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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.
신대용,한상목,최신건,Shin, Dae-Yong,Han, Sang-Mok,Choi, Shin-Geon 한국세라믹학회 2002 한국세라믹학회지 Vol.39 No.9
석탄회와 벤토나이트 및 이스트 분말의 성형체를 800∼1,000$^{\circ}C$에서 1시간 소성하여 수질정화용 미생물 고정화 세라믹 담체를 제조하여 기공${\cdot}$기계적 특성을 조사하였다. 석탄회와 벤토나이트(FB)시편은 벤토나이트의 첨가량과 소성온도가 증가함에 따라 기공특성은 감소하였으나 압축강도는 증가하였다. 800∼1,000$^{\circ}C$에서 소성한 FB시편은 압축강도 89.6∼128.9 kgf/$cm^2$, 부피비중 1.25∼1.43, 겉보기비중 1.61∼1.78, 기공률 27.2∼62.2%, 평균기공경 7.9∼25.6${\mu}m$, 기공용적 8.9∼$22.2{\times}10^{-5}\;cm^3/g$ 및 비표면적 35.2∼134.3 $m^2/g$을 나타내었다. 이스트 분말을 첨가한 FBY시편은 FB시편에 비하여 기공특성이 향상되었으나 압축강도는 감소하였다. 9F1B시편에 10wt%의 이스트 분말을 첨가하여 900$^{\circ}C$에서 1시간 소성한 9F1B1Y시편은 압축강도 98.7 kgf/$cm^2$, 부피비중 1.20, 겉보기비중 1.67, 기공률 68.1%, 평균기공경 48.9 ${\mu}m$, 기공용적 $29.5{\times}10^{-5}\;cm^3/g$ 및 152.2 $m^2/g$의 비표면적을 나타내었으며, 담체의 기공에 S. saprophyticus의 부착특성이 양호하여 수질정화용 미생물 고정화 담체로 이용이 가능하였다. Porous ceramic supports with immobilized microorganisms for the water purifier were synthesized by firing green compacts of mixed powder comprising of fly ash, bentonite and an additive of yeast powder at 800∼1,000$^{\circ}C$ for 1h and the pore and mechanical properties of specimens were investigated. The compressive strength was increased in FB (Fly Ash + Bentonite) specimens while pore properties was decreased with increasing the bentonite content and sintering temperature. The compressive strength, bulk density, apparent density, porosity, mean pore size, pore volume and specific surface area of FB specimens at 800∼1,000$^{\circ}C$ were 89.6∼128.9 kgf/$cm^2$, 1.25∼1.43, 1.61∼1.78, 27.2∼62.2%, 7.9∼25.6 ${\mu}m$, 8.9∼$22.2{\times}10^{-5}\;cm^3/g$ and 35.2∼134.3 $m^2/g$, respectively. The pore properties of FBY (FB+yeast powder) specimens were superior to that of FB specimens, however compressive strength was decreased with increasing yeast powder content. The overall properties of 9F1B1Y (9F1B+10% of yeast powder) specimens at 900$^{\circ}C$ for 1 h were 98.7 kgf/$cm^2$, 1.20, 1.67, 68.1%, 48.9 ${\mu}m$, $29.5{\times}10^{-5}\;cm^3/g$ and 152.2 $m^2/g$, respectively. In this study, it was revealed that 9F1B1Y specimen demonstrated better S. saprophyticus adherence properties n their surface pores. Consequently, the microorganisms immobilized on porous ceramic supports showed better water purifying performance with many pores and adequate strength.
빙핵활성단백질의 N-terminal부분을 이용한 녹색형광단백질의 Zymomonasmobilis세포 표면 발현
이은모(Lee,Eun-Mo ),최신건(Choi,Shin-Geon) 강원대학교 산업기술연구소 2009 産業技術硏究 Vol.28 No.B
Green fluorescent protein (GFPuv) was displayed on the surface of ethanol-producing bacteria Zymomonas mobilis using N-terminaldomain 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 mobili ssuggest that INP anchor motif could be used for future fusion partner in Z. mobili sstrain improvement.
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.
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.