making it a promising functional food material. Therefore, we tried to clone the menB gene encoding DHNA synthase from Leuconostoc mesenteroides CJNU 0147. Based on the genome sequence of Leu. mesenteroides ATCC 8293 (GenBank accession no., CP000414), a primer set (Leu_menBfull_F and Leu_menBfull_R) was designed for the PCR amplification of menB gene of CJNU 0147. A DNA fragment (1,190 bp), including the menB gene, was amplified, cloned into pGEM-T Easy vector, and sequenced. The deduced amino acid sequence of MenB (DHNA synthase) protein of CJNU 0147 had a 98% similarity to the corresponding protein of ATCC 8293. The menB gene was subcloned into pCW4, a lactic acid bacteria - E. coli shuttle vector, and transferred to CJNU 0147. The transcription of menB gene of CJNU 0147 (pCW4::menB) was increased, when compared with those of CJNU 0147 (pCW4) and CJNU 0147 (−). The DHNA was produced from it at a detectable level, indicating that the cloned menB gene of CJNU 0147 encoded a DHNA synthase which is responsible for the production of DHNA, resulting in an increase of bifidogenic growth stimulation activity.

• Journal of Applied Biological Chemistry
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• 제52권3호
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• pp.109-115
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• 2009

Xoo로 부터 aspartate aminotransferase로 추정되는 유전자를 분리한 다음 발현시켜 생화학 특성을 조사하였다. 분리된 유전자는 His6 pET-21(a) 운반체에 삽입시켰으며 E. coli BL21(DE3)에서 발현시켰다. 재조합된 Asp-AT는 affinity chromatography를 이용하여 분리하였으며 SDS-PAGE분석에서 43kDa의 단일 밴드를 나타내었다. 분리된 효소는 amino donor 로서 L-aspartate에 대하여 효소활성도가 가장 높았고, L-leucine 및 L-cysteine에 대하여서도 상당한 활성도를 나타내었다. 효소의 최적 pH는 약 7.5 근처에서 나타났고 효소의 안정성은 산성조건 보다는 알칼리 조건에서 훨씬 높았다. 최적 온도는 약 $35-40^{\circ}C$로 나타났고 $55^{\circ}C$에서 20분간 열처리한 이후의 잔여 활성도는 약 78%로 나타났다. 여러 중금속 중에서 망간 이온에 의해 효소활성이 촉진되었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.31-32
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• 2005

Neoagaro-oligosaccharides are produced only by enzymatic degradation of agarose by ${\beta}-agarase.^{1)}$ Neoagaro-oligosaccharides inhibit the growth of bacteria, slow the rate of degradation of starch, are used as low-calorie additives to improve food quality, and have macrophage-stimulating activity. Furthermore, neoagarobiose is a rare reagent that has both moisturizing effect on skin and whitening effect on melanoma $cells.^{2)}$ An agar-degrading marine bacterium was isolated from the sea water at the northeast coast in Cheju island, Korea. The strain was gram negative, aerobic, and motile rod. The 16S rRNA of the strain had the closest match of 98% homology, with that from Agarivorans albus. On the basis of several phenotypic characters and a phylogenetic analysis, this strain was designated Agarivorans sp. JA-1. In solid agar plate, Agarivorans sp. JA-1 produced a diffusible agarase that caused agar softening around the colonies. Agarivorans sp. JA-1 was cultured for 36 hr in marine broth 2216 (Difco, USA) and the supernatant that containing an extracellular ${\beta}-agarase$ was prepared by centrifugation of culture media. The enzyme exhibited relatively strong activity at $40^{\circ}C$ and was stable up to $60^{\circ}C$. Using PCR primers derived from the ${\beta}-agarase$ gene of Vibrio sp., the gene encoding ${\beta}-agarase$ from Agarivorans sp. JA-1 was cloned and sequenced. The structural gene consists of 2931 bp encoding 976 amino acids with a predicted molecular weight of 107,360 Da. The deduced amino acid sequence showed 99% and 34% homology to $agaA^{2)}$ and $agaB^{2)}$ genes for ${\beta}-agarase$ from Vibrio sp., respectively. The expression plasmid for ${\beta}-agarase$ gene of Agarivorans sp. JA-1 is being constructed and the recombinant enzyme will be biochemically characterized.

• 한국임상수의학회지
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• 제28권4호
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• pp.427-430
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• 2011

6개월령의 잡종 수컷 고양이가 갑작스런 호흡곤란과 식욕부진 증상으로 내원 하였다. 신체검사상 심음의 감소와 함께 확연한 호흡곤란이 확인 되었다. 혈액학적 검사에서 고글로블린 혈증을 동반한 고단백혈증이 관찰되었으며, 다클론성 감마글로블린병증이 확인 되었다. 환축은 흉부 방사선 검사에서 흉수가 확인 되었으며, 흉수는 비감염성 삼출물로, 흉수 역시 다클론성 감마글로블린병증을 동반한 고글로블린증과 감소한 알브민 대 글로블린 비율이 관찰되었다. 또한 흉강 삼출물을 통한 역전사 중합효소연쇄반응에서 고양이 코로나바이러스에 양성반응을 확인하였다. 따라서, 환축은 고양이 전염성 복막염이 강하게 의심되었으며, 재조합 인간 인터페론 알파, pentoxifylline 과 스테로이드제를 이용한 적극적인 치료가 시작 되었다. 환축은 초기 치료에 반응이 좋았으며, 치료 이후 5개월간 재발 증상이 관찰되지 않았다. 결론적으로 본 증례는 재조합 인간 인터페론 알파와 pentoxifylline을 이용한 고양이 전염성 복막염의 치료 반응에 대한 국내 첫 증례보고이다.

• Journal of Microbiology
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• 제37권3호
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• pp.168-174
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• 1999

Escherichia coli O157 is an important pathogenic organism which causes diarrhea, haemorrhagic colitis, and haemolytic ureamic syndrome (HUS) in human. E. coli O157 KNIH317 was isolated form patients suffering with HUS in Korea. We designed a primer set for cloning shiga-like toxin (slt) gene. The amplified PCR product was used to Southern and colony hybridization as a probe. As a result, we cloned 4.5-kb KpnI fragment containing the slt gene encoding shiga-like toxin from chromosomal DNA of E. coli O157 KNIH317. This recombinant plasmid was named pOVT45. E. coli XL1-Blue harboring pOVT45 showed cytotoxicity in Vero cells. We sequenced the slt gene of this strain. The A-subunit gene of the slt was composed of 960 base pairs with ATG initiation codon and TAA terminationcodon. The B-subunit was composed of 270 base paris with ATG initiation codon and TGA termination codon. Nucleotide sequence comparison of the slt gene exhibited 100%, 98.4%, 93.7%, and 93.7% identity with that of shiga-like toxin type II (sltII) of E. coli bacteriophage 933W, variant slt of E. coli, slt of E. coli, and variant sltII of E. coli, respectively. From these results, it was concluded that the cloned slt gene belongs to SltII family and that the strain used in this study may be a lysogeny of E. coli bcteriphage 933W.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1343-1349
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• 2004

A native strain of Pasteurella multocida was isolated from pigs suffering from severe atrophic rhinitis at domestic farms in Gyeonggi Province, Korea, and was identified as capsular serogroup 'D' and somatic serotype '4' by disc diffusion decapsulation and gel diffusion precipitation tests, respectively. The P. multocida (D:4) induced atrophic rhinitis in healthy pigs by the secondary infection. The gene for outer membrane protein H (ompH) of P. multocida (D:4) was cloned in Escherichia coli DH5$\alpha$ by PCR. The open reading frame of the ompH was composed of 1,023 bp, possibly encoding a protein with 341 amino acid residues containing a signal peptide of 20 amino acids at N-terminus, and the gene product with molecular mass of ca. 38 kDa was identified by SDS-PAGE. Hydropathy profiles indicated that there are two variable domains in the OmpH. To express the ompH in E. coli, the gene was manipulated in various ways. Expression of the truncated as well as full-length forms of the recombinant OmpH was fatal to the host E. coli BL21 (DE3). However, the truncated OmpH fused with GST was consecutively expressed in E. coli DH5$\alpha$. A large quantity of the fused polypeptide was purified through GST-affinity chromatography.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1176-1183
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• 2009

Lactobacillus crispatus (L. crispatus) ZJ001 is highly adhesive to epithelial cells and expresses S-layer proteins. In this study, S-S-layer layer genes were sequenced and expressed in E. coli to characterize the function of proteins with this particular strain. L. crispatus ZJ001 harbored two S-layer genes slpA and slpB, and only slpA gene was expressed in the bacterium, as revealed by RT-PCR and immunoassays. The mature SlpA showed 47% amino acid sequence identity to SlpB. The SlpA and SlpB of L. crispatus ZJ001 were highly homologous at the C-terminal region to other Lactobacillus S-layer proteins, but were substantially variable at N-terminal and middle regions. Electron microscopic analysis indicated that His-slpA expressed in E. coli was able to form a sheet-like structure similar to the natural S-layer, but His-slpB formed as disc-like structures. In the cell binding experiments, HeLa cells were able to bind to both recombinant His-slpA and His-slpB proteins to the extent similar to the natural S-layer. The cell binding domains remain mostly in the N-terminal regions in SlpA and SlpB, as shown by high binding of truncated peptides SlpA2-228 and SlpB2-249. Our results indicated that SlpA was active and high binding to HeLa cells, and that the slpA gene could be targeted to display foreign proteins on the bacterial surface of ZJ001 as a potential mucosal vaccine vector.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1076-1080
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• 2008

Glucagon, a peptide hormone produced by alpha-cells of Langerhans islets, is a physiological antagonist of insulin and stimulator of its secretion. In order to improve its bioactivity, we modified its structure at the C-terminus by amidation catalyzed by a recombinant amidase in bacterial cells. The human gene coding for glucagon-gly was PCR amplified using three overlapping primers and cloned together with a rat ${\alpha}$-amidase gene in plasmid pMGA. Both genes were expressed under control of the strong constitutive promoter of aph and secretion signal melC1 in Streptomyces lividans. With Phenyl-Sepharose 6 FF, Q-Sepharose FF, SP-Sepharose FF chromatographies and HPLC, the peptide was purified to about 93.4% purity. The molecular mass of the peptide is 3.494 kDa as analyzed by MALDI TOF, which agrees with the theoretical mass value of the C-terminal amidated glucagon. The N-terminal sequence of the peptide was also determined, confirming its identity with human glucagon at the N-terminal part. ELISA showed that the purified peptide amide is bioactive in reacting with glucagon antibodies.

• Asian-Australasian Journal of Animal Sciences
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• 제20권6호
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• pp.850-855
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• 2007

Leptin is the adipocyte-specific product of the obese gene and plays a major role in food intake and energy metabolism. Leptin research was mainly focused on mammalian species, but understanding of leptin and its function in poultry is very poor. In this study, the duck leptin gene was amplified using the reverse transcription-polymerase chain reaction (RT-PCR) from duck liver RNA. The cDNA fragment was inserted into the pET-28a expression vector, and the resulting plasmid was expressed in Escherichia coli BL21 (DE3). Experimental mice were given an intraperitoneal injection of 10 mg/kg leptin dissolved in phosphate buffered saline (PBS), while the control mice were injected with PBS. The effect of leptin on food intake, body weight and fatty deposition in mice was detected. Sequence analysis revealed that duck leptin had a length of 438 nucleotides which encoded a peptide with 146 amino acid residues. The sequence shares highly homology to other animals. The coding sequence of duck leptin was 84 and 86% identical to human and pig leptin nucleotides sequence. Highest identity was with the rat coding sequence (95%). The identity of the amino acid sequence was 84, 82 and 96% respectively compared to that of the human, pig and rat. Results of SDS-PAGE analysis indicated that a fusion protein was specifically expressed in E. coli BL21 (DE3). The purified product was found to be biologically active during tests. Continuous administration of recombinant duck leptin inhibited food intake. Despite the decrease of food intake, leptin significantly induced body weight and fatty deposition. These changes were accompanied by a significant down-secretion of plasma glucose, cholesterol, triglyceride and insulin levels in mice. The observations provide evidence for an inhibitory effect of leptin in the regulation of food intake and for a potential role of duck leptin in the regulation of lipogenesis.