Cloning And Expression Of Arginine Deiminase In Corynebacterium Crenatum And Its Application In The Efficient Synthesis Of L-Citrulline

L-Citrulline is widely used in the pharmaceutical and healthcare industries because of its efficacy in the treatment of inflammation,sepsis,uremic circulatory disorders,and so on.Arginine deiminase?ADI?can catalyzes the hydrolysis of L-arginine to L-citrulline and has the prospect of treating cancer and leukemia.In this study,the arginine deiminase encoding gene?arcA?from E.fecalis was expressed efficiently in C.crenatum SYPA 5-5.The enzymatic properties of the ADI were studied.The recombinant strain C.crenatum SYPA 5-5/pXMJ19-arcA was cultivated in 5-L fermentor and employed for whole cell transformation to produce L-citrulline,and the transformation conditions were optimized.The main research results were as followed:The gene encoding arginine deiminase from E.fecalis was amplified by PCR and sequenced.The recombinant plasmids pXMJ19-arcA was constructed,and then expressed in C.crenatum SYPA 5-5 subsequently.The enzyme activity of recombinant strain C.crenatum SYPA 5-5/pXMJ19-arcA was 3.40 U,and the specific enzyme activity was 1.39 U·mg^-1.However,the arginine deaminase activity was not detected in original C.crenatum.The recombinant arginine deiminase was purified by Ni-NTA and the characteristic of purified arginine deiminase was investigated.The purified enzyme showed a specific activity of 3.38 U·mg^-1,and an activity of 0.27 U.The optimum pH and temperature of the recombinant arginine deiminase were 6.5 and 37°C,respectively.The recombinant enzyme exhibited Mn²⁺preference and exhibited Cu²⁺ and Zn²⁺ inhibition.The enzyme stability experiment showed that when the pH between 5.0-7.0 and the temperature between 0-37?,the recombinant arginine deiminase owns a high stability.And the Michaelis contant was 12.18 mmol·L^-1 and the maximum velocity was 0.36 ?mol·min^-1·mL^-1.The whole cell of recombinant C.crenatum SYPA 5-5/pXMJ19-arcA were used for bioconversion of L-Cit from L-Arg.Additionally,bioconversion condision was optimized.As the results showed,the optimum bioconversion pH was 6.5 and the optimum bioconversion temperature was 37?.The optimum L-Arg concentration was 300 g·L^-1 and the optimum concentration of bioconversion buffer was 0.02 M.And the whole cell of the recombinant strain C.crenatum was used for the bioconversion of L-Cit under optimal conditions,300.52 g·L^-1 L-Cit was produced in 36 h,the production rate was 8.33 g·L^-1·h^-1,and the mole conversion of L-Cit reached up to 99.6%.The recombinant strain C.crenatum SYPA 5-5/pXMJ19-arcA was cultivated in 5-L fermentor and the inducing condition was optimized.The result showed that the highest recombinant enzyme activity was achieved when recombinant strain was induced for 30 h.Under this condition,the whole cell of the recombinant strain was used for the bioconversion of L-Cit from L-Arg and the whole-cell was recycled for the following bicoconversion.The whole-cell could be used for 6 times,1800 g·L^-1 L-Arg was added and 1685 g·L^-1 L-Cit was obtained within 252 h at the end of the conversion,the overall average L-Cit production rate was 7.14 g·L^-1·h^-1 and the overall mole average conversion was 99.6%.