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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.
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.
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.
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.
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.
( Shin Geon Choi ),( Yong Seok Kim ),( Sun Yeol Paek ) 한국미생물 · 생명공학회 2010 Journal of microbiology and biotechnology Vol.20 No.10
Two angiostatic fusion proteins (hAE and hEA), differing in tandem connection manners, were constructed from human angiostatin (hAS) and endostatin (hES) proteins. These fusion proteins were then evaluated for synergistic antiangiogenic properties. The 65 kDa secreted fusion proteins, expressed in Pichia pastoris, were verified by both mass analysis and Western blotting assay. Luciferase reporter gene assay, using a VEGF promoter, revealed that the angiostatin-endostatin fusion protein (hAE), and its corresponding fusion gene delivery on human microvascular endothelial cells (HMEC-1), resulted in a more potent synergistic antiangiogenic effect than the endostatin-angiostatin fusion protein (hEA). These results suggest that the orientation of the fusion genes in hAS and hES might be an important factor in the development of therapeutic proteins.
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.
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.
수직형 복합 연삭시스템 베드의 동특성 해석에 관한 연구
최승건(Seung-Geon Choi),김성현(Seong-Hyun Kim),최웅걸(Woong-Kirl Choi),신현정(Hyun-Jung Shin),이은상(Eun-Sang Lee),김규동(Kyu-Dong Kim) 한국기계가공학회 2013 한국기계가공학회지 Vol.12 No.5
Machine tools are the cores of industrial development in recent period. It is difficult to develop a system which can do cutting and grinding process in the one system. Hybrid Vertical Grinding System is capable of processing in a single apparatus cutting or grinding. The modal analysis and structural analysis for the development of Hybrid Vertical Grinding System is the first time of domestic work. In this study, Hybrid Vertical Grinding System bed was designed and analyzed by using SS401 and FC300 as materials. And by using Finite Element Methods, the design and material of the bed was analysed. Finally, we can make a better choice of structure and material of the bed by comparing the analysis results.