Molecular Cloning and Expression of Cellulase of Gene of Pseudomonas sp. in Escherichia coli

Chung, Young Chul,Kim, Yang Woo,Kang, shin Kwon,Rho, Jong Su,Sung, Nack Kie 한국산업미생물학회 1990 한국미생물·생명공학회지 Vol.18 No.6

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 형질전환체에서 0.7kb-와 4.6kb-HindⅢ 단편을 함유한 재조합 플라스미드 pLC1과 pLC2를 각각 분리하였다. DNA hybridization 실험에서 pLC1과 pLC2는 Pseudomonas sp. LBC 505와 CYC 10 유래임이 각각 밝혀졌고, lmmunoassay 실험에서도 유사성이 인정되었다. pLC1을 함유하고 있는 대장균은 cellulase의 24%를 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. The genes for cellulases of Pseudomonas sp. LBC505 and CYC10, potent cellulase complex-producing strains, were cloned in Escherichia coli with pUC19. Recombinant plasmids pLC1 and pLC2 were isolated from transformants producing cellulase by congo red staining, and their genes cloned were 0.7 kb and 4.6 kb HindⅢ fragments, respectively. The inserts of pLC1 and pLC2 were hybridized to chromosomal DNAs digested with HindⅢ from Pseudomonas sp. LBC505 and CYC10, respectively. Immunodiffusion assays revealed that pLC1-and pLC2-encoded cellulase showed similarity with that of host strains. About 24% of cellulase activity was observed in the extracellular fraction of E. coli carrying pLC1, and its activity was higher about 1.4 times than that of LBC505. The enzymatic properties of pLC1 and pLC2 encoded cellulase were the same as those of cellulase from host strains. HPLC analysis and substrate specificity showed that cellulases were the same as those of cellulase from host strains. HPLC analysis and substrate specificity showed that cellulases cloned were endocellulase.

Pseudomonas sp.의 Cellulase 유전자의 대장균에의 클로닝 및 발현

정영철,김양우,노종수,성낙계,강신권 한국미생물 · 생명공학회 1990 한국미생물·생명공학회지 Vol.18 No.6

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. The genes for cellulases of Pseudomonas sp. LBC505 and CYC10, potent cellulase complex-producing strains, were cloned in Escherichia coli with pUC19. Recombinant plasmids pLCl and pLC2 were isolated from transformants producing cellulase by Congo red staining, and their genes cloned were 0.7 kb and 4.6 kb HindIII fragments, respectively. The inserts of pLCl and pLC2 were hybridized to chromosomal DNAs digested with HindIII from Pseudomona~ sp. LBC505 and CYC10, respectively. Immunodiffusion assays revealed that pLC1-and pLC2-encoded cellulase showed similarity with that of host strains. About 24% of cellulase activity was observed in the extracellular fraction of E. coli carrying pLC1, and its activity was higher about 1.4 times than that of LBC505. The enzymatic properties of pLC1 and pLC2 encoded cellulase were the same as those of cellulase from host strains. HPLC analysis and substrate specificity showed that cellulases were the same as those of cellulase from host strains. HPLC analysis and substrate specificity showed that cellulases cloned were endocellulase.

Erwinia carotovora 유래의 cellulase 유전자의 클로닝 및 대장균에서의 발현

김세돈,최신건 江原大學校 産業技術硏究所 2009 産業技術硏究 Vol.29 No.B

New cellulase genes, named as CelV2 and CelN1, respectively, were isolated from Erwinia carotovora ATCC15713 and expressed in E. coli. The CelV2 and CelN1 gene were PCR amplified with degenerated primers and PCR products were sequenced and expressed in E. coli. Two new cellulase genes showed 97% homologies with previously reported Erwinia cellulase genes. The recombinant cellulase were purified with Ni-NTA column chromatography and its enzymatic properties were characterized. The optimum temperature of two enzymes were about 50℃ degree and optimum pH were around pH7.0. The newly isolated celluase genes could be used for enhancing substrate range of alcohol-producing bacteria such as Zymomonas mobilis.

Identification of novel pathogenicity-related cellulase genes in <i>Xanthomonas oryzae</i> pv. <i>oryzae</i>

Temuujin, Ulambayar,Kim, Jae-Won,Kim, Jong-Kun,Lee, Byoung-Moo,Kang, Hee-Wan Elsevier 2011 Physiological and molecular plant pathology Vol.76 No.2

<P><B>Abstract</B></P><P>Twelve genes encoding cellulases, including endo- and exoglucanases, were identified from the genomic database of <I>Xanthomonas oryzae</I> pv. <I>oryzae</I> KACC10331. The genes were amplified by polymerase chain reaction (PCR) from <I>X. oryzae</I> pv. <I>oryzae</I> KACC10859 and mutated by transposon insertion; further, marker exchange was performed with the target genes of the wild type strain. Homologous recombination events were confirmed by PCR and Southern hybridization analysis. We found that the mutant strains <I>eglXoA::Tn5</I>, <I>eglXoB</I>::<I>Tn5</I>, and <I>celXoB</I>::<I>Tn5</I> were completely virulence-deficient. In addition, mutants <I>celbXoA</I>::<I>Tn5</I>, <I>bglXoC</I>::<I>Tn5</I>, and <I>bglXoF</I>::<I>Tn5</I> showed attenuated virulence, while the virulence of other mutants was not affected.</P> <P><B>Graphical abstract</B></P><P><ce:figure id='dfig1'></ce:figure></P><P><B>Highlights</B></P><P>► Twelve cellulase genes in <I>X. oryzae</I> pv. <I>oryzae</I> genome were mutated by transposon insertion. ► Pathogenicity and complementation of the mutants were assayed on rice leaves. ► <I>eglXoA, celXoB</I>, <I>celbXoA, bglXoC</I> and <I>bglXoF</I> were identified as novel pathogenicity-related genes.</P>

Erwinia carotovora 유래의 cellulase 유전자의 클로닝 및 대장균에서의 발현

김세돈(Kim, Se-Don),최신건(Choi, Shin-Geon) 강원대학교 산업기술연구소 2009 産業技術硏究 Vol.28 No.B

New cellulase genes, named as CelV2 and CelN1, respectively, were isolated from Erwinia carotovora ATCC15713 and expressed in E. coli. The CelV2 and CelN1 gene were PCR amplified with degenerated primers and PCR products were sequenced and expressed in E. coli. Two new cellulase genes showed 97% homologies with previously reported Erwinia cellulase genes. The recombinant cellulase were purified with Ni-NTA column chromatography and its enzymatic properties were characterized. The optimum temperature of two enzymes were about 50℃ degree and optimum pH were around pH7.0. The newly isolated celluase genes could be used for enhancing substrate range of alcohol-producing bacteria such as Zymomonas mobilis.

고온 알칼리성 Bacillus sp. F204의 Cellulase 유전자의 Escherichia coli 및 Bacillus subtilis에의 Cloning 및 발현

정영철,김양우,강신권,노종수,박재현,성낙계,Chung, Young-Chul,Kim, Yang-Woo,Kang, Shin-Kwon,Rho, Jong-Su,Park, Jae-Hyeon,Sung, Nack-Kie 한국식품과학회 1991 한국식품과학회지 Vol.23 No.1

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. Cellulase genes from thermophilic alkalophilic Bacillus sp. F204 a potent cellulase complex-producing bacterium, were cloned in Escherichia coli with pUC 19. Plasmids pBC191 and pBC192, isolated from transformants forming yellow zone around colony on the LB agar plate containing 0.5% carboxymethyl cellulose and ampicillin, contained 4.6 Kb and 5.8 Kb HindIII fragments, respectively. The 4.6 Kb insert of pBC191 had single sites for BamHI EcoRI, KpnI and pvuII. DNA hybridization and immunodiffusion studies showed that pBC191-encoded cellulase gene was homologous with that of host strain. pKC231, constructed by inserting 4.6 Kb insert of pBC191 at the HindIII site of pKK223-3, E. coli expression vector, and pGC711, constructed by inserting 4.6 Kb insert of pBC191 at the HindIII site of pGR71, E. coli and B. subtilis shuttle vector, had 3.2 times and 2.8 times as much cellulase activity as pBC191, respectively. Substrate specificity analysis showed that cellulases cloned were CMCase.

Cloning and Characterization of a Bifunctional Cellulase-Chitosanase Gene from Bacillus licheniformis NBL420

( In Pyo Hong ),( Hong Ki Jang ),( Shin Young Lee ),( Shin Geon Choi ) 한국미생물생명공학회 2003 Journal of microbiology and biotechnology Vol.13 No.1

Purification, Characterization, and Gene Cloning of Chitosanase from Bacillus cereus H-l

Jang, Hong-Ki,Yi, Jae-Hyoung,Kim, Jung-Tae,Lee, Keun-Eok,Park, Shin-Geon 한국미생물 · 생명공학회 2003 한국미생물·생명공학회지 Vol.31 No.3

새롭게 분리된 Bacillus cereus H-1으로부터 크기가 45-kDa인 chitosanase를 정제하여 특성을 파악하였고 1.3-kb의 chitosanase 유전자(choA)를 대장균에 클로닝하여 발현시켰다. H-1의 chitosanase 단백질(ChoA)은 ammonium sulfate 침전과 CM-sephadex칼럼 크로마토그래피에 의해 정제하였다. 최적 pH는 약 7이었고 pH 안정성은 $50^{\circ}C$에서 4-11로 나타났다. 최적 온도는 약 5$0^{\circ}C$였으며 효소 활성은 $45^{\circ}C$ 아래에서 비교적 안정하였다. H-1 chitosanase는 soluble 또는 glycol chitosan뿐만아니라 carboxymethyl cellulose(CMC)에 대한 활성도 나타내었다. 정제된 ChoA의 MALDI-TOF MS분석에 기초하여 이미 알려진 다른 Bacillus chitosanases와의 데이터베이스 검색을 통해 전체 아미노산 서열을 밝혀내었다. Chitosanase gene에 해당하는 1.6 kb의 PCR 산물을 얻었으며 그의 DNA 서열을 결정하였다. choA의 추정 아미노산은 Bacillus sp. No 7-M과 Bacillus sp. KCTC0377BP의 아미노산과 98%의 유사성을 나타내었다. 재조합 ChoA단백질은 E. coli DH5$\alpha$에서 원 균주와 동일한 크기로 발현되었다. N말단의 추정아미노산서열을 다른 chitosanas리 서열과 비교해 볼때 ChoA는 chitosanase-cellulase 활성을 갖는 family 8에 속하는 미생물 endo-chitosanaseT. 추정되었다. A 1.3-kb of chitosanase gene (choA) encoding 45-kDa polypeptide was cloned, expressed, and characterized from a newly isolated Bacillus cereus H-1. The chitosanase protein (ChoA) of B. cereus H-l was purified to homogeneity by ammonium sulfate precipitation and CM-sephadex column chromatography. Optimum pH was around 7, and stable pH range in the incubation at 50 C was 4-11. Optimum temperature was around 50 C, and enzyme activity was relatively stable below 45 C. ChoA showed the activities toward carboxymethyl cellulose (CMC) in addition to soluble or glycol chitosan. Based on MALDI-TOF MS analysis of purified ChoA, the entire amino acid sequence of ChoA was interpreted by database searching of previously known Bacillus chitosanases. A 1.6 kb of PCR product of corresponding chitosanase gene was obtained and its DNA sequence was determined. The deduced amino acid of choA revealed that ChoA have a 98% homology with those of Bacillus sp. No.7-M strain and Bacillus sp. KCTC0377BP. The recombinant ChoA protein was expressed in E. coli DH5$\alpha$. Deduced amino acid comparison of choA with other chitosanases suggested that it belongs to family 8 microbial endo-chitosanase with chitosanase-cellulase activity.

Molecular Cloning and Expression of Cellulase Gene from Pseudomonas sp. in Escherichia coli

SUNG, NACK KIE,SHIM, KI HWAN,CHUNG, YOUNG CHUL,KIM, YANG WOO 경상대학교 유전공학연구소 1989 遺傳工學硏究所報 Vol.8 No.-

The genes for cellulases of Pseudomonas sp. LBC 505 and CYC 10, a potent cellulase complex-producing strain, were cloned in E. coli with pUC 19. Plasmid pLC 1, pLC 2 and pLC 3, were isolated from the transformants producing cellulase by congo-red staining, and their genes cloned were in 0.5-,2.1-, and 4.6Kb Hind III fragments, respectively. The inserts of pLC1 and pLC3 were hybridized to chromosomal DNAs digested with Hind III from Pseudomonas sp. LBC 505 and CYC 10, respectively. Immunodiffusion assays revealed that pLC 1 and pLC 3-encoded cellulase showed homology with that of host strains. About 82% of cellulase activity was observed in the extracellular fraction of E. coli carrying pLC 1, and its activity was higher about 1.4 times than that of LBC 505. The enzymatic properties of pLC 1, pLC 2 and pLC 3-encoded cellulase were the same as those of cellulase from host strains. HPLC analysis showed that cellulases cloned were endocellulase.

Research articles : Cloning and Sequence Analysis of the Cellobiohydrolase I Genes from Some Basidiomycetes

( Ekachai Chukeatirote ),( Sajeewa S. N. Maharachchikumbura ),( Shannaphimon Wongkham ),( Phongeun Sysouphanthong ),( Rungtiwa Phookamsak ),( Kevin D. Hyde ) 한국균학회 2012 Mycobiology Vol.40 No.2

Genes encoding the cellobiohydrolase enzyme (CBHI), designated as cbhI, were isolated from the basidiomycetes Auricularia fuscosuccinea, Pleurotus giganteus, P. eryngii, P. ostreatus, and P. sajor-caju. Initially, the fungal genomic DNA was extracted using a modified cetyltrimethyl ammonium bromide (CTAB) protocol and used as a DNA template. The cbhI genes were then amplified and cloned using the pGEM-T Easy Vector Systems. The sizes of these PCR amplicons were between 700~800 bp. The DNA sequences obtained were similar showing high identity to the cbhI gene family. These cbhI genes were partial consisting of three coding regions and two introns. The deduced amino acid sequences exhibited significant similarity to those of fungal CBHI enzymes belonging to glycosyl hydrolase family 7.