|
|
|
| Cloning and Expression of S-2-chloropropionic Acid Dehalogenase in Pichia pastoris |
| QIN Ying-chun, YANG Li-rong, XU Gang, WU Jian-ping |
| Department of Chemical Engineering and Biological Engineering, Zhejiang University, Hangzhou 310027, China |
|
|
|
Abstract The primers of S-2-chloropropionic acid dehalogenase (DehII-B2) gene was designed by its gene sequence and was cloned into pPIC9K shuttle plasmid. The recombinant plasmid was linearized by enzyme Sac I and transformed into GS115 yeast strain by electroporation. Screening of multiple copies of the transformants was performed on YPD medium containing different concentrations of geneticin G418. After screening, recombinants with different copies were obtained. The research of copy number and methanol concentrations effects on recombinant DehII-B2 indicated that one obtained highest activity containing 12 copy numbers at the concentration of methanol was 1%. The expressed recombinant DehII-B2 activity was up to 236U/L. Additionally, recombinant DehII-B2 showed the quite good genetic stability. The optimum temperature and pH of the recombinant DehII-B2 was 50℃ and 9.5, respectively.
|
|
Received: 14 May 2013
Published: 25 August 2013
|
|
|
|
|
[1] Littlechild J A, Rye C A. Biochemical and structural studies of a L-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii. Extremophiles, 2009, 13: 179-190.
[2] Nakamura T, Yamaguchi A. Roles of K151 and D180 in L-2-haloacid dehalogenase from Pseudomonas sp. YL: Analysis by molecular dynamics and abinitio fragment molecular orbital calculations. Journal of Computational Chemistry, 2009, 30: 2625-2634.
[3] Markus A, Klages U. Oxidation and dehalogenation of 4-chlorophenylacetate by a two-component enzyme system from Pseudomonas sp. strain CBS3. Journal of Bacteriology, 1984, 160(2): 618-621.
[4] Li Y F, Kurihara T. Mass spectrometric analysis of the reactions catalyzed by L-2-haloacid dehalogenase mutants and implications for the roles of the catalytic amino acid residues. Journal of Molecular Catalysis B-Enzymatic, 2003, 23: 337-345.
[5] Smith J M, Harrison K. Purification and characterization of D-2-haloacid dehalogenase from Pseudomonas putida strain Aj1/23. Journal of General Microbiology, 1990, 136: 881-886.
[6] Ridder I S, Rozeboom H J. Three-dimensional structure of L-2-haloacid dehalogenase from Xanthobacter autotrophicus GJ10 complexed with the substrate-analogue formate. Journal of Biological Chemistry, 1997, 272: 33015-33022.
[7] McFadden J M, Frehywot G L, Townsend C A. A flexible route to (5R)-Thiolactomycin, a naturally occurring inhibitor of fatty acid synthesis. Org Lett, 2002, 4(22): 3859-3862.
[8] 林春娇. C2/C3卤代酸脱卤酶的筛选及克隆表达. 杭州: 浙江大学, 2011. Lin C J. Cloning, screening and expression of C2/C3 Dehalogenation halogenated acid enzymes. Hangzhou: Zhejiang University, 2011.
[9] Tsang J S, Sam L. Cloning and characterization of a cryptic haloacid dehalogenase from Burkholderia cepacia MBA4. Journal of Bacteriology, 1999, 181(19): 6003.
[10] Jiang K Y, Zhang B, Yan Y J. Cloning and expression of Pseudomonas fluorescens 26-2 lipase gene in Pichia pastoris and characterizing for transesterification. Applied Biochemistry and Biotechnology, 2009, 159: 355-365.
[11] 彭毅, 步威, 康良仪. 甲醇酵母表达系统. 生物技术通报, 2000, 1: 38-41. Peng Y, Bu W, Kang L Y. Methanol yeast expression system. Biotechnology Bulletin, 2000, 1: 38-41.
[12] Montesio R, Garcia R, Quintero O, et al. Variation in N-linked oligosaccharide structures on heterologous proteins secreted by the methylotrophic yeast Pichia pastoris. Protein Expression and Purification, 1998, 14(2): 197-207.
[13] Gemmill T R, Trimble R B. Overview of N-and O-linked oligosaccharide structures found in various yeast sprices. Biochimica et Biophysica Acta, 1999, 1426(2): 227-237.
[14] James M, Cregg, Kevin J,et al. Pichia pastoris as a host system for transformations. Molecular and Cellular Biology, 1985, 5(12): 3376-3385.
[15] 项炯华, 吴坚平, 王能强, 等. 2-氯丙酸脱卤酶产酶菌种的筛选及酶学性质研究. 化学反应工程与工艺, 2005, 21(6): 537-541. Xiang J H, Wu J P, Wang N Q, et al. Strains screening and enzymology properties of 2-chloropropionic acid dehalogenase. Chemical Reaction Engineering and Technology, 2005, 21(6): 537-541.
[16] 刘鹏, 林春娇, 杨立荣, 等. S-2-氯丙酸脱卤酶的定向进化及其应用. 中国生物工程杂志, 2012, 32(5): 66-72. Liu P, Lin C J, Yang L R, et al. Directed evolution and application of S-2-chloropropionic acid dehalogenase. China Biotechnology, 2012, 32(5): 66-72.
[17] 黄培堂. 分子克隆实验指南. 第3版. 北京: 科学出版社, 2005. Huang P T. Molecular Cloning (A laboratory Manual). 3 ed, Beijing: Science Press, 2005.
[18] Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227: 680-685.
[19] Chen X L, Cao Y H, Ding Y H, et al. Cloning, functional expression and characterization of Aspergillus sulphureus β-mannanase in Pichia pastoris. Biotechnol, 2007, 128: 452-461.
[20] Gorgenes J F, Van W H, Knoetze J H, et al. Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium. Applied Microbiology and Biotechnology, 2005, 67: 684-691.
[21] KatakuraY, Zhang W, Zhuang G, et al. Effect of methanol concentration on the production of human β2-glycoprotein I domain V by a recombinant Pichia pastoris: A simple system for the control of methanol concentration using a semiconductor gas sensor. Journal of Fermentation and Bioengineering, 1998, 86: 482-487. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
