• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.1047-1051
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• 2004

The phytopathogenic bacterium Pectobacterium chrysanthemi secretes multiple isozymes of plant cell wall disrupting enzyme such as pectate lyase and cellulase. The cel gene, existing in tandem with the pel gene, was isolated previously [10]. The role of Cel5Z and PelL1 in P. chrysanthemi PY35 pathogenicity on potato tissues was assessed by mutagenizing cloned cel gene and pel gene in tandem and recombining them with the chromosomal alleles. Strains with the Km cassette interposon in pelL1 or a double mutant showed a delay in the appearance of symptoms, suggesting that P. chrysanthemi PY35 pectate lyase PelL1 may playa minor role in soft-rot pathogenesis.

• 한국미생물·생명공학회지
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• 제19권3호
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• pp.222-227
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• 1991

Rhizobium fredii USDA193은 대두(Peking)의 근모 세포벽에 침투하여 근류를 형성한다. 본 실험에서는 전보 (1)에서 분리 보고한 R.fredii의 pectate lyase 유전자 clone(SY1)으로부터 $\Omega$변이를 시켰다. 이것을 R.fredii USDA193에 다시 marker-exchange시켜 얻은 변이주(R.fredii USDA193$\Omega$와 R.fredii USDA193omega1)의 pectate lyase 활성이 완전히 block되지 못하였다. R.fredii 내에서는 다른 종류의 pel 유전자가 존재하 것으로 생각되며 pelB 및 pelE의 Rhizobium mutants들은 근류형성 초기단계에서 직접적으로 영향을 미치지 못하였다.

• 한국식물병리학회지
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• 제10권3호
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• pp.157-162
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• 1994

Erwinia rhapontici causes soft-rot disease in a number of plants such as onion, garlic and hyacinth. There has been no report that E. rhapontici produces pectate lyase. Pel gene was cloned from genomic DNA of the parasitic soft-rot E. rhapontici polymerase chain reaction by using synthetic oligonulceotide primers designed from the pel 1 to E. carotovora. The recombinant plasmid pJE101 containing pectate lyase gene, when introduced into E. coli DH5$\alpha$, produced pectate lyase an macerated hyacinth tissue.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.277.1-277.1
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• 2002

The purpose of this study was to develop PEl-based gene carriers with optimal serum stability and reduced cytotoxicity. PEl is an efficient gene transfer agent with the ability of DNA condensation and endosome escape: however; use of the polymer in vivo is hampered by signigicant reduction in transfection activity by the presence of serum. Chitosan is a non-toxic. biodegradable and biocompatible polymer with hydrophilic functional groups so it may provide a physical stability against challenge by serum proteins. (omitted)

• 한국미생물·생명공학회지
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• 제24권5호
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• pp.553-559
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• 1996

We have designed nucleotide sequences of hEGF structural gene to eliminate the N-terminal methionine residue incorporated during the translation initiation step, and constructed recombinant human epidermal growth factor (rhEGF) secretion plasmids pYHB101, and pYHB2 in which pelB signal sequence-hEGF gene was expressed under the control of the T7, and tac promoter, respectively. We also constructed pYHB1 vector which contains rhEGF gene controlled by T7 promoter. The transformant with pYHB101 showed relatively slow growth pattern compared to the transformant with pYHB1. However, we observed that the transformant with pYHB101 secreted rhEGF of 13 mg/l significantly after 5 hr induction with 1 mM IPTG and that the T7 promoter was more effective than tac promoter when connected to pelB signal sequence. The amount of rhEGF was 14 mg/l under the sub-optimized condition.

• Applied Biological Chemistry
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• 제40권5호
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• pp.380-387
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• 1997

Pectate lyase isoenzymes을 분비하는 Erwinia carotovorn subsp. carotovora LY34는 식물조직을 연화시키는 연부균이다. 이 균주로부터 게놈 DNA를 분리하여 Sau3Al 제한효소로 부분 절단한 다음 pBluescript $SK^+$ 벡터에 클로닝하여 pectate Iyase를 분비하는 클론을 분리하였다 분리 결과 4.2 kb크기의 DNA 단편을 가지고 있었으며 이를 다시 재클로닝하여 3.1 kb크기의 pelCI유전자를 함유하는 pLYPA100을 구하였다. 이 유전자의 DNA 염기서열을 분석한 결과 374 개의 아미노산을 구성하는 1,122 bp의 ORF를 확인하였다. 시작코돈과 종결코돈은 ATG와 TAA였으며 초기 서열 22개의 아미노산으로 구성된 전형적인 원핵세포의 signal peptide가 존재하였다. PeICI의 단백질 염기서열을 다른 단백질과 유사성을 분석한 결과 Erwinia carotovera subsp. carotovora Er 균주의 PelIII, Erwinia carotevora subsp. carotovora SCR193 균주의 PeIC 및 Erwinia caretovora subsp. atroseptica C18 균주의 Pel3과 유사하였으며 PLbc family에 속하였다. PeICI의 분자량은 40,507, pI는 7.60으로 계산되었다.

• Journal of Microbiology and Biotechnology
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• 제12권5호
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• pp.753-759
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• 2002

$\beta$-Cyclodextrin glucanotransferase ($\beta$-CGTase) was overproduced extracellularly using recombinant E. coli by transforming the plasmid pECGT harboring a secretive signal peptide. The $\beta$-CGTase gene of alkalophilic Bacillus firmus var alkalophilus was inserted into the high expression vector pET20b(+) containing a secretive pelB signal peptide, and then transformed into E. coli BL2l(DE3)pLysS. The optimum culture conditions fer the overproduction of $\beta$-CGTase were determined to be TB medium containing 0.5% (w/v) soluble starch at post-induction temperature of $25^{\circ}C$. A significant amount of $\beta$-CGTase, up to 5.83 U/ml, which was nine times higher than that in the parent strain B. firmus var. alkalophilus, was overproduced in the extracellular compartment. A pH-stat fed-batch cultivation of the recombinant E. coli was also performed to achieve the secretive overproduction of $\beta$-CGTase at a high cell density, resulting in production of up to 21.6 U/ml of $\beta$-CGTase.

• Journal of Microbiology and Biotechnology
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• 제20권4호
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• pp.670-677
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• 2010

An alkaline pectate lyase, Bsp165PelA, was purified to homogeneity from the culture broth of alkaliphilic Bacillus sp. N16-5. The enzyme showed a specific activity as high as 1,000 U/mg and had optimum activity at pH 11.5 and $50^{\circ}C$. It was composed of a single polypeptide chain with a molecular mass of 42 kDa deduced from SDS-PAGE, and its isoelectric point was around pH 6.0. It could efficiently depolymerize polygalacturonate and pectin. Characterization of product formation revealed unsaturated digalacturonate and trigalacturonate as the main products. The pectate lyase gene (pelA) contained an open reading frame (ORF) of 1,089 bp, encoding a 36-amino acids signal peptide and a mature protein of 326 amino acids with a calculated molecular mass of 35.943 Da. The deduced amino acid sequence from the pelA ORF exhibited significant homology to those of known pectate lyases in polysaccharide lyase family 1. Some conserved active-site amino acids were found in the deduced amino acid sequence of Bsp165PelA. $Ca^{2+}$ was not required for activity on pectic substrates.

• Journal of Microbiology and Biotechnology
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• 제19권8호
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• pp.781-786
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• 2009

Small interfering synthetic double-stranded RNA (siRNA) was applied to suppress the expression of the human cytotoxic-T-Iymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene transformed in transgenic rice cell cultures. The sequence of the 21-nucleotide siRNA was deliberately designed and synthesized with overhangs to inactivate the expression of hCTLA4Ig. The chemically synthesized siRNA duplex was combined with polyethyleneimine (PEl) at a mass ratio of 1:10 (0.33 ${\mu}g$ siRNA:3.3 ${\mu}g$ PEl) to produce complexes. The siRNA complexes (siRNA+PEI) were labeled with Cy3 in order to subsequently confirm the delivery by fluorescent microscopy. In addition, the cells were treated with sonoporation at 40 kHz and 419W for 90 s to improve the delivery. The siRNA complexes alone inhibited the expression of hCTLA4Ig to 45% compared with control. The siRNA complexes delivered with sonoporation downregulated the production of hCTLA4Ig to 73%. Therefore, we concluded that the delivery of siRNA complexes into plant cells could be enhanced successfully by sonoporation.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.277.2-278
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• 2002

Synthetic vectors have been considered as a safer and more versatile alternative to viral-based gene delivery systems. A variety of simple synthetic vector systems such as cationic lipid- and polymer-complexed plasmid DNA were shown to have a significant transfection activity in vitro but their use in vivo has been hampered by the decrease in transfection efficiency mediated by non-specific electrostatic interactions with serum components. (omitted)