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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2016, Vol. 36 Issue (12): 42-48    DOI: DOI:10.13523/j.cb.20161207
    
Functional Expression of a Nitrile Hydratase from Klebsiella oxytoca KCTC 1686 in E. coli
WANG Li-yan1, WANG Yu2, WU Jian-ping1, XU Gang1, YANG Li-rong1
1. Zhejiang University, Department of Chemical and Biological Engineering, Hangzhou 310027, China;
2. Jiangnan University, Wuxi 214122, China
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Abstract  

Nitrile hydratase is consisted of α- and β-subunit and regulated by the activator for its functional expression. To investigate the influence of different proportions of subunits and activator (17k) on recombinant expression, the nitrile hydratase (NHaseK) from Klebsiella oxytoca KCTC 1686 was studied and several recombinant strategies were constructed. Taking the advantage of two T7 promoters (T7 promoter-1 and T7 promoter-2) in pETDuet-1, the three gene segments of NHaseK were inserted into this plasmid in eight different arrangements. When all the three gene segments were expressed under T7 promoter-1, NHaseK showed a specific activity of 0.78 U/mg as α- and β-subunits equivalently expressed, and was 24% higher than at the ratio of α-and β-subunit as 5: 3. With this optimal condition, 17k was added following the T7 promoter-2, which enhanced the expression level of 17k by 2-folds and improved the activity to 0.82 U/mg. Noted that the activity reduced to 10% when subunits were separately in control of different promoters, indicating that the α-and β-subunits needed to be transcribed simultaneously. Further, the influence of plasmid copy number was investigated and the number of 15~20 was appropriate to express NHaseK in E. coli. In conclusion, the equivalent expression of subunits and the abundant expression of 17k were in favor of the functional expression of NHaseK.

Key words： Nitrile hydratase      Two promoters      Plasmid copy number      Functional expression     
Received: 12 June 2016      Published: 25 December 2016
ZTFLH:  Q556+4  
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WANG Li-yan, WANG Yu, WU Jian-ping, XU Gang, YANG Li-rong. Functional Expression of a Nitrile Hydratase from Klebsiella oxytoca KCTC 1686 in E. coli. China Biotechnology, 2016, 36(12): 42-48.

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https://manu60.magtech.com.cn/biotech/DOI:10.13523/j.cb.20161207     OR     https://manu60.magtech.com.cn/biotech/Y2016/V36/I12/42

[1] Asano Y, Tani Y, Yamada H. A new enzyme nitrile hydratase which degrades acetonitrile in combination with Amidase. Agricultural and Biological Chemistry, 1980, 44(9):2251-2252.
[2] Prasad S, Bhalla T C. Nitrile hydratases (NHases):at the interface of academia and industry. Biotechnol Adv, 2010, 28(6):725-741.
[3] Masutomo S, Inoue A, Kumagai K, et al. Enantioselective hydrolysis of (RS)-2-Isopropyl-4'-chlorophenylacetonitrile by Pseudomonas sp.B21C9. Bioscience Biotechnology and Biochemistry, 1995, 59(4):720-722.
[4] 蒋木庚, 邢月华, 王鸣华, 等. 高效立体选择性戊菊酯和氰戊菊酯的合成. 南京农业大学学报, 1990, 13(4):110-116. Jiang M G, Xing Y H, Wang M H, et al. Stereosyntheses of S(+)-5439 and esfenvalerate. Journal of Nanjing Agricultural University. 1990, 13(4):110-116.
[5] Fallon R D, Stieglitz B, Turner I. A Pseudomonas putida capable of stereoselective hydrolysis of nitriles. Applied Microbiology and Biotechnology, 1997, 47(2):156-161.
[6] Wu S, Fallon R D, Payne M S. Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Escherichia coli:activity requires a novel downstream protein. Applied Microbiology and Biotechnology, 1997, 48(6):704-708.
[7] Komeda H, Kobayashi M, Shimizu S. Characterization of the gene cluster of high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93(9):4267-4272.
[8] Petrillo K L, Wu S J, Hann E C, et al. Over-expression in Escherichia coli of a thermally stable and regio-selective nitrile hydratase from Comamonas testosteroni 5-MGAM-4D. Applied Microbiology and Biotechnology, 2005, 67(5):664-670.
[9] Song L, Yuan H J, Coffey L, et al. Efficient expression in E. coli of an enantioselective nitrile hydratase from Rhodococcus erythropolis. Biotechnol Lett, 2008, 30(4):755-762.
[10] Rzeznicka K, Schatzle S, Bottcher D, et al. Cloning and functional expression of a nitrile hydratase (NHase) from Rhodococcus equi TG328-2 in Escherichia coli, its purification and biochemical characteriation. Appl Microbiol Biotechnol, 2010, 85(5):1417-1425.
[11] Guo F M, Wu J P, Yang L R, et al. Overexpression of a nitrile hydratase from Klebsiella oxytoca KCTC 1686 in Escherichia coli and its biochemical characterization. Biotechnology and Bioprocess Engineering, 2015, 20(6):995-1004.
[12] Shin S H, Kim S, Kim J Y, et al. Complete genome sequence of Klebsiella oxytoca KCTC 1686, used in production of 2,3-butanediol. Journal of Bacteriology, 2012, 194(9):2371-2372.
[13] 裴晓林. 腈水合酶基因资源开发及其重组表达体系在制备烟酰胺中的应用. 杭州:浙江大学, 2013. Pei X L. Discovery of nitrile hydratase genes and their recombinant expression for the production of nicotanamide. Hangzhou:Zhejiang University, 2013.
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