Fomitopsis pinicola 균사체로부터 Endoglucanase의 최적생산

구지민 ( Ji Min Gu ),박상신 ( Sang Shin Park ) 한국미생물생명공학회(구 한국산업미생물학회) 2013 한국미생물·생명공학회지 Vol.41 No.2

소나무잔나비버섯(Fomitopsis pinicola MKACC 54347) 의 균사체로부터 endoglucanase를 생산하기 위한 최적 배양조건을 조사하였다. 복합배지 중 MSM이 가장 높은 endoglucanase 활성을 나타내었으며, MSM의 성분과 농도를 각각 2.0% CMC, 2.0% yeast extract, 0.2% KH2PO4, 0.03% MnSO4 및 0.3% trace metal 용액으로 첨가하였을 때 효소활성이 가장 우수하였다. 따라서 F. pinicola로부터 endoglucanase를 생산하기 위한 최적 배지조건은 2.0% CMC, 2.0% yeast extract, 0.2% KH2PO4, 0.03% MnSO4 및 0.3% trace metal 용액이다. 이상의 배지를 사용하여 배양온도 25oC에서 8일 동안 배양하였을 때 최대로 효소 활성이 나타내는 것을 확인하였다. CMC를 기질로 사용한 activity staining의 결과를 통해 F. pinicola 균사체의 endoglucanase 활성여부를 확인할 수 있었으며 그 분자량이 43-70 kDa 임을 확인하였고, 배양액 중의 효소활성의 최적 pH와 온도는 각각 pH 5.0과 55oC인 것으로 나타났다. The culture conditions to maximize the production of endoglucanase (EC 3.2.1.4) from the brown rot fungus Fomitopsis pinicola MKACC 54347 mycelia were investigated. Among the tested media for endoglucanase production, Mandel`s mineral salts medium (MSM; 1% cellulose, 0.1% peptone, 0.14% (NH4)2SO4, 0.03% urea, 0.2% KH2PO4, 0.03% MgSO4·7H2O, 0.03% CaCl2, and 0.1% trace metal solution (19.8 mM FeSO4, 13.0 mM MnSO4, 12.2 mM ZnSO4, and 15.4 mM CoCl2)) produced the highest activity of the enzyme. To optimize the medium composition for enzyme activity, the effects of various carbon, nitrogen, phosphorus, and inorganic sources were investigated in MSM. Maximal enzyme production was accomplished using a medium containing 2% carboxymethyl cellulose (CMC), 2% yeast extract, 0.2% KH2PO4, 0.03% MnSO4, and 0.3% trace metal solution. Different physiological conditions, like incubation period and temperature, were also examined to assess their influence on enzyme production. Enzyme production from F. pinicola reached its highest level after cultivation for 8 days at 25oC. Nondenaturing polyacrylamide gel electrophoresis (PAGE), followed by the endoglucanase activity staining using CMC as the substrate, was performed to identify the endoglucanase under the culture conditions studied. Zymogram analysis of the culture supernatant revealed an endoglucanase band with a molecular mass of 52 kDa. The optimum pH and temperature for enzyme activity were 55oC and pH 5.0, respectively.

Overproduction f Pseudomonas sp. LBC505 Endoglucanase in Escherichia coli and Bacillus Subtilis

Chung, Young chul,Kim, Kyeong Sook,Kim, Yang Woo,Chun, Sung Sik,Sung, Nack Kie 한국미생물 · 생명공학회 1995 Journal of microbiology and biotechnology Vol.5 No.1

Endoglucanase gene of Pseudomonas sp. LBC505 was previously cloned in pUC19 to yield plasmid pLC1. overproduction of endoglucanase was attempted by following ways. First, the endoglucanase gene of Pseudomonas sp. LBC505 cloned in pUC19(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 plasmids showed the higher yield of the endoglucanase. E.coli harboring pKCC30 which had three inserted endoglucanase genes expressed about 12.3 times as much CMCase activity as E.coli harboring pLC1. 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 pLC1 and the endoglucanase produced was entirely secreted into the culture medium.

재조합 효모 세포의 고농도배양을 통한 섬유소와 자일란 분해효소 유전자의 동시 과발현

김연희,허선연,김군도,남수완 한국미생물·생명공학회 2018 한국미생물·생명공학회지 Vol.46 No.4

For the coexpression of endoxylanase and endoglucanase genes in yeast Saccharomyces cerevisiae, the genes were separately inserted downstream of the yeast ADH1 promoters, resulting the plasmid pAGX3 (9.83 kb). In the batch culture on YPD medium of the yeast transformant, S. cerevisiae SEY2102/pAGX3, the total activities of the enzymes reached about 7.91 units/ml for endoxylanase and 0.43 units/ml for endoglucanase. In the fed-batch culture with intermittent feeding of yeast extract and glucose, the total activities of 24.9 units/ml for endoxylanase and 0.84 units/ml for endoglucanase were produced which were about 3.1- fold and 2.0-fold increased levels, respectively, compared to those of the batch culture. Most of endoxylanase and endoglucanase activities were found in the extracellular media. This recombinant yeast could be useful for the development of simultaneous saccharification bioprocess of the cellulose and xylan mixture. Endoxylanase와 endoglucanase 유전자가 ADH1 프로모터 하류에 따로따로 삽입된 pAGX3 플라스미드를 함유한Saccharomyces cerevisiae에서 endoxylanase와 endoglucanase 유전자는 성공적으로 발현되었으며, YPD 배지에서의 회분배양 결과, endoxylanase는 7.91 units/ml, endoglucanase 는 0.43 units/ml에 달하는 총활성을 보였다. Yeast extract 와 포도당을 간헐적으로 공급하는 유가배양에서 endoxylanase 와 endoglucanase의 총활성은 24.9 units/ml과 0.84 units/ ml을 각각 보였으며, 이는 회분배양에서 발현된 각각 활성의 3.1배와 2배에 해당되었다. 또한, 대부분의 endoxylanase 와 endoglucanase 활성은 세포밖 배지에서 측정되어, 향후이 재조합 효모는 섬유소(cellulose)와 xylan 혼합물의 동시당화 바이오공정 개발에 활용될 가능성이 높다 하겠다.

Expression of Clostridium thermocellum Endoglucanase Gene in Lactobacillus bulgaricus and Lactobacillus plantarum and in vitro Survival Characteristics of the Transformed Lactobacilli

Cho, J. S.,Kang, S. H.,Lee, H. G.,Lee, H. J.,Woo, J. H.,Moon, Y. S.,Yang, C. J.,Choi, Y. J. 한국동물자원과학회 2003 한국축산학회지 Vol.45 No.4

다양한 미생물들로부터 유래한 cellulase 중에서, 특히 장내 단백질 가수분해효소에 안정한 Clostridium thermocellum 균주 유래의 endoglu-canase를 선별하였다. 그 후 그 유전자의 자체프로모터에 의해 발현되는 재조합 Lactobacillus용 발현벡터를 구축하였고, 그 발현벡터를 pSD1이라 명명하였다. 이 발현벡터를 L. bulgaricus와 L. plantarum 균주에 각각 전기천공법을 이용하여 형질전환시키는데 성공하였으며 그 재조합 균주들로부터 endoglucanase 효소역가를 조사한 결과 각각 배지 상층액에서 0.12, 0.144U/㎖로 조사되었다. 한편 이들 균주들의 생균제로 갖추어야할 특성인 내산성, 내담즙성 및 항생제내성 여부를 조사한 결과, 이들 균주들은 모두 pH3과 같은 산성 조건하에서도 안정하였으며, 내담즙성에 있어서는 특히 L. plantarum 균주의 경우 0.3, 1%의 oxgall에서도 안정하였다. 또한 항생제 내성을 조사한 결과 두 균주 모두 amikacit gentamicin, kanamycif colistin에 저항성이 높은 것으로 나타났다. Endoglucanase A from Clostridium thermocellum which is resistant to pancreatic proteinase was selected out of numbers cellulases then were expressed in lactobacilli. Recombinant lactobacilli expression vector, pSD1, harboring the endoglucanase gene from C. thermocellum under control of its own promoter, was constructed. Both L. bulgaricus and L. plantarum were electrotransfomed with pDS1, the eddoglucanase activities of 0.120 and 0.144 U/㎖ were found in culture media of L. bulgaricus and L. plantarum containing pSD1, respectively. In vitro survival characteristics of the transformed lactobacilli were tested. Both L. bulgaricus and L. plantarum showed a similar resistance to low ? 3. Moreover, L. plantarum showed a rather homogenous resistant pattern against the tested antibioties. Both of the strains were resistant to amikacin, gentamicin, streptomycin, kanamycin, and colistin.

Saccharomyces cerevisiae에서 Trichoderma Endoglucanase의 발현과 분비

남수완,김병우,신동하,김재범,신지원,정대균,정춘수 동의대학교 기초과학연구소 1999 基礎科學硏究論文集 Vol.9 No.1

The endoglucanase gene, egl6, of Trichoderma sp. was connected with the yeast ADH1 promoter, and the resultant plasmid, pVT-C4, was introduced into three S. cerevisiae host strains (YNN27, 2805, and SEY2102). Among each 80 transformants, the cell growth and expression level of endoglucanase were compared in test-tube cultivation, and three respective transformants for each host cells showing the highest expression level and cell growth were selected. When three recombinant yeast cells were batchwise cultivated for 48 hr in flask, the total activities of endoglucanase expressed were about 1140 unit/1 with 2805/pVT-C4, 1020 unit/l with SEY2102/pVT-C4, and 590 unit/l with (YNN27/pVT-C4. Irrespective of host strain, about 80% of the expressed endoglucanase was detected in the extracellular medium. In addition, it was also found that the recombinant enzyme was secreted into the culture medium as two major forms of lightly and heavily glycosylated proteins.

Cloning of the Endoglucanase Gene from Actinomyces sp. 40 in Escherichia coli and Some Properties of the Gene Products

Min, Hae Ki,Choi, Yun Jaie,Cho, Kwang Keun,Ha, Jong Kyu,Woo, Jung Hee 한국미생물 · 생명공학회 1994 Journal of microbiology and biotechnology Vol.4 No.2

The β-1,4-endoglucanase gene from Actinomyces sp. 40 was cloned into Escherichia coli DH5α with pUC19. Chromosomal DNA from Actinomyces sp. 40 was cleaved with the restriction enzyme Sau3Al and ligated into pUC19 for the transformation of Escherichia coli DH5α. Positive clones of β-1,4-endoglucanase gene were detected as the clear zones on a medium supplemented with carboxymethylcellulose (CMC). This transformant possessed a single plasmid, designated pDS1, which contained the vector DNA and a 3.5 kilobase (kb) Sau3Al insertion fragment encoding endoglucanase. The size of the cloned fragment was reduced to 2.0 kb. The endoglucanase activity produced by the E. coli DH5a (pDS6) was higher than that of Actinomyces sp. 40 strain. The optimum pH and temperature of the cloned enzyme were pH 4.0∼5.0 and 55℃, respectively. The cloned enzyme was stable at 55℃ or below and in buffer ranging from pH 4.0 to 7.0. The enzyme degraded CMC but did not degrade xylan, cellobiose, and methyl-umbelliferylcellobiopyranoside (MUC).

Pseudomonas sp. 유래 Endo-1,4-β-Glucanase 및 β-1,4-Glucosidase 유전자의 안정성 개선

김양우,전성식,정영철,노종수,성낙계 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.6

이미 분리된 Pseudomonas 유래 endoglucanase와 β-glucosidase 유전자의 안정성을 개선하기 위하여 plasmid pDR1453에 존재하는 recA promoter의 downstream에 endoglucanase와 β-glucosidase 유전자를 각각 subcloning 하여 재조합 plasmid pDRE10과 pDRG20을 구축하였다. 이 재조합체를 E. coli 1100의 염색체 DNA 상에 존재하는 recA gene에 도입시켜 plasmid 안정성을 항생물질 저항성으로 조사하였을때 안정성은 완전히 증가하였음이 확인되었다. 또한 pDRE10과 pDRG20을 함유하고 있는 형질전환체 E. coli 1100은 recA promoter에 의해 발현이 용이하여고 nalidixic acid의 유도로 각각 endoglucanese와 β-glucosidase 생성을 촉진시켰다. 염색체 DNA에 재조합 plasmid의 도입으로 인한 plasmid 안정성 및 효소활성 개선은 대량배양에 의한 물질생산에 하나의 모델로서 사용할수 있을 것으로 예상된다. To improve stability of recombinant DNA pLC1 encoding endoglucanase gene and pGL1 encoding β-glucosidase gene. DNA fragments of genes coding endoglucanase and β-glucosidase were cloned within the recA gene on a pDR1453, and the pDRE10 and pDRG20 of recombinant plasmids were integrated into the recA gene one the E. coli 1100 chromosomal DNAs. The stability of inheritance was completely maintained in E. coli 1100; Transformants E. coli 110/pDRE10 and pDRG20 were expressed well by recA promoter and increased endoglucanase and β-glucosidase activities. This method can be used as a model to improve the stability of recombinant plasmid in large scale culture.

Saccharomyces cerevisiae에서 Clostridium thermocellum Endoglucanase 유전자의 구성적 발현

남수완,정대균,정봉현 동의대학교 기초과학연구소 1998 基礎科學硏究論文集 Vol.8 No.1

To develop an effective and powerful yeast probiotics, Saccharomyces cerevisiae strains producing cellulolytic enzymes were genetically engineered. We constructed two plasmids in which the endoglucanase A gene, celA, of Clostridium thermocellum was connected in frame with ADH1 or GAPDH promoter. These plasmids were transformed into various S. cerevisiae host strains, and then the cell growth, expression level and plasmid stability between the transformed yeast cells were examined in the flask culture. The difference in genetic background of host strains did affect significantly the cell growth and expression level of endoglucanase. Based on the higher levels of cell growth(5.4∼5.9 g-DCW/L) and plasmid stability(79∼85%), three host cells(YNN27, 2805 and SEY2102) and ADH1 promoter were selected as optimal host-vector systems for the constitutive expression of celA. The recombinant yeast produced about 200 unit/L of endoglucanase as a growth-associated manner in the batch fermentation, due to the increased cell growth of 11∼13 g-DCW/L. In addition, 25∼38% and 53∼64% of the endoglucanase activity were detected in the culture medium and periplasmic space, respectively. These results indicate that the signal sequence of celA functioned well in S. cerevisiae cells and the recombinant yeast cells can be employed for the production of probiotics.

Cel8H, a Novel Endoglucanase from the Halophilic Bacterium Halomonas sp. S66-4: Molecular Cloning, Heterogonous Expression, and Biochemical Characterization

Xiaoluo Huang,Fei Huang,Hui Wang,Zongze Shao,Yuzhi Hong,Ling Lin,Chanjuan Li,Ziduo Liu 한국미생물학회 2010 The journal of microbiology Vol.48 No.3

A recombinant Escherichia coli clone expressing an endoglucanase was identified from a genomic library of the halophilic bacterium Halomonas sp. S66-4, and the enzyme was designated Cel8H. The cel8H gene consisted of 1,053 bp and encoded 350 amino acids sharing the highest identity of 48% to other known endoglucanases. The protein was expressed in E. coli BL21 (DE3) and purified to homogeneity. The purified recombinant enzyme had an optimal activity of 4.9 U/mg at pH 5 and 45°C toward the substrate carboxymethylcellulose. It exhibited extraordinary properties which differed from endoglucanases reported previously at the point of high salt tolerance above 5 M, simultaneously with high pH stability at pH 4-12 and high temperature stability at 40-60°C. Various substrate tests indicated that the enzyme hydrolyzes β-1,4-glucosidic bonds specifically.

Whole-genome de novo sequencing of wood rot fungus Fomitopsis palustris (ATCC62978) with both a cellulolytic and ligninolytic enzyme system

Hong, C.Y.,Lee, S.Y.,Ryu, S.H.,Kim, M. Elsevier Science Publishers 2017 Journal of biotechnology Vol.251 No.-

<P>Fomitopsis palustris is a model brown rot fungus causing destructive wood decay based on the cellulase system. Endoglucanase secreted by F. palustris hydrolyzes cellulose in both the crystalline and amorphous form. In this study, whole-genome sequencing was conducted to identify genes related to F. palustris cellulose degradation and their functions. We determined the 43-Mb complete draft genome of F. palustris (ATCC 62978), comprising 14,592 predicted gene models. Gene annotation provided crucial information about the location and function of protein-encoding genes. Three types of endoglucanases were expressed: endo-1,3-beta-glucanase, endo-1,4-betaD- glucanase, and endoglucanase. In addition, various ligninolytic enzymes such as laccase, aromatic compound dioxygenase, and aryl alcohol dehydrogenase were expressed in F. palustris (ATCC 62978). Colony polymerase chain reaction (PCR) indicated that the endo-1,4-beta-D-glucanase gene comprises 732 bp. Optimization of the expression conditions of endoglucanase by real-time PCR revealed that endoglucanase was highly expressed after 7 days in all conditions, which was secreted during the secondary metabolism. Studies for large-scale cellulase production from this fungus and investigation of its ligninolytic system will promote its extensive use in various applications. The genomic information determined herein provides a basis for molecular genetics studies to understand the genome functions of F. palustris (ATCC 62978).</P>