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| The Study on Expression,Purification and Characterization of α-amylase from Solanum tuberosum in P.pastoris |
| WANG Yong-gang1, MA Yan-lin1, MA Jian-zhong1, MA Xue-qing2, REN Hai-wei1 |
1. College of Life Science and Engingeering, Lanzhou University of Technology, Lanzhou 730050 China;
2. State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China |
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Abstract The gene of α-amylase was amplified by through RT-PCR with Solanum tuberosum cv.Shepody cDNA as template, which was subcloned into vector pPIC9k and the recombinant plasmid pPIC9K-amy was linearzed with Sac II, then transformed into P.pastoris GS115 by electroporation. The high expressed amylase active strain GSamyA5 was attained by Trypan blue-BMMY medium screening and induced to express the enzyme with 0.5% methanol for 3 days under the 30℃, and fermentation broth was purified by Ni2+-NTA agarose. the enzyme characterization was studied. The result showed that The enzyme activity was stable between 40~50℃ with maximum activity at 45℃ and the activity hold 92.6% keeping 50min. The enzyme activity was stable between pH6.0~7.0 with maximum activity at pH6.0. Ca2+ and K+ had a stimulating effect on the recombinant amylase. The relative enzyme activity was up to 125% with Ca2+, Cu2+, Fe2+, Fe3+ and Zn2+ had an significantly inhibitory effect on the recombinant amylase.
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Received: 11 February 2014
Published: 25 September 2014
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[1] 谷军. α-淀粉酶的生产与应用. 生物技术, 1994, 4(3): 1-5. Gu J. The production and application of alpha amylase. Biotechnology, 1994, 4(3): 1-5.
[2] Antje C. α-amylse: In:Anjet C(ed):Springer Handbook of Enzymes. NewYork: Springer-Varlag, 2003. 1-39.
[3] 刘逸寒, 李玉, 路福平,等. 耐酸性高温α-淀粉酶突变基因在大肠杆菌中的表达及酶学性质研究.食品发酵与工业, 2007, 33(2): 36-41. Liu Y H, Li Y, Lu F P, et al. Expression of the acid-resistant and heat-stable α-amylase mutation gene in Escherichia coli and study on characterization of the enzyme. Food and Fermentation Industries, 2007, 33(2): 36-41.
[4] Zhang J, Zeng R. Molecular cloning and expression of an extracellular α-amylase gene from an Antarctic deep sea psychrotolerant Pseudomonas stutzeristrain 7193. World Journal of Microbiology and Biotechnology, 2011, 27(4): 841-850.
[5] 刘洋, 沈微, 石贵阳,等. 中温α-淀粉酶的酶学性质研究. 食品科学. 2008, 29(9): 373-377. Liu Y, Shen W, Shi G Y, et al. Enzymatic properties of mesophilic α-amylase from Bacillus amyloliquefaciens M23. Food Science, 2008, 29(9): 373-377.
[6] Dandan Niu, Zhirui Zuo, Gui-Yang Shi,et al. High yield recombinant thermostable α-amylase production using an improved Bacillus licheniformis system. Microb Cell Fact, 2009, 8:58.
[7] Hakata M, Kuroda M, Miyashita T, et al. Suppression of α-amylase genes improves quality of rice grain ripened under high temperature. Plant Biotechnology Journal, 2012, 10(9): 1110-1117.
[8] Lee C C, Along D W, Robertson G H. An E.coli expression system for the extracellular secretion of barley alpha-amylase. J Protein Chem., 2001, 20(3): 233-237.
[9] 董晨, 曹娟, 张迹, 等. 耐高温α-淀粉酶基因在枯草芽孢杆菌中的高效表达. 应用与环境生物学报, 2008,14(4):534-538. Dong C, Cao J, Zhang J, et al. High-level expression of thermostable α-amylase in Bacillus subtilis. Chin J Appl Environ Biol, 2008,14(4)534-538.
[10] Ying Q, Zhang C, Guo F, et al. Secreted expression of a hyperthermophilic α-amylase gene from Thermococcus sp. HJ21 in Bacillus subtilis. Journal of Molecular Microbiology and Biotechnology, 2013, 22(6): 392-398.
[11] 陈陶声. 酒精发酵研究. 北京: 科学出版社, 1987.
[12] 周广麒, 余伟民. 马铃薯酒精发酵工艺的研究. 中国酿造, 2008, 8: 36-38. Zhou G Q, Yu W M. Study on the technology of potato ethanol fermentation. China Brew, 2008, 8: 36-38.
[13] 林俊涵. 毕赤酵母高密度发酵工艺的研究. 中国生物工程杂志, 2009, 29(5): 120-125. Lin J H. High density fermentation control of Pichia pastoris. China Biotechnology, 2009, 29(5): 120-125.
[14] 陈苗, 商汉桥.毕赤酵母表达操作手册. 美国: Invitrogen公司.
[15] 马向东, 马立新, 薛征峰,等. 一种鉴定α-淀粉酶活性及其产生菌的新方法. 华中农业大学学报, 2000, 19(5): 456-460. Ma X D, Ma L X, Xue Z F, et al. A new method to identify α-amylase activity and its producing bacteria. Journal of Hua zhong Agricultural University, 2000, 19(5): 456-460.
[16] 王秀奇, 秦淑媛, 高天慧,等. 基础生物化学实验. 北京: 高等教育出版社, 2006. 276-277.
[17] 王永刚, 马建忠, 马雪青, 等. 马铃薯淀粉酶基因的克隆及生物信息学分析. 食品科学, 2010, 19: 048. Wang Y, Ma J, Ma X, et al. Cloning and sequence analysis of potato α-amylase gene. Food Science, 2010, 19: 048.
[18] Juge N, Nøhr J, Le Gal-Coëffet M F, et al. The activity of barley α-amylase on starch granules is enhanced by fusion of a starch binding domain from Aspergillus niger glucoamylase. Biochimica et Biophysica Acta, 2006, 1764:275-284.
[19] Shahhoseini M, Ziaee A A, Ghaemi N. Expression and secretion of an alpha-amylase gene from a native strain of Bacillus licheniformis in Escherichia coli by T7 promoter and putative signal peptide of the gene. J Appl Microbiol,2003,95: 1250-1254.
[20] Ghang D M, Yu L, Lim M H, et al. Efficient one-step starch utilization by industrial strains of Saccharomyces cerevisiae expressing the glucoamylase and alpha-amylase genes from Debaryomyces occidentalis, Biotechnol. Lett.,2007, 29(8): 1203-1208.
[21] 周春海. 小麦β-淀粉酶生产啤酒用糖浆糖化工艺条件的优化. 现代食品科技, 2012, 28(3): 297-299. Zhou C H. Optimization of saccharification conditions of syrup for brewing produced by wheat β-amylase. Modern Food Science and Technology. 2012, 28(3): 297-299.
[22] 王永刚. 马铃薯α-淀粉酶基因和黑曲霉γ-淀粉酶基因的分子克隆及其在毕赤酵母菌. 兰州, 兰州理工大学, 2010.
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