| Nitrile Hydratase（NHase,EC 4.2.1.84）is a metallic enzyme that can catalyze the hydration of nitrile compounds into amides.Nitrile hydratases are usually composed ofαandβsubunits,and there is an activation protein gene downstream of the gene cluster,which helps the enzyme to take up metal ions to complete post-translational modification.Two potential nitrile hydratases at Roseomonas stagni DSM 19981 were found in the public gene pool,and both contained the cobalt-binding amino acid motif Cys-Thr-Leu-Cys-Ser-Cys of cobalt-type nitrile hydratases.The first potential NHase（Fs NHase）is composed of three subunits,one of which is similar to theαsubunit of conventional nitrile hydratase,and the other two subunits are similar to the C and N terminal of theβsubunit of conventional nitrile hydratase.Another potential NHase（αp14k）genotype is more specific in that the gene encoding theαsubunit naturally merges with the gene encoding the activating protein into a single gene,but does not contain the gene encoding theβsubunit.In this study,these two potential nitrile hydratases were heterologous expressed in Escherichia coli genetically engineered bacteria.Their properties were determined and their interactions were explored.The main research results are as follows:（1）Express and purify Fs NHase then measure its specific enzyme activity to nicotinitrile,acrylonitrile and potassium thiocyanate,and analyze the mechanism of interaction between the activities of hydrating nitrile and hydrolyzed potassium thiocyanate.Fs NHase was successfully expressed in Escherichia coli BL21（DE3）and obtained by ammonium sulfate precipitation,anion exchange chromatography and gel filtration chromatography.The optimum p H of Fs NHase was 7,the optimum temperature was 40℃.Under optimal conditions,the specific enzyme activity of nicotinitrile,acrylonitrile and potassium thiocyanate were 17.8±1.3 U·mg-1,30.6±1.9 U·mg-1,13.5±0.9 U·mg-1,respectively.Conventional nitrile hydratases do not have thiocyanate hydrolase activities.Based on the three-dimensional structure comparison,the different amino acids located at the top of the substrate binding pocket of Fs NHase may be the reason for the interaction of nitrile hydratase and thiocyanate hydrolase activities.（2）Heterologous expression and purification of the natural fusionαp14k complex,then explore whether whether the naturalαp14k withoutβsubunit can independently function as nitrile hydratase.High purityαp14k complex was obtained by heterologous expression and purification.It was confirmed by gel filtration chromatography thatαp14k existed mainly in the form of monomer in solution.The enzymatic properties ofαp14k complex were characterized.The optimum temperature and p H ofαp14k for nicotinitrile catalysis were 45℃,7.4,and the specific enzyme activity of nicotinitrile catalysis was 12.77±0.18 U·mg-1.Inductively coupled plasma mass spectrometry（ICP-MS）showed that each fusedαp14k complex could bind 0.47 cobalt ions.The in vivo/in vitro activation results of Fs NHase andαp14k showed thatαp14k did not activate Fs NHase in vivo or in vitro,suggesting that the natural fusionαp14k complex could function as nitrile hydratase independently.（3）By constructing a mutant library,the dominant mutants with improvedαp14k catalytic ability were screened.A small and refined mutant library was constructed by virtual screening,a mutant R250A,whose catalytic nicotinitrile activity was 3.6 times higher than that of the wild type was obtained,and its activity reached 30.01±0.6 U·mg-1.The role of the histidine-rich linker linking the alpha subunit and p14k was studied.The mutant M1 was constructed to delete linker and the mutant M2 was constructed to replace linker into five amino acids GGGGS.The catalytic activity of M1 was 44.15±0.37 U·mg-1,and that of M2was 41.31±0.31 U·mg-1.The results indicated that linker could inhibit the hydration activity. |
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