Candida albicans KNIH10으로부터 Enolase의 분리 및 면역진단의 응용

박용춘(Yong Chjun Park),유재일(Jae Il Yoo),이영선(Yeong Seon Lee),신종희(Jong Hee Shin),김봉수(Bong Su Kim) 大韓微生物學會 2000 大韓微生物學會誌 Vol.35 No.2

Cloning and Nucleotide Sequence of the recA Gene from Shigella sonnei KNIH104S Isolated in Korea

Park, Yong-Chjun,Shin, Hee-Jung,Kim, Young-Chang Korean Society for Biochemistry and Molecular Biol 1999 Journal of biochemistry and molecular biology Vol.32 No.5

Shigella sonnei is an important cause of human enteric infections. S. sonnei KNIH104S was previously reported to be isolated from Korean shigellosis patients. We cloned a 2.8-kb KpnI fragment containing the recA gene encoding a recombinase from the chromosomal DNA of S. sonnei KNIH104S. This recombinant plasmid was named pRAK28. E. coli HB101, a recA mutant, cannot grow on Luria-Bertani medium in the presence of the alkylating agent methylmethane sulfonate, however, E. coli HB101 harboring pRAK28 was found to grow on this medium. As far as we know, we are the first to sequence the recA gene from S. sonnei. This gene is composed of 1062 base pairs with an ATG initiation codon and a TAA termination codon. Nucleotide sequence comparison of the S. sonnei recA gene exhibited 99.7% and 99.5% identity with those of S. flexneri and E. coli, respectively.

Cloning and Nucleotide Sequence Analysis of Verotoxin Gene from Escherichia coli O157 KNIH317 Isolated in Korea

Park, Yong-Chjun,Shin, Hee-Jung,Kim, Young-Chang The Microbiological Society of Korea 1999 The journal of microbiology Vol.37 No.3

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.

16S rRNA를 이용한 다금바리, 자바리, 능성어 판별법 개발

박용춘(Yong-Chjun Park),정용현(Yong-Hyun Jung),김미라(Mi-Ra Kim),신준호(Joon-Ho Shin),김규헌(Kyu-Heon Kim),이재황(Jae-Hwang Lee),조태용(Tae-Yong Cho),이화정(Hwa-Jung Lee),이상재(Sang-Jae Lee),한상배(Sang-Bae Han) 한국식품과학회 2013 한국식품과학회지 Vol.45 No.1

다금바리, 자바리 및 능성어는 농어목(Perciformes Order) 바리과(Serranidae Family)에 속하며, 고급횟감으로 인기가 높은 어종이다. 자바리 및 능성어의 경우 몸통부분에 줄무늬가 선명하게 있으나 성어가 되면서 점차 불분명해지는 특징이 있어 무늬의 유무로 인하여 다금바리와 구별하기는 쉽지 않다. 또한 자바리는 제주도에서 다금바리라는 명칭으로 불리고 있으며, 일부 유통업자들이 자바리 및 능성어를 다금바리로 유통시키는 사례가 있어 종 특이 프라이머를 이용한 판별법을 마련하게 되었다. 종 특이 프라이머를 설계하기 위하여 유전자은행(www.ncbi.nlm.nih.gov)에 등록되어있는 다금바리(Accession No. AY947575), 자바리(Accession No. JN603832), 능성어(Accession No. AY947559), 우럭(Accession No. DQ678295) 등의 16S DNA부위의 염기서열을 대상으로 하였으며 비교 및 분석에는 소프트웨어인 BioEdit ver.7.0.9.0 프로그램을 사용하였다. 그 결과 다금바리, 자바리, 능성어를 판별할 수 있는 각각의 NS-003-F/NS-005-R(136 bp), EB-001-F/EB-002-R(181 bp), ES-001-F/ES-001-R(123 bp) 프라이머를 개발하였으며, PCR 조건을 확립하였다. 또한 적용성 검토를 위하여 우럭, 참돔을 대상으로 실시한 결과 비 특이적 밴드가 형성되지 않는 것을 확인하였다. 따라서 본 연구에서 개발된 다금바리 등에 대한 판별법은 시중에 불법적으로 유통 가능성이 있는 제품을 신속하고 과학적으로 판별할 수 있어 식품안전관리에 활용도가 매우 클 것으로 기대된다. Niphon spinosus, Epinephelus bruneus, and Epinephelus septemfasciatus are involved in the Perciformes Order and Serranidae Family. When E. bruneus and E. septemfasciatus are fully grown, the striped pattern on the body gradually disappears. Therefore, morphological classification of adult fishes is quite difficult to identify the differences to N. spinosus. In this study, we investigate the method to differentiate those using PCR. To design the primers, 16S rRNA region of N. spinosus, E. bruneus, and E. septemfasciatus registered in the GeneBank (www.ncbi.nlm.nih.gov) have been used and for the analysis, Bio Edit ver. 7.0.9.0 was used. As a result, it was design NS-003-F/NS-005-R (136 bp), EB-001-F/EB-002-R (181 bp), and ES-001-F/ES-001-R (123 bp) primers for the differentiation of each 3 different fishes. Therefore, the species-specific primer sets would be a useful tool for scientific and speedy differentiation against the illegal distribution for consumer protection.

Cephalosporin C Acylase 생산균주의 분리 및 특성

박용춘,김욱현,임재윤,김영창 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.5

7-aminocephalosporanic acid(7-ACA)에 내성이 있으나 cephalosporin 계 항생제에는 민감한 Micrococcus luteus ATCC 9341을 지시균으로 사용하고, D-(α)-phenylglycine methylester와 7-ACA, 도는 glutaric acid dimethyl ester와 7-ACA를 기질로 사용한 bioassay 방법을 acylase 활성에 의하여 집락 주위에 지시균의 생장 저지환을 생성하는 20 종류의 세균을 선발하였다. 환 생성균들 중에서 cephalosporin C, glutaryl 7-ACA에는 내성이 있으나 7-ACA에는 감수성이 있는 SS5 균을 이용한 bioassay 방법과 HPLC 분석을 통하여 cephalosporin C acylase 활성이 있는 APS20 균주를 최종적으로 선발하였으며, 이 균주는 Bacillus macerans로 동정되었다. B. macerans APS20은 cephalosporin C에 대한 β-lactamase 활성이 없었으나, 100 ㎍/㎖ 농도의 cephalosporin C에 내성을 나타내었다. 그러나 penicillin G의 경우에는 50 ㎍/㎖에서도 생장하지 못하였다. Twenty microbial strains producing the acylase were isolated from soil by using Micrococcus luteus ATCC 9341 as an indicator strain, using either D-(α)-phenylglycine methylester and 7-aminocephalosporanic acid (7-ACA) or glutaric acid dimethylester and 7-ACA as substrates. Among the isolates, only one strain was turned out to be the 7-ACA producer from either cephalosporin C or glutaryl 7-ACA as the substrates by using the overlay of 7-ACA sensitive strain (SS5). 7-ACA produced from cephalosporin C by an isolate (APS20) was detected by high performance liquid chromatography. The isolated strain (APS20) was identified to Bacillus macerans on the basis of cellular fatty acid profile by gas chromatography. Bacillus macerans APS20 had no β-lactamase activity on cephalosporin C, and that is very important for the enzymatic production process of 7-ACA. However, this strain was resistant up to 100 ㎍/㎖ of cephalosporin C.

Cephalosporin C 내성과 7-Aminocephalosporanic Acid 감수성을 지닌 균주의 선발 및 특성

김욱현,박용춘,임재윤,김영창 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.5

A strain which showed cephalosporin C resistance and 7-aminocephalosporanic acid sensitivity was isolated from nature. Among the isolates, SS5 was sensitive to cephalosporin C, penicillin G, ampicillin, 7-aminocephalosporanic acid, 6-aminopenicillanic acid, and 7-aminodeacetoxy cephalosporanic acid at concentrations of 1,000 ㎍/㎖, 2,000 ㎍/㎖, 3,000 ㎍/㎖, 30 ㎍/㎖ 100 ㎍/㎖ and 100 ㎍/㎖, respectively. But SS5 was sensitive at very how concentration of chloramphenicol, kanamycin, neomycin, streptomycin and tetracycline. Since SS5 was sensitive to 7-ACA (30 ㎍/㎖) and didn’t have β-lactamase activity on the cephalosporin C, SS5 could be useful as an indicator strain for the production of 7-ACA, which is an important precursor for the synthesis of many semisynthetic cephalosporins.

Pseudomonas sp. Strain DJ77에서 Rieske-Type의 Ferredoxin을 암호화하는 phnR 유전자의 구조

김성재,박용춘,김치경,임재윤,이기성,민경희,김영창 한국산업미생물학회 1997 한국미생물·생명공학회지 Vol.25 No.4

phenanthrene을 cis-phenanthrene dihydrodiol 형태로 바꾸는데 관여하는 효소는 방향족고리에 두 개의 수산기를 붙이는 dioxygenase이다. phenanthrene과 biphenyl을 분해하는 능력이 있는 Pseudomonas sp. strain DJ77에서 클로닝한 재조합 플라즈미드 pUPX5로부터 전보의 2,3-dihydroxybiphenyl 1,2-dioxygenase을 암호화하는 phnQ의 하류방향으로 327개의 open reading frame을 발견하였다. 이 ORF, PhnR을 분석한 결과 dioxygenase의 Rieske-type ferredoxin 성분을 암호화하고 있는 것으로 밝혀졌다. PhnR은 108개의 아미노산으로 구성되어 분자량 11355의 단백질을 만들어내며, Sphingomonas BN6의 naphthalenesulfonic acid dioxygenase ferredoxin 단위체와 79.2%의 상동성을 나타내었고, Pseudomonas sp. strain C18의 DoxA, Pseudomonas sp. strain NCIB9816의 NdoA와 NahAb 등과는 46.7%의 상동성을 보였다. One of the three components of the phenanthrene dioxygenase which is required for conversion of phenanthrene to cis-phenanthrene dihydrodiol, Rieske-type ferredoxin encoded by phnR has been cloned and sequenced from Pseudomonas sp. strain DJ77. The gene phnR is positioned at the downstream of phnQ encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase. The PhnR ferredoxin contains 108 amino acids with a Mr of 11,355. The deduced amino acid sequence of the PhnR ferredoxin is 35-79% identical to those of homologous ferredoxins encoded by various genes.

Cloning and Nucleotide Sequence of the recA Gene from Shigella sonnei KNIH104S Isolated in Korea

Shin, Hee-Jung,Park, Yong-Chjun,Kim, Young-Chang The Korea Science and Technology Center 1999 BMB Reports Vol.32 No.5

Shigella sonnei is an important cause of human enteric infections. S. sonnei KNIH104S was previously reported to be isolated from Korean shigellosis patients. We cloned a 2.8-kb KpnI fragment containing the recA gene encoding a recombinase from the chromosomal DNA of S. sonnei KNIH104S. This recombinant plasmid was named pRAK28. E. coli HB101, a recA mutant, cannot grow on Luria-Bertani medium in the presence of the alkylating agent methylmethane sulfonate, however, E. coli HB101 harboring pRAK28 was found to grow on this medium. As far as we know, we are the first to sequence the recA gene from S. sonnei. This gene is composed of 1062 base pairs with an ATG initiation codon and a TAA termination codon. Nucleotide sequence comparison of the S. sonnei recA gene exhibited 99.7% and 99.5% identity with those of S. flexneri and E. coli, respectively.

Pseudomonas sp. Strain DJ77에 존재하는 Glutathione S-Transferase 아미노 말단잔기의 Site-directed Mutagenesis

우희종,박용춘,김성재,정용제,정안식,김영창 한국산업미생물학회 1997 한국미생물·생명공학회지 Vol.25 No.4

Pseudomonas sp. DJ77로부터 분리된 glutathione S-transferase(GST)의 N-말단 아미노산 서열은 MKLFISPGACSL로 전체 아미노산 서열의 상동성은 E. coli와는 45%, Haemophilus influenzae와는 20%로 알려져 있다. N-말단의 tyrosine 잔기는 박테리아 뿐 아니라 알려진 모든 세포성 GSTs에 존재하며 포유동물의 α, μ, π유형의 GSTs에서는 기능적인 활성자리라고 알려져 있다. 이런 점 때문에 Pseudomonas sp. DJ77의 GST에서 N-말단에 tyrosine 대신에 Phe-4와 Ile-5가 존재하는 것은 매우 특이한 일이다. Pseudomonas sp. DJ77 GST의 반응기작을 이해하기 위한 한 방법으로 site-directed mutagenesis를 이용하여 이 두 아미노산들을 각각 tyrosine으로 바꾸었다. 이 두 돌연변이주의 CDNB에 대한 효소 활성은 야생형와 비교해 거의 비슷한 활성을 나타내었고 in vivo에서의 CDNB에 의한 성장 저해 분석을 통해서도 같은 결과를 얻을 수 있었다. 이러한 결과들은 Pseudomonas sp. DJ77 GST가 적어도 포유류의 GST와는 다른 반응기작을 가진다는 것을 의미하는 것이다. Glutathione S-transferase (GST) was purified from Pseudomonas sp. DJ77, and its N-terminal sequence was determined to be MKLFISPGACSL. A specific tyrosyl residue in the vicinity of the N terminus is conserved in all the known cytosolic GSTs and has been shown to function as a catalytic residue in α, μ, π class GSTs from mammals. However, Pseudomonas sp. DJ77 GST has the Phe-4 and Ile-5 instead of Tyr in N-terminus. Its replacement with tyrosine did not significantly affect the enzyme activity. Results from in vitro biochemical analyses were confirmed by the in vivo activity-based CDNB growth inhibition analyses. Our results clearly indicate that GST of Psedomonas sp. DJ77 has a novel reaction mechanism different from that of mammalian GSTs.

Development of Detection Method for Economically Motivated Adulteration Red Pepper Powder by Molecular Approach

Sang-Ho Baik,Yong-Chjun Park,Jong-Hui Kim 한국식품영양과학회 2012 한국식품영양과학회 학술대회발표집 Vol.2012 No.10