黑曲霉内切葡聚糖酶AnCel5A的表达纯化与晶体优化
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摘要
来源于黑曲霉(Aspergillus niger)的β-1,4-内切葡聚糖酶(AnCel5A)是糖苷水解酶第5家族成员,水解纤维素分子无定形区的β-1,4-糖苷键,在纤维素的降解过程中发挥关键作用。本研究通过对AnCel5A的结晶条件初筛,获得并优化AnCel5A晶体以期解析其蛋白质晶体结构。将截去N端信号肽的AnCel5A蛋白在Pichia pastoris X33/pPICZαA中进行表达,发酵液上清透析除盐并进行Endo H去糖基化酶切,后续依次通过离子交换层析、疏水层析,获得高纯度蛋白。通过坐滴气相扩散法对AnCel5A的结晶条件进行初筛,在CryoI screen kit (A6)孔中长出了初始晶体,经优化获得了更好的晶体。蛋白晶体于台湾新竹同步辐射中心(NSRRC)BL15A1线站进行了X射线衍射数据收集,结果显示其分辨率达到1.8?。
The β-1,4-endoglucanase from Aspergillus niger(AnCel5 A) is a cellulase which is classified to glycoside hydrolases subfamily(GH) 5. It can hydrolyze the amorphous region of cellulose long chains by endo-action,which makes it one of the key enzymes in cellulose hydrolysis.This research aims at determination the crystal structure of AnCel5 A by screening and optimization its protein crystals. The signal peptide truncated AnCel5 A is successfully expressed in P. pastoris X33. Purified proteins are obtained by using ion-exchange and hydrophobic chromatography. The crystal screen of AnCel5 A is completed by using sitting-drop vapor diffusion method and screen kits bought from the Hampton Research company. Initial crystals are found in the reservoir solution of cryoI kit(A6). After further optimization,better crystals are obtained. The crystals diffracted to 1.8 ? and dataset is collected at beam line BL15A1 of the National Synchrotron Radiation Research Center(NSRRC,Hsinchu,Taiwan).
The β-1,4-endoglucanase from Aspergillus niger(AnCel5 A) is a cellulase which is classified to glycoside hydrolases subfamily(GH) 5. It can hydrolyze the amorphous region of cellulose long chains by endo-action,which makes it one of the key enzymes in cellulose hydrolysis.This research aims at determination the crystal structure of AnCel5 A by screening and optimization its protein crystals. The signal peptide truncated AnCel5 A is successfully expressed in P. pastoris X33. Purified proteins are obtained by using ion-exchange and hydrophobic chromatography. The crystal screen of AnCel5 A is completed by using sitting-drop vapor diffusion method and screen kits bought from the Hampton Research company. Initial crystals are found in the reservoir solution of cryoI kit(A6). After further optimization,better crystals are obtained. The crystals diffracted to 1.8 ? and dataset is collected at beam line BL15A1 of the National Synchrotron Radiation Research Center(NSRRC,Hsinchu,Taiwan).
引文
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[10]ZHANG Huimin,LI Jianfang,WU Minchen,et al. Expression of a thermostable xylanase gene in Pichia pastoris and its enzymatic characterization[J]. Journal of Food Science and Biotechnology,2013,32(2):124-128.(in Chinese)
[11]LI P,ANUMANTHAN A,GAO G X,et al. Expression of recombinant proteins in Pichia pastoris[J]. Applied Biochemistry and Biotechnology,2007,142(2):105-124.
[12]CARTER C W J,CARTER C W. Protein crystallization using incomplete factorial experiments[J]. Journal of Biological Chemistry,1979,254(23):12219-12223.
[13]CHAYEN N E,STEWART P D S,MAEDER D I. An automated system for micro-batch protein crystallization and screening[J].Journal of Applied Crystallography,1990,23(4):297-302.
[14]VEDADI M,NIESEN F H,ALLALI-HASSANI A,et al. Chemical screening methods to identify ligands that promote protein stability,protein crystallization,and structure determination[J]. Proceedings of the National Academy of Sciences,2006,103(43):15835-15840.
[15]FAN Haifu,LIANG Dongcai. The diffraction-phase problem in structural genomic[J]. Chinese Bulletin of Life Science,2003,15(2):65-69.(in Chinese)
[2]WARREN R A. Microbial hydrolysis of polysaccharides[J]. Annual Review of Microbiology,1996,50(23):183-212.
[3]TANG Zizhong,LIU Shan,HAN Xueyi,et al. Improving endoglucanase activity from Bacillus subtilis by error-prone PCR[J].Journal of Food Science and Biotechnology,2013,32(7):754-761.(in Chinese)
[4]QIN Y,WEI X,SONG X,et al. Engineering endoglucanase II from Trichodermareesei to improve the catalytic efficiency at a higher pH optimum[J]. Journal of Biotechnology,2008,135(2):190-195.
[5]ZHANG J,SHI H,XU L,et al. Site-directed mutagenesis of a hyperthermophilic endoglucanase Cel12B from Thermotogamaritima based on rational design[J]. PLoS One,2015,10(10):1-2.
[6]TISHKOV V I,GUSAKOV A V,CHERKASHINA A S,et al. Engineering the pH-optimum of activity of the GH12 family endoglucanase by site-directed mutagenesis[J]. Biochimie,2013,95(2):1704-1710.
[7]TOMME P,WARREN R A J,GILKES N R. Cellulose Hydrolysis by Bacteria and Fungi[M]//POOlE R K. Advances in Microbial Physiology. Academic Press. 1995:1-81.
[8]DAVIES G,HENRISSAT B. Structures and mechanisms of glycosyl hydrolases[J]. Structure,1995,3(9):853-859.
[9]HONG J,TAMAKI H,AKIBA S,et al. Cloning of a gene encoding a highly stable endo-beta-1,4-glucanase from Aspergillusniger and its expression in yeast[J]. Journal of Bioscience and Bioengineering,2001,92(5):434-441.
[10]ZHANG Huimin,LI Jianfang,WU Minchen,et al. Expression of a thermostable xylanase gene in Pichia pastoris and its enzymatic characterization[J]. Journal of Food Science and Biotechnology,2013,32(2):124-128.(in Chinese)
[11]LI P,ANUMANTHAN A,GAO G X,et al. Expression of recombinant proteins in Pichia pastoris[J]. Applied Biochemistry and Biotechnology,2007,142(2):105-124.
[12]CARTER C W J,CARTER C W. Protein crystallization using incomplete factorial experiments[J]. Journal of Biological Chemistry,1979,254(23):12219-12223.
[13]CHAYEN N E,STEWART P D S,MAEDER D I. An automated system for micro-batch protein crystallization and screening[J].Journal of Applied Crystallography,1990,23(4):297-302.
[14]VEDADI M,NIESEN F H,ALLALI-HASSANI A,et al. Chemical screening methods to identify ligands that promote protein stability,protein crystallization,and structure determination[J]. Proceedings of the National Academy of Sciences,2006,103(43):15835-15840.
[15]FAN Haifu,LIANG Dongcai. The diffraction-phase problem in structural genomic[J]. Chinese Bulletin of Life Science,2003,15(2):65-69.(in Chinese)
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