| Nitrile hydratase is a highly efficient and green biocatalyst that catalyzes the formation of amide compounds from organic nitrile compounds.It has been widely used in the industrial production of amide products due to the mild reaction conditions,high yield,and environmental friendliness.In the industrial process,the thermostability,and substrate/product tolerance of the nitrile hydratase are the key issues affecting the economy of the process.Acrylamide is the largest species biosynthesized by nitrile hydratase.In this paper,researches were carried out to excavate new nitrile hydratase and modify the thermostability and product tolerance to improve the catalytic performance,as well as to promote the technological advance and economy in the industrial productio n of acrylamide.The main contents are as follows:(1)A small enzyme library of nitrile hydratase was constructed by gene mining,and acrylonitrile was used as a model substrate for catalytic performance screening.Among them,NHAB derived from Bordetellapetrii DSM 12804 showed good solubility and the highest enzyme activity of 570.2 U/mg DCW(Dry cell weight),but its thermostability was very poor,and no residual enzyme activity could be detected after incubation at 50℃ for 1 h.While NHAM derived from Aurantimonas manganoxydans S185-9A1 showed high activity among the thermostable strains,and its residual enzyme activity retained 77.1%after 1 h of incubation at 50℃.(2)A computational strategy using Synergetic Energy and Correlated-configuration for Redesigning Enzyme(SECURE)was proposed and NHAB was selected as a model enzyme.The strategy included a rational pruning of the potential mutations and the assembling of effective single point mutations.The best mutant A6M/B4M(α-subunit:S30T,A71D,A74D,A78R,S8 1T,A133P;β-subunit:L25F,G27Y,N59P,A173N)showed an increase in Tm by 13.2℃,an 865-fold increase in the half-life at 50℃(14.4 h),and an 11.2%increase in specific enzyme activity(1697 U/mg protein),compared with the wild type.Molecular dynamics simulations showed that the overall structural enhancement and newly formed intermolecular interaction were responsible for the enhanced thermostability of the mutant.(3)A high-throughput method for the screening of NHase based on the nitrile hydratase/amidase cascade reaction was established.Based on this method,a total of 90 key positions on the protein surface,subunit interface,and at the substrate/product access tunnel of NHAM were selected for saturation mutagenesis.The key sites affecting product tolerance and catalytic activity were obtained.a-R98 located at both the subunit interface and the access tunnel,β-G41,β-S51,and β-R55 located at the access tunnel.Single-point mutants α-R98I,β-G41D,βS51E,and β-R55C showed elevated residual enzyme activities after incubation in 300 g/L acrylamide solution for 1 h,76.6%,65.1%,70.8%and 66.8%.respectively,which were 2.0-fold,1.7-fold,1.8-fold and 1.7-fold that of the wild type(38.5%),and the catalytic activity increased 1.4-fold,1.3-fold,1.2-fold and 1.1-fold,respectively.(4)Multi-point mutants with significantly improved catalytic activity and product tolerance were further obtained by iterative saturation mutagenesis based on tunnel engineering.Among them,mutant D2 exhibited the highest specific enzyme activity of 2944.2 U/mg protein,which was 5.0-fold higher than wild-type(490.5 U/mg protein).The catalytic efficiency kcat/Km of mutant D3 was 19.4-fold higher than that of the wild type,reaching 11.28 mM-1s-1.Besides,all mutants exhibited a broader substrate spectrum.Structural analysis showed that the distorted conformation of the tunnel was straightened.Meanwhile,the diameter at the bottleneck was increased and the length was shortened,which facilitated the substrate entry and binding.Besides,the spatial distribution of acrylamide in the mutant protein was significantly less than that of the wild type,which made the mutant more stable in high concentrations of product solutions.(5)Researches on production of nitrile hydratase by Escherichia coli and the catalytic synthesis of acrylamide were carried out to reduce the manufacturing cost and improve the conversion efficiency.D2-J strain expressed in E.coli-J host was obtained.The induction conditions were 22℃,0.15 g/L CoCl6H2O,3.0 g/L lactose.TB medium was replaced with an industrial complex organic nitrogen source composed of wheat peptone and yeast extract LM902.The final OD600 was 220.6 and the enzyme activity reached 26533 U/mL after 48 h in a 15 L-fermenter,which was 17.4 times and 29.5 times of that in shaking flask fermentation,respectively.The fed-batch catalytic process was operated in pure water system at initial pH 8.8 with 100000 U input.The product concentration of 49.9%could be reached in 200 min,and the substrate acrylonitrile could be converted into acrylamide within 7 h,reaching a product concentration of 52%.The efficient production of acrylamide was realized,which laid the foundation for the industrialization of D2J strain. |
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