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Research Article


 

Over-expression of NAD kinase in Corynebacterium crenatum and its Impact on L-Arginine Biosynthesis

Muhammad Masfiqur Rahman1, Zhao Qin Qin1, Wenfang Dou1, Rao Zhiming1 and Zhenghong Xu1,2*

1Laboratory of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, 2The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology,  Jiangnan University, Wuxi 214122, P R China

*For correspondence: Email: zhenghxu@jiangnan.edu.cn  Tel: +86-510-85918206; Fax: +86-510-85918206

Received:  8 June 2012                                                  Revised accepted: 6 November 2012

Tropical Journal of Pharmaceutical Research, December 2012; 11(6): 909-916

http://dx.doi.org/10.4314/tjpr.v11i6.6  

Abstract

 

Purpose: To improve the biosynthesis of L-arginine by overexpressing homologous NAD kinase (ppnk) in Corynebacterium crenatum SYPA5-5 and to study its impact in presence of high (HOS) and low oxygen supply (LOS).

Methods: A recombinant plasmid (pJC1-tac-ppnK) harboring homologous NAD kinase (ppnk) was constructed in a shuttle vector pJC1 and transferred in L-arginine producing strain Corynebacterium crenatum SYPA5-5. Furthermore, fermentation was performed by shake flask method with consecutive determination of cell growth and glucose concentration. NAD+ kinase activity was studied by stop method and NADP(H) concentrations were determined by spectrophotometric enzymatic cycling method. To check the biosynthesis of amino acids, HPLC method was used to determine extracellular amino acid concentrations.

Results: In HOS condition, NAD+ kinase activity increased by 116 %, while intracellular concentrations of NADP+ and NADPH increased by 7.3 and 36.8 %, respectively. Whereas, in LOS condition , NAD+ kinase activity increased  49 % , with intracellular 14.67 and 15 % increases in NADP+ and NADPH respectively. More importantly, recombinant strain could produce 26.47 and 11.36 g/L L-arginine in HOS and LOS respectively, which is higher than control strain value of 24.29 and 7.58 g/L respectively. 

Conclusion: These results suggest that altering the concentration of co-enzymes by NAD kinase in Corynebacterium crenatum is an effective way to increase NADP+ with concurrent production of NADPH for further enhanced L-arginine biosynthesis in Corynebacterium crenatum in both conditions of high and low oxygen supply.

 

Keywords: NAD kinase, PpnK, L-arginine, Corynebacterium crenatum.

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