Catalytic Modification Of Nitrile Hydratase For Industrial Use

Nitrile hydratase（EC 4.2.1.84）is a key enzyme in nitrile metabolism.NHase can catalyze the hydration of nitrile substrates to produce amide products.It is mainly used in the industrial production of niacinamide,acrylamide and 5-cyanopentamide.Different from the chemical synthesis,biocatalyst has the advantage of mild reaction conditions and high specificity.Since the hydration reaction of nitrile is exothermic,the enzymatic production of amides has a high requirement for the stability of NHase.Rhodococcus rhodochrous J1contains two nitrile hydratases（H-NHase and L-NHase）of high molecular weight and low molecular weight.H-NHase has high catalytic activity for acrylonitrile and shows high stability,which has been applied in the industrial production of acrylamide.However,although H-NHase had high stability,it preferred to catalyze aliphatic nitrile,and had low catalytic activity for aromatic nitrile substrates such as nicotinitrile.In addition,NHase generally has low catalytic activity for small nitrile substrates such as acetonitrile,while niacinamide and acetamide products corresponding to acetonitrile have high application value in biomedicine,chemical antifreezing and other fields.Therefore,the modification of H-NHase based on high stability has certain theoretical significance and application value for the catalytic performance of aromatic nitrile and small volume acetonitrile substrates.In this study,H-NHase was used as the research object.The key amino acids of the substrate channel were semi-designed by combining molecular docking,virtual screening and site-specific saturation mutation methods,so as to maintain stability and improve its performance in catalytic synthesis of nicotinamide,acetamide and other amide compounds with high application value,and to characterize the enzymatic properties of the optimal mutants.On this basis,the amide production technology of genetically engineered bacteria was established by high density fermentation technology of Escherichia coli and adding strategy of stabilizer.The main research contents are as follows:（1）A mutant library was constructed by semi-rational design to screen the dominant mutants with improved catalytic ability.The hot spots were mutated by molecular docking,virtual screening and site-specific saturation mutation,andβW48Y was obtained with increased catalytic nicotinitrile activity.Its enzyme activity was 1198 U·mg^-1,6.20 times that of H-NHase,the activity of NHase was higher than that of other strains.And the half-life ofβW48Y was similar to that of H-NHase at 50℃,which was 30 min.The half-life ofβW48Y was 6 min and H-NHase was 7 min under 60℃treatment,and that of mutantβW48Y was 42min and H-NHase was 32 min under 1 mol·L^-1 nitrile substrate treatment.The specific enzyme activity ofαQ83E was 1645 U·mg^-1,1.77 times that of the H-NHase,and the half-life ofαQ83E was similar to that of H-NHase at 50℃for 30 min.The half-life ofαQ83E and H-NHase were 28 min and 50 min respectively when 40%（v/w）acetamide was used.By protein structure analysis,the conformation ofβW48Y substrate entrance was wider.The distance between the substrate and cobalt ion changes from 5.1（?）to 3.5（?）.The free energy of substrate binding to the active center(-3.7 to-3.4 kcal·mol^-1)was lower than that of H-NHase(-3.6 to-2.9 kcal·mol^-1).The above factors synergically improved the catalytic performance ofβW48Y for nicotinitrile.（2）The whole cell catalytic performance ofβW48Y E.coli dominant mutation was verified after high density fermentation culture in 5 L fermenter.The incubation time was 27hours,the cell density reached the maximum OD₆₀₀=129.0,and the enzyme activity of nicotinitrile catalyzed by cell liquid was 11195 U·m L^-1.On this basis,the whole cell catalytic production of niacinamide was studied,and the optimal catalytic method was determined to be the batch addition of substrate.When the catalytic reaction was carried out for 105 min,the product concentration ofβW48Y catalyzed niacinamide from niacinamide reached 440 g·L^-1.The concentration of niacinamide was higher than that of wild type H-NHase under the same catalytic conditions(390 g·L^-1).（3）The effects of different stabilizers and immobilization methods on the storage stability ofβW48Y were studied.The stability ofβW48Y was improved by adding 10%PEG200 or 10%PEG400 with nicotinitrile as substrate.After being stored at 25℃for 2 days,the relative enzyme activity was 1.35 times and 1.41 times of that without stabilizer,and 1.75times and 1.40 times of that without stabilizer after 6 days.When acetonitrile was used as substrate,βW48Y pure enzyme with 5%PEG200 or 5%PEG400 was added and stored at25℃for 3 days,the relative enzyme activity was 1.19 times and 1.37 times of the control group.The dominant mutantβW48Y recombinant E.coli was immobilized by sodium alginate,polyvinyl alcohol and calcium chloride.The catalytic efficiency of the immobilized cells was close to that of the control group（βW48Y recombinant cells）,and the activity of the immobilized cells was 40%of the initial cell activity.