Corynebacterium glutamicum-Escherichia coli Shuttle Vector 개발과 C.glutamicum 의 Homoserine Dehydrogenase Gene Cloning

최신건,박종현,신현경 한국미생물 · 생명공학회 1991 한국미생물·생명공학회지 Vol.19 No.1

Tn5의 kanamycin 저항성 유전자를 가진 pBEL1 plasmid와 C.glutamicum cryptic plasmid인 pSR1으로 7.5kb의 새로운 plasmid를 만든 후, 이를 pCE1301이라 명명하였다. 이 pCE1301은 PEG1301은 PEG-protoplast법으로 C.glutamicum을 형질전환하였을 때 효율이 약 $3.0\times 10^3$형질전환제/$\mu g$이었으며 SalI과 EcoRI 제한효소 절단부위가 1개씩이었다. 또 Km이 없는 배지에서 25세대까지 안정하게 유지되었으며 B.flavum, E.coli에서 복제되었다. 이 pCE1301을 이용하여 C.glutamicum 의 homoserine dehydrogenase 유전자를 cloning하였다. A 7.5 kilobases hybrid plasmid, designated as pCE1301, was constructed by combining Eschurichia cwli plasmid pBELl which carries the kanamycin resistance gene of Tn5 with a cryptic plasmid, pSRl of Corynebacterium glutamicum. pCE1301 was transformed C. glutaicum by PEG-mediated protoplast method and its transformation efficiency was about $3.0\times 10^3$ transformants per $\mu g$ of the hybrid plasmid DNA. The physical map reveals that pCE1301 has single restriction sites for SalI and EcoRl, respectively. 'The kanamycin resistance of pCE1301 was stably maintained in C. glutamicum up to 25 generations and any segregation was not detected. pCI31301 was also introduced into Brevibacterium flavum and E coil, and replicated in those strains. pCE1301 was proved to be useiul in cloning the homoscrine dehydrogenase gene from C. glutamicum.

Intein을 이용한 대장균에서의 Trichoderma reesei 유래의 Cellobiohydrolase I 섬유소 결합 도메인의 발현

최신건(Choi, Shin-Geon) 강원대학교 산업기술연구소 2016 産業技術硏究 Vol.36 No.1

Cellulose binding domains (CBDs) of cellulases are thought to assist in the hydrolysis of insoluble crystalline cellulose. To gain sufficient amount of CBDs, the self-cleavable intein tag was used for expression and purification of Trichoderma reesei cellobiohydrolase I CBD in E. coli . Synthetic CBD genes, CBD or linker-CBD were cloned into expression vector pTYB11. Recombinant CBDs were successfully purified by intein mediated purification with an affinity chitin-binding domain. The final yields of recombinant CBD and linker-CBD were 3.2 mg/L and 1.4 mg/L, respectively. The functional bindings of recombinant CBDs were confirmed by Avicel binding experiments. The simple and easy purification method using self-cleavable intein tag can be further used in pretreatment of crystalline cellulose or characterization of engineered CBDs.

Bacillus sonorensis KCTC13918로부터 새로운 laccase유전자 ( soncotA )의 클로닝과 대장균에서의 발현

최신건(Shin-Geon Choi),윤현종(Hyeonjong Yoon) 강원대학교 산업기술연구소 2017 産業技術硏究 Vol.37 No.1

A new putative laccase gene (soncotA) which show 78% homology with that from Bacillus licheniformis (liccotA) was isolated from draft genome sequence of Bacillus sonorensis KCTC 13918. A 1,545 bp of PCR product corresponding 514 amino acids was cloned into NdeI-NotI site of pET21c and expressed as soluble form in E. coli. About 59 kDa size of recombinant laccase was purified into homogenity by Ni-NTA column and laccase activity was confirmed by zymography. The enzymatic properties of recombinant laccase were characterized. The specific activity of B. sonorensis laccase was 0.033 fold lower than that of Bacillus licheniformis laccase. The finding of new laccase gene broadened the enzymatic diversity of Bacillus species laccases.

담자균류의 배양에 의한 폴리페놀 함유 농산물의 전환 효과

변광우,홍인표,정재현,최신건,이현용,이신영 한국산업식품공학회 2002 산업 식품공학 Vol.6 No.4

Construction of a Corynebacterium glutamicum-Escherichia coli Shuttle Vector and Cloning the Homoserine Dehydrogenase Gene from C. glutamicum

Choi, Shing Geon,Park, Jong Hyun,Shin, Hyun Kyung 한국산업미생물학회 1991 한국미생물·생명공학회지 Vol.19 No.1

Tn5의 kanamycin 저항성 유전자를 가진 pBEL1 plasmid와 C. glutamicum cryptic plasmid인 pSR1으로 7.5kb의 새로운 plasmid를 만든 후, 이를 pCE1301이라 명명하였다. 이 pCE1301은 PEG-protoplast법으로 C. glutamicum을 형질전환하였을 때 효율이 약 3×10 exp(3) 형질전환체/㎍이었으며 SalI과 EcoRI제한효소 절단부위가 1개씩이었다. 또 Km이 없는 배지에서 25세대까지 안정하게 유지되었으며 B. flavum, E. coli에서 복제되었다. 이 pCE1301을 이용하여 C. glutamicum의 homoserine dehydrogenase 유전자를 cloning하였다. A 7.5 kilobases hybrid plasmid, designated as pCE1301, was constructed by combining Escherichia coli plasmid pBEL1 which carries the kanamycin resistance gene of Tn5 with a cryptic plasmid, pSR1 of Corynebacterium glufamicum. pCE1301 was transformed C. glutamicum bu PEG-mediated protoplast method and its transformation efficiency was about 3.0×10 exp(3) transformants per ㎍ of the hybrid plasmid DNA. The physical map reveals that pCE1301 has single restriction sites for SalI and EcoRI, respectively. The kanamycin resistance of pCE1301 was stably maintained in C. glutamicum up to 25 generations and any segregation was not detected, pCE1301 was also introduced into Brevibacterium flavum and E. coli, and replicated in those strains. pCE1301 was proved to be useful in cloning the homoserine dehydrogenase gene from C. glutamicum.

Enhancement of chitinolytic activity of by co-expression of endochitinase and chitobiosidase genes

Kim, Jungtae,Choi, Shin-Geon 江原大學校 産業技術硏究所 2010 産業技術硏究 Vol.30 No.B

Chitinolytic activity was enhanced by coexpression of endo-chitinase gene (chiA) and chitobiosidase gene (chiB) from Serratia marcescens KFRI314 using constitutive expression vector, pHCEIA, in E. coli. Coexpression vector was constructed by inserting ribosome binding site (RBS) into junction between two chitinase genes. SDS-PAGE analyses showed that two chitinase were constitutively expressed while E. coli clones expressing two chitinases simultaneously increased halo size on colloidal chitin plate. Furthermore, the chitinolytic activities were much enhanced in coexpressed clones when degradation patterns of substrate analogues such as 4-MU-(NAG), 4-MU-(NAG)₂,4-MU-(NAG)₃ were used. Consequently, the combined use of endochitinase and chitobiosidase greatly increased overall chitinolytic activities on recombinant E. coli clones

빙핵활성단백질의 N-terminal 부분을 이용한 녹색형광단백질의 Zymomonas mobilis 세포 표면 발현

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

Green fluorescent protein (GFPuv) was displayed on the surface of ethanol-producing bacteria Zymomonas mobilis using N-terminal domain of ice nucleation protein (INP) as an anchoring motif. To evaluate the ice nucleation protein as plausible anchor motif in Z. mobilis, GFPuv gene was subcloned into Zymomonas expression vector yielding pBBR1MCS-3/pPDC/INPN/GFPuv plasmid., INP-GFPuv fusion protein was expressed in Z. mobilis and its fluorescence was verified by confocal microscopy. The successful display of GFPuv on Zymomonas mobilis suggest that INP anchor motif could be used for future fusion partner in Z. mobilis strain improvement.

맥주오염미생물의 동정과 specific PCR primer의한 신속한 검출 방법

이택인,최신건 江原大學校 産業技術硏究所 2008 産業技術硏究 Vol.28 No.A

Several contaminated bacteria such as Lactobacillus brevis and Pediococcus damnosus in beer production cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. Recently, two contaminated strains were isolated from vessel of beer production and identified as Lactobacillus species by API kit identificaton as well as 16S-23S ITS sequencing analyses. Two isolated strains were named as Lactobacillus sp. HLAl and Lactobacillus HLB2, respectively. A polymerase chain reaction (PCR) method was developed for the rapid and specific detection of Lactobacillus sp.. Two sets of primer pairs (HLA1-F/HLA1-R and HLB2-F/HLB2-R) were designed for the amplification of a 1576 base pair (bp) fragment of the HLA1 16S-23S rRNA gene and 1888 bp fragement of the HLB2 16S-23S rRNA. Amplified PCR products were highly specific to detect corresponding bacteria when other contaminated strains were used as PCR templates. However, detection of both strains were limited when 100 ㎕ of cultured samples were mixed with 100 ㎕ of beer sample in arbitrary manner. The sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

Construction of bifunctional xylanase-cellulase fusion protein from Bacillus licheniformis NBL420 and its expression in E. coli

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

The bifunctional Xylanase-Cellulase hybrid protein was constructed by gene fusion. Two genes corresponding to endoxylanase gene (xylS) and endocellulase gene (celA) were amplified by PCR from Bacillus licleniformis NBL420. It was then linked through splicing by overlap extension (SOE) by PCR method. The two resulting fused hybrids, xyl/cel and cel/xyl, which differ by its orientation, were confirmed by its nucleotide sequencings. One of two fusion genes, xyl/cel was successfully expressed into pET22b(+) vector (pxyl/cel) with bifunctional xylanase-cellulase activity. On the contrary, the other cel/xyl fusion protein showed only cellulase activity with much decreased xylanase activity. Enzymatic properties of Xyl/Cel fusion protein were investigated regarding optimum pH, optimum temp, thermostability, and pH stability. It was revealed that Xyl/Cel fusion protein retained the bifunctional xylanase-cellulase activities eventhough two enzymes were connected with each other directly. These informations could be useful for construction of other hybrid proteins as well as increased range of substrate utilization.

Cloning and Characterization of Xylanase Gene from Bacillus licheniformis NBL420

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

The gene encoding endoxylanase (xylS) was isolated from a genomic library of Bacillus licheniformis NBL420. Two positive clones, which harbor 1.5 kb and 0.8 kb inserts respectively, were screened on RBB dyed-xylan plates and the recombinant plasmids were named as pBX3 and pBX5. The nucleotide sequencings of two inserts revealed the existence of common 639 bp of open reading frame which encode 232 amino acids. The xylS gene was successfully subcloned into pET22b(+) vector and overexpressed. Enzymatic properties including optimum pH, optimum temp, thermostability and pH stability were investigated. Activity staining of XylS was identical with that of original Bacillus licheniformis NBL420.