| Nitrile hydratase?NHase;EC:4.2.1.84?can catalyze the formation of amides by combining nitric acid with water under normal temperature and neutral conditions.It is a key enzyme to replace traditional chemical catalytic process to achieve green manufacturing of amide products.NHase is a metalloenzyme containing?-and?-subunits.The maturation of the enzyme follows the self-subunit swapping mechanism.However,the thermal stability of the enzyme and its tolerance to organic solvent are poor,and it is difficult to meet the application requirements of the industrial production for enzymatic properties.Therefore,it is urgent to discover new NHase and improve the catalytic performance of the enzyme.Inspired by the newly discovered protein structure of Monosiga brevicollis monopeptide NHase,this study performed subunit fusion on NHase derived from Pseudomonas putida 5B/NRRL-18668.The thermal stability and product tolerance of the newly constructed NHase improved in large extent;We also studied the activation mechanism of subunit fused NHases from P.putida 5B and Rhodococcus rhodochrous J1,revealing the mechanism by which the regulatory protein activates the fusion nitrile hydratase.The main findings are as follows:?1?The NHase from P.putida 5B was studied and the subunit fusion NHases with greatly improved activity and stability was obtained.Based on the reported genotype of the eukaryotic single-chain NHase,the subunit-fusion strategy was used to fuse the two subunits of NHase from P.putida 5B and acquired two fusion NHases with greatly improved activity and stability.The fusion NHase-?BA?P14K possesses 54%higher specific activity than that of the original enzyme,and the NHase-?BAP14K?possesses 39%higher specific activity than that of the original enzyme,and the half-life of the two fusion NHases at 50°C were respectively 2.8 times and 2 times as that of the original enzyme.Besides,the product tolerance of the two fusion NHases were also improved compared with that of the original enzyme.?2?Further improve the stability of NHase based on a semi-rational modification strategy.Using NHase-?BA?P14K as the starting protein,a highly efficient small mutant library was established by combining point mutant scan program and molecular dynamics simulation,and three mutants with improved activity and stability were screened out,they are B-M150C,B-T173Y and B-S189E.The catalytic efficiencies of the three mutants were 1.1 times,1.5 times and 2.2 times higher than that of the starting protein,and the corresponding half-lives at at 50°C were respectively 32%,7%and 107%higher than that of the starting protein.?3?The mature activation mechanism of the fused NHase was analyzed.The NHase activators P14K and NhlE derived from P.putida 5B and R.rhodochrous J1 were obtained by optimizing the expression strategy.On this basis,the activation mechanism of NHase was explored.The activated NHase is obtained by mixing activator with homologous cobalt-free fused NHase in vitro;in addition,partial activation can also be achieved by using activator to activate NHase from different sources.The results indicate that the activator protein acts as a metal chaperone to assist in the transportation of cobalt ions to NHase;in addition,the activator can activate not only the homologous NHase but also the heterologous NHase with different activation efficiency.?4?The NHase of M.brevicollis?MbNHase?was expressed successfully.The Escherichia coli was selected as a host to express MbNHase,and its specific activity was determined as 88.4U·mg-1.According to the conclusion that the activator can activate the noncognate NHase,two plasmids respectively co-expressing MbNHase with P14K and NhlE were constructed.After purification,the enzymic activity assay showed that the specific activity of Mb NHaseP14K and MbNHaseE were 220.2 U·mg-1 and 127.0 U·mg-1,which were 2.5 times and 1.4 times higher than that of Mb NHase,indicating that two prokaryotic activatos can assist to increase the activity of eukaryotic fusion NHase. |
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