• 한국미생물·생명공학회지
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• 제21권6호
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• pp.549-555
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• 1993

The production of cellulase by Pseudomonas sp. LBC505 isolated was under the strict genetic and biochemical control mechanisms such as catabolit repression and induction. These biochemical control reduced cellulase production. Thus LBC505 was mutated to increase enzyme yields. Cells growth and cellulase production were inhibited by the addition of 2-deoxy glucose (2-DG), which is presumed to function as repressor for the selection of high cellulase yielding mutant.

• 한국미생물·생명공학회지
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• 제18권6호
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• pp.633-639
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• 1990

Cellulase 복합체와 xylanase를 동시에 분비하는 Pseudomonas sp. LBC 505와 CYC 10의 cellulase 유전자를 pUC19를 사용하여 E.coli에 클로닝시켰다. Congo red 염색시 노란색 환을 형성하는 대장균 형질전환에서 7.0Kb-와 4.6Kb-HindIII 단편을 함유한 재조합 플라스미드 pLC1과 pLC2를 가각 분리하였다. DNA hybridization 실험에서 pLC1 과 pLC2는 Pseudomonas sp. LBC 505와 CYC 10 유래임이 각각 밝혀졌고, Immunoassay 실험에서도 유사성이 인정되었다. pLC1을 함유하고 있는 대장균은 cellulas의 24를 세포외로 분비하였고, 효소활성은 모균주에 비해 1.4배 증가하였다. pLC1과 pLC2의 효소학적 성질도 모균주와 동일하였으며, 기질특이성과 HPLC로 유리당을 분석한 결과, 클로닝된 유전자는 endo type인 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제5권1호
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• pp.14-17
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• 1995

Endoglucanase gene of Pseudomonas sp. LBC505 was previously cloned in pUC19 to yield plasmid pLCl. The Pseudomonas sp. LBC505 endoglucanase gene was subcloned in a temperature-regulated Es-cherichia coli expression vector, pAS1, containing the leftward promoter $P_L$ of bacteriophage lambda. The level of gene expression was controlled by the thermal inactivation of the heat-sensitive lambda cI857 repressor. Best yield of endoglucanase was obtained by lowering the incubation temperature to $37^{\circ}C$ after induction at $42^{\circ}C$ for 1h. Under these conditions enzyme production continued for about 5h at a gradually decreasing rate. Ecoli harboring recombinant plasmid pASC10 expressed 4.3 times as much CMCase activity as E.coli containing pLCl. To enhance the expression level of endogl, ucanase gene, we have also changed the presumptive Shine-Dalgamo sequence (AGAGGT) of the gene to consensus sequence (AGGAGGT) by site-directed mutagenesis. The genes mutated were subcloned in pASl resulting in the formation of recombinant plasmid pASS50. E.coli harboring the plasmid pASS50 expressed 6.2-fold higher levels of CMCase activity than that of E.coli harboring pLC1.

• Journal of Microbiology and Biotechnology
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• 제5권1호
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• pp.18-21
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• 1995

Endoglucanase gene of Pseudomonas sp. LBC505 was previously cloned in pUCl9 to yield plasmid pLC1. overproduction of endoglucanase was attempted by following ways. First, the endoglucanase gene of Pseudomonas sp. LBC505 cloned in pUCl9(pLC1) was tandemly inserted, step by step, into a expression vector pKK223-3 in a directly repeated form to enhance productivity of endoglucanase. Escherichia coli containing pKCC30 among the resulting plasm ids showed the higher yield of the endoglucanase. Ecoli harboring pKCC30 which had three inserted endoglucanase genes expressed about 12.3 times as much CMCase activity as Ecoli harboring pLCl. Second, the endoglucanase gene was subcloned into Bacillus subtilis expression vector pgnt41 for both overproduction and extracellular secretion of the endoglucanase. A resulting plasmid pgntc15 in Bacillus subtilis expressed 4.3-fold higher levels of CMCase activity than that of E.coli harboring pLCl and the endoglucanase produced was entirely secreted into the culture medium.

• 한국미생물·생명공학회지
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• 제19권4호
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• pp.331-336
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• 1991

섬유성 물질을 발효기질로서 사용하기 위하여, 분리균 Pseudomonas sp. LBC-505의 cellulase를 이용하여 여러 종류의 천연 섬유성 물질에 대한 당화실험을 행하였다. Cellulase 복합체의 생산은 glucose에 의하여 저해되었고 CMC, avicel, 밀기울, 볏짚 등의 섬유성 물질에 의해 유도되었으며, 55(w/v) 밀기울 배지에서 최대 효소활성을 나타내었다. CMCase 와 xylanase의 최적 효소 반응온도는$50^{\circ}C$였으며, $\beta$-glucosidase는 $55^{\circ}C$ 였다. 또한, 이들 효소의 최적 반응 pH 는 모두 6.6이었다. 조효소 단독처리에 의한 천연섬유소의 당화율은 낮게 나타났으나, 5%(v/v) HCl로 실온에서 24시간 전처리한 후 효소반응을 행한 결과 가수분해율이 18.4%(w/w)로 볏집이 가장 양호하였으며, 구성당은 주로 glucose, xylose 및 cellobiose 였다.

• 한국미생물·생명공학회지
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• 제21권2호
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• pp.113-118
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• 1993

Fro the purpose of producing glouse from cellobiose or oligo saccharide and obtaining genetic information of beta-1,4-glucosidase gene, alpha beta-1,4-glucosidase gene of Pseudomonas sp. LBC505, potent cellulase complex and xylanase producing strain, was cloned in Esherichia coli and Bacillus subtilis into pUC19 and pBD64, respectively. Recombinant plasmid pGL1 contained 1.2kb EcoRI fragment was isolated from transformants forming blue color around colony on LB agar plate containing 20 ng/ml of 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside(X-glu) and ampicillin.