• Korean Journal of Microbiology
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• v.30 no.1
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• pp.1-7
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• 1992

We cloned a gene cluster encoding phenanthrene-degrading enzymes on a 6.8-kb Xhol fragment from the Pseudomonas sp. DJ77 chromosomal DNA into the vector pBLUESCRIPT SIC(+). The resultant clone, containing the recombinant plilsmid pHENX7, was able to convert 3-methylcatechol to a yellow mela-cleavage compound. Since the pHENX7R in which the DNA insert was cloned in the opposite orientation lacked extradiol dioxygenase activity. the direction of transcription was established. Four polypeptides, PhnC (24 kDa). PhnD (31 kDa), PhnE (34 kDa). and PhnF (15 kDa), were identified in E coli JM101 transformed with several pHENX7-derived plasmids. The locations and extents of ~ndividual genes were determined by subcloning. The gene order was phnC-phnD-phnE-phnF-phnG, and phnC, phnD, phnE, and phnG genes encoded glutathione S-transferase, mrta-cleavage compound hydrolase, extradiol dioxygenase, mera-cleavage compound dehydrogenase, respectively.

• Korean Journal of Microbiology
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• v.33 no.2
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• pp.86-91
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• 1997

Pseudomonas sp. DJ77로부터 클로닝된 glutathione S-transferase 유전자(phnC)의 염기서열을 결정하였다. 603bp의 open reading frame(ORF)이 존재하였고 개시코돈 앞에서 Shine-Dalgarno sequence를, 종결코돈 뒤에서는 terminator sequence를 발견하였다. phnC 유전자에서 만들어지는 phnC 단백질은 21,416 Da으로 SDS-polyacrylamide gel 전기영동 결과와 일치하였다. PhnC는 Bulkholderia cepacia LB400, Cycloclasticus oligotrophus RB1의 GST와 각각 53.7%, 49%의 높은 상동성을 나타냈다. 아미노산 서열의 상동성과 필수잔기들의 존재유무로 판단할 때 PhnC GST는 theta class GSTs와 진화적으로 유연관계가 높았지만 alpha, mu, pi, sigma class GSTs에서 구조적, 기능적으로 중요하다고 알려진 아미노산 잔기들이 PhnC GST에도 보존되어 있었다. 또한, phnC 유전자의 위치가 C. oligotrophus RB1, B. cepacia LB400 등의 GST 유전자 위치와 유사하다는 점에서 PhnC 효소는 난분해성 방향족 탄화수소의 분해에 관여하는 것으로 생각된다.

• Korean Journal of Microbiology
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• v.30 no.1
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• pp.8-14
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• 1992

Nucleotide Sequence of phnE Gene Encoding Extradiol Dioxygenase fromPseudomonas sp. Strain DJ77Kim, Young-Chang'.", Myeong-Su Shin1, Kil-Sang Younl, Young-Soon Park1, andUg-Hyeon Kim'.' (Department of Microbiology, C'hungbuk National University.Cheongju 360-763, KOREA. and 'Research Center for Molecular Microbiology,Seoul National University)nal University)

• Korean Journal of Microbiology
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• v.29 no.6
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• pp.392-396
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• 1991

Mu d1(Ap lac) bacteriophage can be used to search for genes which are members of a common regulatory network without having to know the functions of the genes in advance. Aim was for obtaining the loci in the SOS network as well as temperature inducible loci. For this purpose, recA441 allele was used. This allele encodes a thermosensitive recA gene product; thus, the recA441 allele can be activated upon temperature upshift without by external DNA damage. Approximately 10, 000 colonies were screened, and then searched for the colonies which expressed .betha.-galactosidase higher level at 42.deg.C than at 30.deg.C. The strains identified fell into two dlasses; (i) ones in which the increased expression was $recA^{+}$ $lexA^{+}$ -dependent, that is, din(damage-inducible) genes which were due to the activation of recA441 allele and (ii) ones in which the increased expression was $recA^{+}$ $lexA^{+}$ -independent and only temperature-inducible, tin genes. Rough mapping position was obtained for these genes.

• Korean Journal of Microbiology
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• v.33 no.2
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• pp.92-96
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• 1997

The 6.8 kb Xhol fragment of chromosomal ONA of Pseudomonas sp. 0177 contains the phnDEFG genes involved in the degradation of polyaromatic hydrocarbons and chlorinated aromatics. Here, we report the nucleotide sequence of the ORF encoding a polypeptide consisted of 143 amino acids with a Mr of 13,859. The nucleotide sequence of the ORF is 99% and 68.6% identical to the downstream region of catE of Sphingomonas sp. strain HV3 and the ORF between xylE and xylG of Sphingomonas yanoikuyae Bl, respectively. The deduced amino acid sequence of the PhnF has 62.3% identity with the amino acid encoded hy orfY region of Citrobacter freundii DSM30040. We now confirm that the ORF is located between the catechol 2,3-dioxygenase (C230), phnE, and 2-hydroxymuconic semialdehyde dehydrogenase (2HMSO), phnG.

• Journal of Microbiology and Biotechnology
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• v.12 no.5
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• pp.729-734
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• 2002

Radiorespirometric analysis revealed that Pseudomonas sp. strain KKI isolated from a soil contaminated with petroleum hydrocarbons was able to catabolize polycyclic aromatic hydrocarbons such as phenanthrene and naphthalene. The rate and extent of phenanthrene mineralization was markedly enhanced when the cells were pregrown on either naphthalene or phenanthrene, compared to the cells grown on universal carbon sources (i.e., TSA medium). Deduced amino acid sequence of the Rieske-type iron-sulfur center of a putative phenanthrene dioxygenase (PhnAl) obtained from the strain KKI shared significant homology with DxnAl (dioxin dioxygenase) from Spingomonas sp. RW1, BphA1b (biphenyl dioxygenase) from Spingomonas aromaticivorans F199, and PhnAc (phenanthrene diokygenase) from Burkholderia sp. RP007 or Alcaligenes faecalis AFK2. Northern hybridization using the dioxygenase gene fragment cloned from KKI showed that the expression of the putative phn dioxygenase gene reached the highest level in cells grown in the minimal medium containing phenanthrene and $KNO_3$, and the expression of the phn gene was repressed in cells grown with glucose. In addition to the metabolic change, phospholipid ester-linked fatty acids (PLFA) analysis revealed that the total cellular fatty acid composition of KKI was significantly changed in response to phenanthrene. Fatty acids such as 14:0, 16:0 3OH, 17:0 cyclo, 18:1$\omega$7c, 19:0 cyclo increased in phenanthrene-exposed cells, while fatty acids such as 10:0 3OH, 12:0, 12:0 2OH, 12:0 3OH, 16:1$\omega$7c, 15:0 iso 2OH, 16:0, 18:1$\omega$6c, 18:0 decreased.

• The Journal of Natural Sciences
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• v.11 no.1
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• pp.65-73
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• 1999

C-P compounds(Pn; phosphonate) such as glyphosate(GPS), aminoethylphosphonate(AEPn) and methyl-phosphonate(MPn) biodegrading genes were cloned from Pseudomonas sp. strain #A1 Which assimilated GPS as sole phosphorous source. Carrying out the in vivo molecular cloning by means of Mini-Mu plasmid, the size of clones($AEPn^+$, $MPn^+$, $GPS^+$) for the gene to degrade C=P compounds are 10-19Kb, 10Kb, and 12-18 Kb, respectively. Moreover, they expressed the phenotype for each Pn when they were transformed into $\Delta phn$ mutants. Hence, it is postulated that Pseudomonas sp.#A1 has three kind of Pn degradative pathway, separately. The phn clones($AEPn^+$, $MPn^+$, $GPS^+$) are verified as the members of PHO regulon because of their phoBR-dependent characteristics.

• Journal of Microbiology
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• v.33 no.4
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• pp.328-333
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• 1995

$\^$31/ P-NMR experiment was performed to detect phophonates (Pn) utilization and degradation in the several different C-P lyase mutants of E. coli and in E. aerogenes and the recombinants. The relative peak intensity (RPI) for the standard samples of 0.5 mM methylphosphonate (MPn) and 1.0 mM aminoethylphosphonate in glucose-MOPS medium showed 0.5 : 1.0 ratio. In the case of BW14329 (.DELTA.phnC-P, .delta.phoA), RPI did not change significantly after 24 hrs culturing, which means it nearly could not utilize Pn. In vivo $\^$31/ P-NMR spectrum of E. aerogens (BWKL 16627) during 3 hrs starvation showed two intense peaks at 0-2 ppm and at near-10 ppm which indicate intracellular orthophosphate (Pi) and pyrophosphate (PPi), respectively. Both of them might be released by degradation of inorganic polyphosphate pool. When MPn is supplied to the medium as an unique P source, Pi content in the cell has the constant, but PPi seems to be slightly decreased. Recombinants (BWKL 16954) grew slower than E. aerogenes in the glucose-MOPS media with various P sources. In vivo $\^$31/ P-NMR spectrum of recombinant did not show any intense signal in the cell. Surprisingly, under the cultivation adding with MPn, a few intense peaks in the region of Pi AND phospate monoester were detected.

• Microbiology and Biotechnology Letters
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• v.47 no.2
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• pp.278-288
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• 2019

In the present investigation, we attempted to design a protocol to develop a hybrid protein with better bioremediation capacity. Using in silico approaches, a Hybrid Open Reading Frame (Hybrid ORF) is developed targeting the genes of microorganisms known for degradation of organophosphates. Out of 21 genes identified through BLAST search, 8 structurally similar genes (opdA, opd, opaA, pte RO, pdeA, parC, mpd and phnE) involved in biodegradation were screened. Gene conservational analysis categorizes these organophosphates degrading 8 genes into 4 super families i.e., Metallo-dependent hydrolases, Lactamase B, MPP and TM_PBP2 superfamily. Hybrid protein structure was modeled using multi-template homology modeling (3S07_A; 99%, 1P9E_A; 98%, 2ZO9_B; 33%, 2DXL_A; 33%) by $Schr{\ddot{o}}dinger$ software suit version 10.4.018. Structural verification of protein models was done using Ramachandran plot, it was showing 96.0% residue in the favored region, which was verified using RAMPAGE. The phosphotriesterase protein was showing the highest structural similarity with hybrid protein having raw score 984. The 5 binding sites of hybrid protein were identified through binding site prediction. The docking study shows that hybrid protein potentially interacts with 10 different organophosphates. The study results indicate that the hybrid protein designed has the capability of degrading a wide range of organophosphate compounds.