Bacillus subtilis BS 62의 γ-Glutamyltranspeptidase 유전자

이태은(Tae-Eun Lee),윤민호(Min-Ho Yoon),최우영(Woo-Young Choi) 충남대학교 농업과학연구소 2007 Korean Journal of Agricultural Science Vol.34 No.2

Poly(γ-glutamic acid) 및 levan의 생성균주로 알려진 Bacillus subtilis BS 62의 γ-GTP(ggt) 유전자를 해석하기 위하여 PCR 반응에 의해 BS62의 염색체 DNA로부터 약 2.5 kb의 γ-GTP(ggt) 유전자 분획을 얻어 그 PCR 산물의 염기서열을 분석하여 기왕에 보고된 기타의 ggt 유전자와 비교 분석한 결과, B. subtilis γ-GTP 유전자(BSU49358)와 98%의 높은 상동성을 보였으며, Pseudomonas sp. A14(S63255)와는 37%, 방선균인 Streptomyces avermitils(AP005028)의 게놈 DNA와는 38%의 상동성을 나타냈다. BS 62의 γ-GTP 유전자의 open reading frame은 587개의 amino acid로 구성된 polypeptide의 것으로 해석되었으며, N-terminal의 28개 아미노산은 B. subtilis 펩타이드의 전형적인 형태를 보였고, 전형적인 리보솜의 부착부위는 개시코돈 ATG의 위쪽 7번에서 12번 염기(AGGAGG)에 위치하였고, 그리고 종지코돈 다음에서는 stem-loop 구조, ORF의 위쪽 약 50 bp 지점에서는 cataboliteresponsive element가 발견되었다. 또한 B. subtilis 효소의 촉매자리로 추정되는 467번 잔기는 threonine으로서, 다른 박테리아의 serine, 포유동물의 cysteine과는 구별되는 것이었다. To characterize γ-glutamyltranspeptidase ( γ-GTP or ggt; EC 2. 3. 2. 2.) gene of Bacillus subtilis BS 62, the γ-GTP gene of BS 62 was prepared from PCR products amplified with the chromosomal DNA. The γ-GTP gene of about 2.5 kb was sequenced, and its homology was compared with the other ggt genes which were reported previously. The base sequence of the gene appeared to have an open reading frame of 1,758 bp encoding a protein of 62,175 Da. The coding region was flanked by putative ribosome binding site - AGGAGG of 7th to 12th upstream - and the stem-loof sequence was followed by transcription terminator codon. Homology of the amino acid residues sequence consisting of 587 amino acid residues was found as 98% with Bacillus subtilis gene (BSU49358), 97.4% with that of Bacillus subtilis KX 102, 37% with Pseudomonas sp. A14 (S63255) and 38% with Streptomyces avermitils (AP005028).

Role of bacterial γ-glutamyltranspeptidase as a novel virulence factor in bone-resorbing pathogenesis

Jinmoon Kim,장성일,Aeryun Kim,Hanfu Su,Niluka Gunawardhana,Yeong-Eui Jeon,Eun Jung Bak,김지혜,차정헌 한국미생물학회 2016 The journal of microbiology Vol.54 No.5

Mammalian γ-glutamyltranspeptidase (GGT) has been identified as a bone-resorbing factor. Since GGT of Bacillus subtilis exhibits similarity in their primary structure and enzymatic characteristics with mammalian GGTs, the bone-resorbing activity of bacterial GGT was examined in this study. Osteoclastogenesis was performed in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. A conditioned medium from GGT-overproducing B. subtilis culture showed significantly higher activity of osteoclast formation than a conditioned medium from wild-type B. subtilis culture. Recombinant GGT (rGGT) of wild-type B. subtilis and an enzymatic activity-defected rGGT of B. subtilis 2288 mutant were expressed in Escherichia coli and purified using His tag. Both purified rGGTs induced similar levels of osteoclastogenesis, suggesting that B. subtilis GGT possesses virulent boneresorbing activity and its activity is probably independent of its enzymatic activity. Furthermore, a recombinant protein of B. subtilis GGT heavy subunit (Bs rGGT/H) showed strong activity of osteoclastogenesis while the light subunit failed to show strong activity, suggesting that the bone-resorbing activity is mainly located at the heavy subunit. More importantly, the GGT enzymatic activity may not be required for this virulence activity since the light subunit contains the catalytic pocket. In addition, B. subtilis rGGT stimulated mRNA expressions of receptor activator of nuclear factor kappa-B ligand (RANKL) and cyclooxygenase-2 (COX-2), while an osteoprotegerin inhibited the osteoclast formation induced by Bs rGGT/H. This is the first demonstration that bacterial GGT itself is sufficient to act as a bone-resorbing virulence factor via RANKL-dependent pathway. Therefore, it can be hypothesized that GGT of periodontopathic bacteria may play an important role as a virulence factor in bone destruction.

Bacillus subtills BS 62의 γ-Glutamyltranspeptidase 유전자

이태은,윤민호,최우영 충남대학교 농업과학연구소 2007 농업과학연구 Vol.34 No.2

To characterize γ-glutamyltranspeptidase (γ-GTP or ggt; EC 2. 3. 2. 2.) gene of Bacillus subtilis BS 62, the γ-GTP gene of BS 62 was prepared from PCR products amplified with the chromosomal DNA. The γ-GTP gene of about 2.5 kb was sequenced, and its homology was compared with the other ggt genes which were reported previously. The base sequence of the gene appeared to have an open reading frame of 1,758 bp encoding a protein of 62,175 Da. The coding region was flanked by putative ribosome binding site - AGGAGG of 7th to 12th upstream - and the stem-loof sequence was followed by transcription terminator codon. Homology of the amino acid residues sequence consisting of 587 amino acid residues was found as 98% with Bacillus subtilis gene (BSU49358), 97.4% with that of Bacillus subtilis KX 102, 37% with Pseudomonas sp. A14 (S63255) and 38% with Streptomyces avermitils (AP005028). Po1y(γ-glutamic acid) 및 levan의 생성균주로 알려진 Bacillus subtilis BS 62의 γ-GTP(ggt) 유전자를 해석하기 위하여 PCR 반응에 의해 BS 62의 염색체 DNA로부터 약 2.5 kb의 γ-GTP(ggt) 유전자 분획을 얻어 그 PCR 산물의 염기서열을 분석하여 기왕에 보고된 기타의 ggt 유전자와 비교 분석한 결과, B. subtilis γ-GTP 유전자(BSU49358)와 98%의 높은 상동성을 보였으며, Pseudomonas sp. A14(S63255)와는 37%, 방선균인 Streptomyces avermitils(AP005028)의 게놈 DNA와는 38%의 상동성을 나타냈다. BS 62의 γ-GTP 유전자의 open reading frame은 587개의 amino acid로 구성된 polypeptide의 것으로 해석되었으며, N-terminal의 28개 아미노산은 B. subtilis 펩타이드의 전형적인 형태를 보였고, 전형적인 리보솜의 부착부위는 개시코돈 ATG의 위쪽 7번에서 12번 염기(AGGAGG)에 위치하였고, 그리고 종지코돈 다음에서는 stem-loop 구조, ORF의 위쪽 약 50 bp 지점에서는 catabolite-responsive element가 발견되었다. 또한 B. subtilis 효소의 촉매자리로 추정되는 467번 잔기는 threonine으로서, 다른 박테리아의 serine, 포유동물의 cysteine과는 구별되는 것이었다.

Bacillus subtilis BS 62의 γ-Glutamyltranspeptidase 유전자

이태은(Tae-Eun Lee),윤민호(Min-Ho Yoon),최우영(Woo-Young Choi) 충남대학교 농업과학연구소 2007 농업과학연구 Vol.34 No.2

Poly(γ-glutamic acid) 및 levan의 생성균주로 알려진 Bacillus subtilis BS 62의 γ-GTP(ggt) 유전자를 해석하기 위하여 PCR 반응에 의해 BS62의 염색체 DNA로부터 약 2.5 kb의 γ-GTP(ggt) 유전자 분획을 얻어 그 PCR 산물의 염기서열을 분석하여 기왕에 보고된 기타의 ggt 유전자와 비교 분석한 결과, B. subtilis γ-GTP 유전자(BSU49358)와 98%의 높은 상동성을 보였으며, Pseudomonas sp. A14(S63255)와는 37%, 방선균인 Streptomyces avermitils(AP005028)의 게놈 DNA와는 38%의 상동성을 나타냈다. BS 62의 γ-GTP 유전자의 open reading frame은 587개의 amino acid로 구성된 polypeptide의 것으로 해석되었으며, N-terminal의 28개 아미노산은 B. subtilis 펩타이드의 전형적인 형태를 보였고, 전형적인 리보솜의 부착부위는 개시코돈 ATG의 위쪽 7번에서 12번 염기(AGGAGG)에 위치하였고, 그리고 종지코돈 다음에서는 stem-loop 구조, ORF의 위쪽 약 50 bp 지점에서는 cataboliteresponsive element가 발견되었다. 또한 B. subtilis 효소의 촉매자리로 추정되는 467번 잔기는 threonine으로서, 다른 박테리아의 serine, 포유동물의 cysteine과는 구별되는 것이었다. To characterize γ-glutamyltranspeptidase ( γ-GTP or ggt; EC 2. 3. 2. 2.) gene of Bacillus subtilis BS 62, the γ-GTP gene of BS 62 was prepared from PCR products amplified with the chromosomal DNA. The γ-GTP gene of about 2.5 kb was sequenced, and its homology was compared with the other ggt genes which were reported previously. The base sequence of the gene appeared to have an open reading frame of 1,758 bp encoding a protein of 62,175 Da. The coding region was flanked by putative ribosome binding site - AGGAGG of 7th to 12th upstream - and the stem-loof sequence was followed by transcription terminator codon. Homology of the amino acid residues sequence consisting of 587 amino acid residues was found as 98% with Bacillus subtilis gene (BSU49358), 97.4% with that of Bacillus subtilis KX 102, 37% with Pseudomonas sp. A14 (S63255) and 38% with Streptomyces avermitils (AP005028).

Nitrogen Depletion Causes Up-Regulation of Glutathione Content and γ-Glutamyltranspeptidase in Schizosaccharomyces pombe

Seung-Hyun Song,임창진 한국미생물학회 2008 The journal of microbiology Vol.46 No.1

This work aims to elucidate the relationship between nitrogen depletion and Glutathione (GSH) level in Schizosaccharomyces pombe. The total GSH level was much higher in the Pap1-positive KP1 cells than in the Pap1-negative TP108-3C cells, suggesting that synthesis of GSH is dependent on Pap1. When the Pap1-positive KP1 cells were transferred to the nitrogen-depleted medium, total GSH level significantly increased up to 6 h and then slightly declined after 9 h. Elevation of the total GSH level was observed to be much less with the Pap1-negative cells. However, glucose deprivation was not able to enhance the GSH level in the KP1 cells. Activity of γ-glutamyltranspeptidase (γ-GT), an enzyme in the first step of GSH catabolism, also increased during nitrogen depletion. The total GSH level was more significantly enhanced in the KP1 cells overexpressing γ-GT2 than γ-GT1 during nitrogen starvation. Reactive oxygen species (ROS) levels were not changed during nitrogen starvation in both Pap1-positive and Pap1-negative cells. Collectively, nitrogen depletion causes up-regulation of GSH synthesis and γ-GT in a Pap1-dependent manner.

Isolation and Characterization of a New γ-Polyglutamic Acid Producer, Bacillus mesentericus MJM1, from Korean Domestic Chungkukjang Bean Paste

( Xin Qing Zhao ),( Kwan Hyong Park ),( Ying Yu Jin ),( In Hyung Lee ),( Young Yell Yang ),( Joo Won Suh ) 한국미생물생명공학회 2005 Journal of microbiology and biotechnology Vol.15 No.1

肝癌細胞의 γ-Glutamyltranspeptidase에 對한 Monoclonal antibody에 關한 硏究

金明坤,柳總根 고려대학교 의과대학 1992 고려대 의대 잡지 Vol.29 No.2

This study was devised to produce monoclonal antibody against hepatocelullar carcinoma speclfic γ-glutamyltranspeptidase (HCC GGT), which proceeded by first purifying the HCC GGT using ion exchange chromatography and immunoaffinity chromatography. Subsequently, a mouse was immunized with the purified enzyme, and then spleen cells taken from the immunized mouse was fused with mouse myeloma cells (SP2-Ag 14) to form the hybridoma. The following results were obtained by characterization of the monoclonal antibody of the hybridoma using immunoglobulin isotyping and immunoblot. 1. The purification of HCC GGT by means of ion exchange chromatography and immunoaffinity chromatogiaphy produced a yield of 29.41%, a purification fold of 167.87, and final specific activity of 21units per mg of protein. 2. Direct enzyme linked immunoadsorbent assay (ELISA) method was used to measure the antibody level against HCC GGT. An antibody titration of the blood sample taken from the mouse immunized with the above enzyme showed value over 1:5, 120. 3. The cell fusion of the mouse myeloma cell and the spleen cell of the immunized mouse was generated 14 positive wells in 221 wells. (specific fusion efficacy of 6.33%) Only one IgG2a monoclonal antibody was determined by both repetitive cloning procedures and immunoglobulin subisotyplng. 4. A antigen-antibody cross matching after the western blot revealed a specific reaction between the HCC GGT antigen at the 40Kd band and the monoclonal antibody of the hybridoma. And the monoclonal antibody did not react with GGT derived from normal liver or kidney.

Degradation of Poly-γ-Glutame into L-Glutamate via exo-Hydrolytic Actions of γ-Glutamyltranspeptidases

Hye Bin KIM,Damee PARK,Ye-Rin JU,Beom Soo SHIN,Tae-Jip KIM 한국생물공학회 2022 한국생물공학회 학술대회 Vol.2022 No.4

Enzymatic Synthesis of Theanine with L-glutamine-Zn(II) Complexes

Hao-Qi Wang,Zhong Yao,Zhi Zhou,Yun Sun,Ping Wei,Ping-Kai Ouyang 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.6

Theanine, a unique amino acid found in tea plants, has many important physiological functions. Theanine can be enzymatically synthesized via the γ-glutamyltranspeptidation reaction. In this study, we described a new method of theanine synthesis using the L-glutamine-Zn(II)(Zn(Gln)2) complex instead of glutamine as the donor,which successfully reduced the side autotranspeptidation reaction and led to higher yield of theanine. We prepared the Zn(Gln)2 complexes and showed that they are stable in liquid bulk under 9.0 pH. After using the Zn(Gln)2 in the γ-glutamyltranspeptidation reaction, we utilized HPLC and Mass spectrometry analysis to demonstrated that Zn(Gln)2was an more effective γ-glutamyl donor than glutamine. The autotranspeptidation reaction was restrained effectively. As a result, the theanine yield and the conversion rate for glutamine were vastly improved. In a reaction mixture containing 48 mM of Zn(Gln)2, 1.6 M ethylamine, and 0.5 U/mL GGT, the final concentration of theanine obtained was 61.3 mM after incubation at 37oC for three hours. The conversion rate for glutamine was 63.8%, which showed a 16.9% increase as compared to when using glutamine alone as the donor substrate.

Protease and γ-Glutamyltranspeptidase Activities of Bacillus spp. Isolated from Rice Straw

Joo-Hee Yang,In-Su Kim,오철환,오남순 한국식품과학회 2012 Food Science and Biotechnology Vol.21 No.1

Bacillus strains were isolated from the rice straw of 7 districts in Korea, and almost all were identified as either B. amyloliquefaciens, B. pumilus, or B. subtilis. The correlation between the protease activity and γ-glutamyltranspeptidase activity produced by the identified Bacillus spp. was positively correlated (R2≒0.747). Statistically, the B. subtilis species might be the most potent producer of protease and γ-glutamyltranspeptidase of 3 identified Bacillus strains.