Modification Of Catalytic Activity Of Highly Thermally Stable Nitrile Hydratase Based On Substrate Access Tunnel Engineering

Nitrile hydratase（NHase,EC 4.2.1.84）is one type of metalloenzyme that catalyzes the formation of amides from nitriles under mild conditions.It is used in the biological production of acrylamide and nicotinamide in industry due to its low amount of by-products and high catalytic efficiency compared to chemical methods.However,the exothermic hydration reaction places high demands on the thermal stability of NHase.NHase from Caldalkalibacillus thermarum TA2.A1（Cal.t NHase）and NHase from Pseudonocardia thermophila JCM3095（Pt NHase）possesses excellent thermal stability,while the enzyme activity is far behind the requirements of industrial production.Based on this,this study uses computational biology and multi-subunit protein expression strategies to modify the NHase from thermophilic microorganisms to improve its enzyme activity,and carry out amplification verification in a 5L fermenter.The main results are as follows:（1）A computational biology approach,which combines molecular modeling and molecular dynamics simulations,was used to improve the catalytic efficiency of enzymatic reactions by mutating N47 and N181 sites at the substrate channel entrance of Cal.t NHase,and E50 and R82 sites at the substrate channel entrance of Pt NHase.The representative amino acid mutations disrupted the hydrogen bonds or salt bridges around the substrate channel entrance,improved the flexibility of the substrate channel entrance,enhanced the channel entrance conformational variability,and subsequently improved the catalytic efficiency of the enzymatic reaction.The dominant mutant Cal.t NHaseβN47F,obtained from the study,increased acrylonitrile catalytic activity to 592.92 U·mg^-1,which was 3.57 times of the wild type.The dominant mutant Pt NHaseβE50L,increased nicotinonitrile catalytic activity to 353.53 U·mg^-1,which was 5.83 times of the wild type.（2）Based on the highly stable and active Pt NHaseβE50L,the plasmid p ET-24a（+）-AB,p RSFDuet-1-BA,p RSFDuet-1-AB was constructed with the aim of adjusting the expression order and expression intensity ofαsubunit andβsubunit.The positive recombinant plasmid p RSFDuet-1-BA was screened,and the whole-cell enzymatic activity of this recombinant with nicotinonitrile as substrate was 1.86-fold higher than that of p ET-24a（+）-BA,alleviating the imbalance in the expression of the two subunits.（3）The recombinant E.coli containing p RSFDuet-1-BA and p ET-24a（+）-BA were cultured in a 5 L fermenter for high density fermentation.E.coli containing p RSFDuet-1-BA reached a maximum OD₆₀₀=130.40 after 34.50 h of fermentation,and the highest enzyme activity for nicotinamide production was 10099.31 U·m L^-1.The whole-cell enzyme activity per unit volume was elevated to 1.29 times that of the E.coli containing initial plasmid.The whole-cell catalytic production of nicotinamide was achieved using high-density fermentative organisms with different densities,and when the cell density was OD₆₀₀=5,the concentration of nicotinamide reaches 502.10 g·L^-1 after 2 h.