• BMB Reports
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• 제29권1호
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• pp.17-21
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• 1996

Ile91 of restriction endonuclease EcoRV, which has not been known to take part directly in catalytic activity, was substituted with Leu by site-directed mutagenesis. The Ile91Leu mutant shows over 1000-fold less activity than the wild type EcoRV under standard reaction condition. The metal ion dependency of the reaction was altered. In contrast to the wild type EcoRV, the mutant prefers $Mn^{2+}$ to $Mn^{2+}$ as the cofactor. In $Mn^{2+}$ buffer the mutant is as active as the wild type enzyme in $Mn^{2+}$ buffer. Like the wild type enzyme, the mutant shows an unspecific binding of DNA in gel shift experiments. In contrast to the wild type enzyme, the mutant did not cleave at noncognate sites of DNA under star condition.

• Journal of Microbiology and Biotechnology
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• 제5권5호
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• pp.269-273
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• 1995

A thermotolerable restriction endonuclease. Svil, found in Streptomyces violochromogenes D2-5 was purified. For the purification, streptomycin sulfate and ammonium sulfate precipitation was used. Ph osphocellulose P-ll, DEAE-Cellulose and Sephacryl-S200 HR colum chromatography were also performed. The purified enzyme was found to be homogeneous and the molecular weight of the enzyme estimated by polyacrylamide gel electrophoresis containing 0.1$%$ SDS was about 32, 000 daltons. The recognition sequence and cleavage site of the enzyme were determined to be $5^1$-$TT\downarrow CGAA$-$3^1$ which is the same sequence as that of Asull. Unlike Asull, however, the Svil shows high thermal stability.

• 한국미생물·생명공학회지
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• 제49권3호
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• pp.316-319
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• 2021

Restriction endonucleases play an important role in molecular cloning, clinical diagnosis, and pharmacological drug studies. In this study, DNA-templated copper nanoclusters (DNA-CuNCs) were used to detect AluI endonuclease activity due to their high fluorescence emission and rapid synthesis of DNA-CuNCs under ambient conditions. Results showed that AluI activity was detected in a highly sensitive manner at low concentrations of AluI endonuclease by the fluorescence intensity of DNA-CuNCs. Additionally, its inhibition was monitored in the presence of daidzein under optimal conditions.

• KSBB Journal
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• 제9권1호
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• pp.63-71
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• 1994

During the last decade enzyme reaction in organic solvent has been studied to show that specificity in buffer is different from that in organic solvent. The specificity of restriction enzyme was effected by various factors such as ionic strength, salt organic solvent and temperature. In this study, restriction enzyme PvuII which is used most frequently in genetic engineering and the substrate was vector pGEM3 whose sequence was already known were used. As a result the recognition sequence site was changed in the presence of organic solvents whose Log P are -1.5∼0. Their specificities were contrast with activities were contrasted. Specificities were not changed in organic solvent easily in inactivating enzyme. We think that the enzyme recognition site was not changed randomly but by preferential order. A recombinant vector which does not contain typical cleavage site CAG↓CTG was cleaved in 20% ethanol solution. This result might show that restriction enzyme could be used to cleave at unusual sites by changing the reaction conditions.

• 미생물학회지
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• 제24권1호
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• pp.18-23
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• 1986

Breνibacterium divaricatum FERM 5948 균주로 부터 Bdi I 제한효소를 ammonium sulfate분획, DEAE-cellulose chroma tograph 그리고 heparin agarose chromatograph 방법을 거쳐 부분정제하여 그 효소적 특성을 관찰하였다. 분리한 Bdi I 제한효소는 pBR 322와 ${\lambda}$ DNA를 이용하여 인지부위를 알아본 결과 5' ATCGAT3'을 인지하는 Cia I과 isoschizomer였다. 또한 CIa I 으로 자른 ${\lambda}$ DNA가 Bdi I 으로 자른 pBR 322에 클로닝 됨으로 보아 Bdi I 제한효소는 T와 C사이를 잘라(5' AT CGAT 3') 5' pGG를 가지는 cohesive end로 만듬을 알 수 있었다. 이 효소의 최적활성온도는 $37^{\circ}C$ 였고 50 - 100 mM의 NaCl 농도에서 활성이 높았으여 150 mM이상의 농도에서는 활성이 저해되었다.

• Restorative Dentistry and Endodontics
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• 제22권1호
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• pp.413-427
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• 1997

Porphyromonas endodontalis and Prevotella intermedia are black-pigmented anaerobic gram negative rods which have been isolated from infected root canals and submucous abscesses of endodontic origin. And they are associated with clinical symptoms such as pain, percussion, and foul odor. It has been reported that there are 3 serotypes according to capsule membrane in P. endodontalis and 2 DNA homology groups and 3 serotypes in P. intermedia, but there is no data available regarding genetic diversity for the species P. endodontalis and P. intermedia. The purpose of this study is to investigate genetic diversities between individual strains of P. endodontalis and P. intermedia which are indistinguishable by serotyping and biotyping using bacterial DNA restriction endonuclease analysis. 45 teeth with at least one clinical symptoms, with single canal, and with pulp necrosis were sampled. For sampling bacteria, access cavity was prepared after disinfecting tooth and its surroundings. Then the paper point was inserted to the apex of the canal, leave there for 15 seconds, and finally it was placed into PRAS Ringer's solution and PBS solution. P. endodontalis and P. intermedia were identified by biochemical test and IIF after subculturing black and brown colonies which were produced after 7 days of incubation on BAP in anaerobic chamber. P. endodontalis and P. intermedia strains were grown in BHI broth and whole genomic DNA was extracted by phenol-chloroform extraction technique and digested by restriction endonuclease, Eco RI and Pst I. The resulting DNA fragments were separated by agarose gel electrophoresis, stained with EtBr and photographed under UV light. The results were as follows : 1. In both P. endodontalis and P. intermedia, different serotypes could be found within a root canal of same patient. 2. There were obvious genetic heterogeneity within a patient and within a serotype in both P. endodontalis and P. intermedia. 3. P. endodontalis serotype c, isolated from different patients, exhibited limited genotypic diversity.

• Journal of Microbiology
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• 제45권2호
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• pp.175-178
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• 2007

An endonuclease from Xanthomonas oryzae pathovar oryzae KACC 10331, XorII, was recombinantly produced in Escherichia coli using a T7 system. XorII was purified using a combination of ion exchange and immobilized metal affinity chromatography (IMAC). An optimized washing protocol was carried out on an IMAC in order to obtain a high purity product. The final amount of purified XorII was approximately 2.5 mg/L of LB medium. The purified recombinant XorII was functional and showed the same cleavage pattern as PvuI. The enzyme activity tested the highest at $25^{\circ}C$ in 50 mM NaCl, 10 mM Tris-HCl, 10 mM $MgCl_{2}$, and 1 mM dithiothreitol at a pH of 7.9.

• 미생물학회지
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• 제26권1호
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• pp.1-5
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• 1988

The esolation and characterization of a new type II restriction endounclease, BamI, from Bacellus macerans ATCC 8244 were described. BmaI endonuclease was partially purified by procedures of ammonium sulfate fractionation, DEAE-cellulose and phosphocellulose chromatographies. This enzume recognized one site on pBR322 DNA, two sites on Bluescribe DNA, three sites on $\lambda$DNA and no site on SV 40 DNA. The same cleavage patterns for vareius DNAs as PvuI indicated that BamI is an isoschisomer of PvuI whose recognition sequence is 5'-CGATCG-3'. The optimal pH for the BmaI endonuclease activity was about 7.0 and optimal NaCl concentration was about 100mM. Manganese ion could partially replace magnesium as a cofactor, but calcium could not at all.

• 대한수의학회지
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• 제32권4호
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• pp.597-603
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• 1992

To elucidate the molecular genetical properties of the canine parvoviruses isolated from the diseased puppies in the regions of Kyunggi and Chungnam provinces, the replicative form (RF) DNA of four field isolates were compared with those of two attenuated vaccine strains and a reference strains of CPV by restriction endonuclease analysis (REA). REA by Hinf I showed that three CPV isolates except CPV-V15 had an identical banding pattern with two vaccine strains, one standard strain and feline panleukopenia virus (FPLV). In CPV-V15 strain the fourth fragment of DNA with 800 bp was deleted. REA by Bgl II and Pst I indicated that CPV-V15 and FPLV had a bigger second fragment than those of the other strains of CPV. Meanwhile REA by Bam HI revealed that all the field isolates and vaccine strains used in this experiment showed similar banding patterns.

• 미생물학회지
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• 제32권4호
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• pp.285-290
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• 1994

자연계에서 새로운 제한효소 생산균을 검색하여 한 균주를 선발하고, 형태학적, 생리학적, 생화학적 특성들을 조사하여 Alcaligenes sp.로 동정하고 제한효소의 특성을 조사하였다. Alcaligenes sp. J-482가 생산하는 제한효소를 AspJI으로 명명하였다. AspJI은 pBR322, Adenovirus 2-DNA, ${lambda}$ DNA 등에 대한 절단양식이 AatII와 같아 AatII의 isoschizomer로 추정 되었으며, 효소활성에 12.5mM 이상의 $MgCl_2$를 필요로 하였으며, NaCl에 의하여 저해되었다. AspJI의 반응 최적 온도는 $37^{circ}C$, 최적 pH는 7.5로 확인 되었으며, 내열성을 조사한 결과 $85^{circ}C$이상에서 15분처리 할 때 안전히 실활되는 것으로 관찰되었다.