• 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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• 제2권1호
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• pp.50-55
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• 1992

A genomic library of a mesophilic cellulolytic anaerobe, Clostridium sp. KCTC 8440 DNA was constructed in Escherichia coli using plasmid pUC9. Clones of E. coli exhibiting carboxymethyl cellulose-hydrolyzing activity (CMCase) were isolated and divided into seven types based on the restriction enzyme patterns of recombinant plasmids. E. coli strains carrying type A genes showed activity on carboxymethyl cellulose about 7-8 times greater than clones carrying genes of other types. Restriction maps of the cloned DNA fragments were determined, and homologies between them were investigated. The results suggest that Clostridium sp. KCTC 8440 has seven distinct CMCase genes.

• 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.

• Journal of Microbiology
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• 제42권3호
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• pp.205-210
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• 2004

The sequence coding for carboxymethylcellulase (CMCase, CelC) was isolated from the DNA of Salmonella typhimurium URl. Comparison between the deduced amino acid sequence of CelC (368 amino acid residues, Molecular mass 41 kDa) and that of the previously published CMCase revealed that this enzyme belongs to the cellulase family 8 and D. The protein was overproduced in Escherichia coli using T7 expression system, and its activity was confirmed by CMC-SDS-PAGE. When the overexpressed CelC protein was tested on cellulose-type substrates, the recombinant protein is able to degrade cellulose-type substrates, such as CM-cellulose, xylan, avicel, lichenan, and laminarin. Optimal temperature and pH for enzyme activity were found to be 50$^{\circ}C$ and pH 6.5, respectively.

• 한국미생물·생명공학회지
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• 제15권5호
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• pp.346-351
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• 1987

Clostridium thermocellum의 cellulase 유전자를 pBR322를 이용하여 Escherichia coli에 cloning하였다. 삽입된 Hind III 분해 DNA 단편의 크기는 약 1. 8kb였으며 이미 알려진 C. themocellum의 cellulase gene과는 다른 제한효소 분해위치를 가진 새로운 cellulase gene으로서 E, coli에서 CMCase와 FPase 활성을 나타내었다. 이 유전자는 생육의 전시기를 통해 표현되었고 고온성 cellulase의 구조적, 기능적 특성이 그대로 유지되었을 뿐만 아니라 상당량이 세포밖으로 분비되었다.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.524.3-524
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• 1986

Carboxymethyl cellulase (CMCase) produced by cloned B. megaterium was found to contain 5.2% carbohydrate but no metal ion. The enzyme was isoelectric at pH 7.23 and was high is basic amino acids. The N-terminal of the enzyme was glutamic acid. The cellulolytic activity of this enzyme was extended to the small molecular substrates such as from cellotriose to cellopentaose. In additon, the enzyme showed transglycoslation activity. The pK values of the enzyme we estimated to be 4.4 and 6.7, andthat of the enzyme-substrate complex were 4.2 and 7.2, respectively. The enzyme was not affected by the treatment with iodoacetic acid, but the modification of enzyme with carbodiimide and diethyl pyrocarbonate resulted in a marked loss of the enzyme activity. These results suggest that the active site of enzyme essentially contains carboxylic and imidazole group of amino acid residues.

• 한국식품과학회지
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• 제23권1호
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• pp.31-36
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• 1991

고온, 알칼리성 Bacillus sp. F204의 CMCase 유전자를 pUC19의 HindIII부위에 연결하여 전이된 E.coli 형질전환체 중 2개의 재조합 플라스미드 즉, pBC191과 pBC192를 선발하였는데, 이들은4.6 Kb와 5.8 Kb HindIII 절편을 각각 함유하고 있었다. pBC191의 4.6 Kb HindIII 절편을 BamHI, EcoRI, KpnI, PvuII 부위가 각각 1개씩 존재하였다. Dioxigenin-labeled deoxyuridin-triphosphate에 4.6 Kb 절편을 표식한 것을 probe로 하여 상동성을 검정한 결과 모균주와 강한상동성이 없었고, 면역학적 실험에서도 Bacillus sp. F204 유래임이 인정되었다. pBC191의 4.6 Kb 절편을 E.coli의 발현 벡타인 pKK223-3과 Bacillus vector인 pGR71에 연결시켜 구축한 pKC231과 pGC711은 각각 pBC191에 비하여 효소활성이 3.2배와 2.8배정도 증가되었으며, 그리고 E.coli에서는 대부분 세포내와 periplasmic 분획에서 검출되었다. 기질 특이성을 조사한 결과에 의하면 pBC191과 pBC192는 CMCase gene을 코딩하고 있는 것으로 나타났다.

• 한국식품영양학회지
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• 제6권4호
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• pp.314-321
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• 1993

We have cloned the Bacillus cellulyticus K-12 avicelase (Avi, E.C.3.2.1.4) gene (ace A) In E. coli. This was accompanied by using the vector PT7T3U 19 and Hind W -Hind m libraries of Bacillus cellulyticus K-12 chromosomal inserts created in 5.cofi. The Libraries were screened for the expression of avicelase by monitoring the immunoreaction of the anti-avicelase (immunoscreening). Positive clones (Ac-3, Ac-5, and Ac-7) contained the identical 3.5kb Hind III fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constituvely using its own promoter in the heterologous host. From the immunoblotting analysis, a polypeptide which showed a CMCase activity with an Mr of 54000 was detected.

• Journal of Microbiology and Biotechnology
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• 제22권3호
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• pp.390-399
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• 2012

Endoglucanase gene egIV was cloned from Trichoderma viride AS 3.3711, an important cellulose-producing fungus, by using an RT-PCR protocol. The egIV cDNA is 1,297 bp in length and contains a 1,035 bp open reading frame encoding a 344 amino acid protein with an estimated molecular mass of 35.5 kDa and isoelectronic point (pI) of 5.29. The expression of gene egIV in T. viride AS 3.3711 could be induced by sucrose, corn straw, carboxymethylcellulose (CMC), or microcrystalline cellulose, but especially by CMC. The transcripts of egIV were regulated under these substrates, but the expression level of the egIV gene could be inhibited by glucose and fructose. Three recombinant vectors, pYES2-xegIV, $pYES2M{\alpha}$-egIV, and $pYES2M{\alpha}$-xegIV, were constructed to express the egIV gene in Saccharomyces cerevisiae H158. The CMCase activity of yeast transformants $IpYES2M{\alpha}$-xegIV was higher than that of transformant IpYES2-xegIV or $IpYES2M{\alpha}$-egIV, with the highest activity of 0.13 U/ml at induction for 48 h, illustrating that the modified egIV gene could enhance CMCase activity and that $MF{\alpha}$ signal peptide from S. cerevisiae could regulate exogenous gene expression more effectively in S. cerevisiae. The recombinant EGIV enzyme was stable at pH 3.5 to 7.5 and temperature of $35^{\circ}C$ to $65^{\circ}C$. The optimal reaction condition for EGIV enzyme activity was at the temperature of $55^{\circ}C$, pH of 5.0, 0.75 mM $Ba^{2+}$, and using CMC as substrate. Under these conditions, the highest activity of EGIV enzyme in transformant $IpYES2M{\alpha}$-xegIV was 0.18 U/ml. These properties would provide technical parameters for utilizing cellulose in industrial bioethanol production.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2002년도 제37회 국제학술심포지움 및 추계학술대회
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• pp.65-65
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• 2002