• 한국미생물·생명공학회지
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• 제24권6호
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• pp.657-663
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• 1996

Pseudomonas sp. P20 isolated from the polluted environment is capable of degrading biphenyl and 4-chlorobiphenyl. The pcbABCD genes responsible for degradation of biphenyl and 4-chlorobiphenyl were cloned using pBluescript SK(+) from the chromosomal DNA of Pseudomonas sp. P20 to construct pCK1 and pCK102, harbouring pcbABCD and pcbCD, respectively. The 2, 3-DHBP dioxygenase gene, pcbC, was cloned again from pCK102 by using pKT230 which is known as a shuttle vector and pKK1 hybrid plasmid was constructed. The E. coli KK1 transformant obtained by transforming the pKK1 into E. coli XL1-Blue showed 2, 3-DHBP dioxygenase activity. The specific 2, 3-DHBP dioxygenase activity of E. coli KK1 was similar to that of the E. coli CK102, but much higher than those of the natural isolates, Pseudomonas sp. DJ-12 and Pseudomonas sp. P20.

• 미생물학회지
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• 제28권1호
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• pp.41-46
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• 1990

자연환경으로부터 분리한 DJ-12 균주는 4CBA 및 4CB를 비롯하여 그 대사산물인 4OHBA와 PCA를 분해하여 단일 탄소원으로 이용하였다. DJ-12 균주에서 4CBA 및 4CB분해유전자는 약 65kb 크기의 plasmid인 pDJ121에 존재하였으며, 이 pDJ121은 ExoRI, HindIII, SalI 그리고 PslI의 절단부위를 각각 9, 11, 10 그리고 19개씩 가지고 있었다. EcoRI으로 처리한 pDJ121 절편을 pKT230에 ligation 시켜 재조합 vector인 pDK450을 만들었으며, 이를 Pseudomonas putida KT2440에 transformation 시켜 얻은 cloned cell 에서는 4CBA 분해유전자가 잘 발현되었다.

• 한국미생물·생명공학회지
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• 제18권5호
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• pp.454-459
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• 1990

식물근부균 Fusarium solani의 생육을 강력히 길항하는 생물방제균 Pseudomonas stutzeri YPL-1에 외부유전자 도입을 통한 유전공학적 육종방법의 기초를 확립하고자 하였다. 이를 위해 plasmid pKT230을 vector로 하여 형질전환의 가능성을 조사하였으며 이때, 혈질전환에 필요한 최적조건을 조사한 결과 P.stutzeri YPL-1의 형질전환에는 대수증식기 초기의 균체가 가장 적합하였고, 20mM RbCl과 100mM $CaCl_2$를 함유한 냉각용액에 1${\mu}g$/ml의 plasmid DNA를 첨가하였을 때 최대의 형질전환 빈도를 나타내었다. 또한 plasmid DNA와 competent cell를 혼합한 후 $0^{\circ}C$에서 60분간 처리하는 것이 가장 효과적이었으며 이와 같은 조건에서 형질전환 빈도는 2~$6 \times 10^{-6}$으로 나타났다.

• 한국미생물·생명공학회지
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• 제17권5호
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• pp.447-453
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• 1989

항생물질 내성유전자를 유전자 조작기법으로 재조합시켜 만든 세균에서 그 유전자들이 전이되는 특성을 하폐수의 자연환경에서 연구하기 위하여, 자연계로부터 분리한 Gram 음성세균 중에서 kanamycin (Km), chloramphenicol (Cm), streptomycin (Sm), sulfadiazine(Su)에 내성을 띄는 균주로 DK1을 선발하였다. DK1 균주는 4개의 plasmid를 가지고 있으나 그 중 크기가 약 68kb인 pDK101 plasmid에 Km$^{${\gamma}$}$ Cm$^{${\gamma}$}$ 유전자를 가지고 있었다. 이 pDK101 plasmid에는 EcoRI site가 16개, PstI site가 32개, SalI site가 6개씩 있었다. pDK101 plasmid 와 vector로 사용한 pKT230을 E. coli으로 처리하여 얻은 절편들로부터 Km$^{${\gamma}$}$Cm$^{${\gamma}$}$ 유전자를 가지는 pDT309와 pDTS29 등의 chimeric piasmid를 제조하였다. 이들을 다시 E. coli C600과 E. coli HB101 에 형질전환 시킴으로써, DKC601, DKC602, DKH 102 그리고 DKH103 등의 재조합 균주를 얻었다. 이들 재조합 균주에서는 모두 Km$^{${\gamma}$}$Cm$^{${\gamma}$}$유전자가 잘 발현되었으며, 그 중 DKC601과 DKH103에서는 각각 pDT529와 pDT309의 chimeric plasmid가 포함되어 있는 것이 전기영동 방법으로 명확히 확인되었다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권1호
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• pp.56-60
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• 2001

The pcbC gene encoding (4-chloro-)2,3-dihydroxybiphenyl dioxygenase was cloned from the genomic DNA of Pseudomonas sp. P20 using pKT230 to construct pKK1. A recombinant strain, E. coli KK1, was selected by transforming the pKK1 into E. coli XL1-Blue. Another recombinant strain, Pseudomonas sp. DJP-120, was obtained by transferring the pKK1 of E. coli KK1 into Pseudomonas sp. DJ-12 by conjugation. Both recombinant strains showed a 23.7 to 26.5 fold increase in the degradation activity to 2,3-dihydroxybiphenyl compared with that of the natural isolate, Pseudomonas sp. DJ-12. The DJP-120 strain showed 24.5, 3.5, and 4.8 fold higher degradation activities to 4-chlorobiphenyl, catechol, and 3-methylcatechol than DJ-12 strain, respectively. The pKK1 plasmid of both strains and their ability to degrade 2,3-dihydroxybiphenyl were stable even after about 1,200 generations.

• Journal of Microbiology and Biotechnology
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• 제1권4호
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• pp.240-245
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• 1991

We have constructed a promoter-probe vector pKU20 using pKT230, a derivative of broad-host-range plsmid RSF1010, as a base. The pKU20 contains structural gene for aminoglycoside phos-photransferase (aph), without promoter, and a multiple cloning site upstream the aph. Using this vector, a 412base pairs (bp) PstI fragment showing strong promoter activity both in Escherichia coli LE392 and Pseudomonas putida KCTC1644 has been cloned from Pseudomonas fluorescens chromosomal DNA on the basis of streptomycin resistance. The nucleotide sequence of the 412 bp fragment has been determined and the putative - 35 and -10 region was observed. Insecticidal protein gene of Bacillus thuringiensis subsp. kurstaki HD-73 inserted on downstream of the promoterlike DNA fragment was efficiently expressed in E. coli and P. putida. The toxin protein was efficiently synthesized in an insoluble form in both strains.

• 한국미생물·생명공학회지
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• 제17권4호
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• pp.295-300
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• 1989

B. thuringiensis가 생산하는 살충성 독소 단백질의 생태학적 응용방법을 개발하기 위한 목적으로 우선 독소 단백질 유전자를 옮겨 발현시키기에 적합한 숙주 미생물의 분리작업을 수행하였다. 국내 주요농산물인 고추, 감자, 무우 등 7가지 농작물의 뿌리부근에 군락을 형성하는 35종의 형광성Pseudomonas들을 분리하였고 독소 단백질 유전자를 함유하는 재조합 plasmid에 대한 숙주로서의 이응가능성을 검토해 보기 위하여 분리균주 35주에 대한 형질전환을 실시한 결과 4주에 독소 단백질 유전자의 도입이 가능하였고 생물검정과 면역학적인 방법 등에 의한 결과 BT 독소 유전자의 발현을 확인하였다.

• Journal of Microbiology
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• 제35권1호
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• pp.53-60
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• 1997

Pseudomonas sp. P20 and Pseudomonas sp. DJ-12 isolated from the polluted environment are capable of degrading biphenyl and 4-chlorobiphenyl (4CB) to produce benzoic acid and 4-chlorobenzoic acid (4CBA) respectively, by pcbABCD-encoded enzymes. 4CBA can be further degraded by Pseudomonas sp. DJ-12, but not by Pseudomonas sp P20. However, the meta-cleavage activities of 2, 3-dihydroxybiphenyl (2, 3-DHBP) and 4-chloro-2, 3-DHBP dioxygenases (2, 3-DHBD) encoded by pcbC in Pseudomonas sp. P20 were stronger than Pseudomonas sp. DJ-12. In this study, the pcbC gene encoding 2, 3-DHBD was cloned from the genomic DNA of Pseudomonas sp. P20 by using pKT230. A hybrid plasmid pKK1 was constructed and E. coli KK1 transformant was selected by transforming the pKK1 hybrid plasmid carrying pcbC into E. coli XL1-Blue. By transferring the pKK1 plasmide of E. coli KK1 into Pseudomonas sp. DJ-12 by conjugation, a recombinant strain Pseudomonas sp. P20, Pseudomonas sp. DJ-12, and the recombinant cell assay methods. Pseudomonas sp. DJ12-C readily degraded 4CB and 2, 3-DHBP to produce 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoic acid (HOPDA), and the resulting 4CBA and benzoic acid were continuously catabolized. Pseudomonas sp. DJ12-C degraded 1 mM 4CB completely after incubation for 20 h, but Pseudomonas sp. P20 and Pseudomonas sp. DJ-12 showed only 90% and Pseudomonas sp. DJ-12 had, but its degradation activity to 2, 3-DHBP, 3-methylcatechol, and catechol was improved.

• 한국약용작물학회지
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• 제17권6호
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• pp.434-438
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• 2009

For the development of a new antidementia functional food or alternative drug using agricultural products, Job's tears (Coix lachrymajobi L.), which shows high acetylcholinesterase (AChE) inhibitory activity (55.1%) was selected and the extraction conditions of AChE inhibitor were optimized. AChE inhibitor of Job's tears was maximally extracted when it was treated with 60% methanol at $40^{\circ}C$ for 6 h. The AChE inhibitor of the methanol extracts was partially purified by systematic solvent extraction, thin layer chromatography, silica gel chromatography and reverse-phase HPLC and the partial purified AChE inhibitor with inhibitory activity ($IC_{50}$) of $0.608\;{\mu}g$ was obtained. The partial purified AChE inhibitor was soluble in methanol and hexane, and insoluble in water. Its maximum absorption spectra was 230 nm and also it was stable in the range of $30^{\circ}C$ and $70^{\circ}C$ and pH 4.0-8.0 for 1 h.