Mining Of High-performance Nitrile Hydratase And Modification Using Semi-rational Design Approach

Nitrile hydratase（NHase,EC 4.2.1.84）is a metalloenzyme that can catalyze the formation of amides from nitriles.It is usually composed of two subunits,αandβ,and there is usually an activator downstream of the gene cluster that helps the incorporation of metal ion,which can form a complex with theαsubunit and promote the maturation of NHase.NHase has been widely used in the production of acrylamide and nicotinamide.However,poor stability and narrow substrate spectrum still hinder the industrialization of the reported NHase.Improving the stability of NHase can effectively increase the utilization batch of the enzyme and the concentration of the product,thereby saving energy and reducing consumption,and broadening the substrate spectrum of NHase can improve the synthesis ability of the enzyme for various amides production,which have a wider range of applications of this enzyme.This study focuses on the modification of existing NHase and the mining of new NHase in order to obtain high-performance NHases with improved stability and expanded substrate spectra.Firstly,the stability of existing highly active NHase was significantly improved through subunit fusion strategies,and the molecular mechanisms was analyzed.At the same time,NHases from extremophilic microorganisms,which typically exhibit excellent enzymatic properties,were screened from thermophilic actinomycetes and halophilic archaea to obtain NHases with high stability and tolerance.Activity is improved and substrate spectra expanded through semi-rational modification.The main findings are as follows:（1）NHase was subunit-fused using a linker peptide,its stability was significantly improved and the mechanism behind the increased stability was analyzed.A variety of NHase with highly activity have been reported and used in industrial production,but poor thermostability is still a key problem limiting the industrial application of NHase.In this study,Pseudomonas putida NRRL-18668-derived NHase（P.p NHase）was used as the research subject.Three types of linker peptides,namely,helical（A）,flexible（B）,and rigid（C）,were selected,each with different repeating units（A:（EAAAK）n;B:（GGSG）n;C:（PA）n）and lengths（n=1,2,3,4,6,8,16）,to construct subunit-fused P.p NHase.Within a certain linker length range,the fusion P.p NHase showed higher specific activity and stability compared to the wild-type enzyme.The longer the linker,the higher the thermal stability of the fusion P.p NHase.Helical-type linker was more suitable for improving the stability of NHase compared to other types of linkers,and the optimal mutant P.p NHase-A8 had a half-life 7.7 times that of the wild-type at 50℃.Structural simulations and molecular dynamics analyses showed that the increased stability of the fusion P.p NHase was mainly due to the increase in the number of salt bridges and hydrogen bonds,as well as the increase in subunit interface area caused by linker insertion.It was verified that the subunit-fusion strategy for improving NHase stability has some universality.（2）A highly thermophilic NHase derived from an extreme thermophilic bacterial strain was discovered.Through semi-rational engineering,the catalytic activity of the enzyme was improved,and its substrate spectrum was expanded.Enzymes derived from extremophiles usually show excellent enzymatic properties,based on bioinformatics analysis,NHase from the extremely thermophilic actinomycete Carbonactinospora thermoautotrophica（C.t NHase）was mined,and its enzymatic properties were determined.The half-life of C.t NHase at 60°C reached 265 min,making it the most stable NHase reported to date.The 3D structure model of C.t NHase was predicted and analyzed,and the substrate tunnel was reconstructed using semi-rational design.A mutantβL48D with significantly improved catalytic ability for 3-cyanopyridine was obtained,and its specific activity was 566.18±18.86 U·mg^-1,7.7 times higher than that of the wild-type(73.80±5.76U·mg^-1).Molecular dynamics analysis showed that the mutation ofβL48D widened the substrate tunnel entrance of the NHase and increased the opening and closing frequency of the substrate tunnel during the dynamic binding process of the substrate.Based on this,the optimal mutant for catalyzing different nitrile substrates was screened,and an efficient C.t NHase"toolbox"for catalyzing multiple nitrile substrates was established.By screening the optimal mutant for catalyzing 4-cyanopyridine,the“toolbox”of C.t NHase was further expanded and applied for whole-cell catalytic production of nicotinamide with a yield of 352 g·L^-1,showing great potential for industrial application.The activation effects of activators on C.t NHase were studied,and the results showed that the activation effects of NHase activators from different sources were significantly different,while the tandem expression of two activators helped to increase the cobalt ion content of C.t NHase and thus improve enzyme activity.（3）Based on gene mining methods,NHase from a halophilic archaeon was obtained.This enzyme showed significantly higher tolerance to high organic solvents compared to NHases from other sources.High tolerance to organic solvents is an important requirement for the industrial application of NHase.High substrate-product tolerance can increase product yield and reduce the subsequent extraction steps of the amide product.NHase（archaeon A07HB70,A.r NHase）from a halophilic archaeon growing in high salt environments was discovered from the NCBI database.Significantly different from NHase from other sources,theαsubunit of this enzyme is fused with the activating protein.Attempts were made to express it in different vectors and hosts,and successful heterologous expression of A.r NHase was achieved in E.coli BL21（DE3）host using a dual-promoter vector p RSFDuet,and its expression level was improved by adding an SKIK tag.The tolerance of A.r NHase to substrate and product was determined,and the results showed that the enzyme still maintained 89.14%of its activity after treatment with 4.0M substrate and 97.52%of its activity after treatment with 4.0 M product,which was significantly higher than NHases from other sources.