Heterologous Expression Of Nitrile Hydratase From Pantoea Sp.At-9b In Escherichia Coli Cells

Nitrile hydratase?NHase,EC 4.2.1.84?are a group of promising biocatalysts,catalyzing the formation of corresponding amides from nitriles.The nitrile hydratase catalytic process has the advantages of high catalytic efficiency,mild reaction conditions,and no by-products.It has been approved to be a promissing alternative to traditional chemical method for green and efficient synthesis of high value-added amide compounds.At present,nitrile hydratase-producing strains such as Rhodococcus,Pseudonocardia and Pseudomonas putida are employed as biocatalysts for synthesis of amide compounds.However,the drawbacks such as time-consuming cultivation,low expression and cumbersome purification procedure strongly restrict their industrial application.Escherichia coli overexpress system was prominent for easy cultivation,fast cell growth and high level protein expression.In this study,a novel nitrile hydratase NHase Ps from Pantoea sp.At-9b was exploited by gene mining,and heterologous expressioned in E.coli cells.By optimizing the secondary structure of the translation initiation region m RNA,the expression of?subunit is enhanced.Moreover,the soluble expression of?subunit is increased by the fusion tag protein strategy,which successfully improved the expression and activity of NHase Ps in E.coli.?1?Based on the conserved region of nitrile hydratase,six novel nitrile hydratase gene from Rhodococcus fascians A76,williamsia Sterculiae strain CPCC 203464,Amycolatopsis thermoflava,Streptomyces thermoautotrophicus,Pantoea sp.At-9b and Rhizobiales bacterium YIM 77505 were exploited and overexpressed in E.coli.The heterologous expression level of recombinant nitrile hydratase and its catalytic activity towards acrylamide were investigated.Among them,the highest activity was obtained by nitrile hydratase NHase Ps from Pantoea sp.At-9b,reaching 31 U/mg dry cell weight?DCW?.Using recombinant E.coli cells as biocatalyst,acrylamide accumulation reached50 g/L in 6.5 h with a cell loading of 0.3 g/L DCW.?2?The heterologous expression of recombinant nitrile hydratase NHase Ps in E.coli was optimized.Firstly,the linker sequence between?subunit and?subunit was designed,and the free energies?G of different linkers were calculated by the software“RNAstructure”.The higher free energy leads to looser m RNA structure,which can be easier translated by ribosomes.By screening different linker sequences,the secondary structure of m RNA in the translation initiation region of the?subunit gene was optimized.NHase Ps M2?-16.8 kcal/mol?with the highest?G value was obtained with a highest activity of 160 U/mg DCW.Secondly,multiple SD sequence optimization strategy was used to improve ribosome binding efficiency.Using the SD-ATG sequence in the translation initiation region of NHase Ps M2 as a template,multiple SD sequences of different combinations were designed.Among them,the optimal NHase Ps M2-2 was achived with an activity of 270 U/mg DCW.Finally,the effects of fusion tag on the soluble expression of?subunit were investigated.The fusion expression of?subunit was improved by fusing 6 kinds of tag proteins at the N terminal of?subunit.NHase Ps?PGB1??with a PGB1 fusion tag was obtained with a highest activity of 558U/mg DCW,which not only enhanced the expression level of?subunit but also made it completely soluble,and the enzyme activity reaches.?3?The expression conditions and enzymatic properties of recombinant NHase Ps?PGB1??were investigated,and the whole-cell catalyzed hydration of acrylonitrile to acrylamide was performed.The results showed that the optimal expression of recombinant NHase Ps?PGB1??was obtained with induction temperature of 26?,final inducer IPTG concentration of 1.0 m M,and cobalt ion concentration of 0.2 m M.The optimal temperature was 30?,the optimal p H was 7.0,respectively.The half-life of recombinant NHase Ps?PGB1??at 30?and 40?was 14 h and 3.7 h,respectively.The kinetics analysis of recombinant NHase showed that the calculated K_m value of NHase Ps?PGB1??,NHase Ps M2-2 and NHase Ps were 34.74,26.86 and 18.91 m M,respectively.the K_cat/K_m was 0.108?0.074?0.067,respectively.Using the whole cell catalytic as biocatalyst,a final concentration of 202 g/L acrylamide was achieved using3.10 g/L DCW whole cells.