Screening,molecular Modification And Application Of Stereoselective Nitrilase

Nitrilases(EC 3.5.5.1)are important industrial enzymes used to convert nitriles directly into corresponding carboxylic acids and ammonia in a single step with mild conditions.It was reported that one of the important industrial applications of nitrilases is the conversion of(R,S)-mandelonitrile(R,S-MN)to(R)-(-)-mandelic acid(R-MA).This reaction exhibits excellent enantioselectivity,low-cost starting material,and most especially a possibility of performing a dynamic kinetic resolution of racemic substrate which provides theoretically 100% yield of the product.R-MA has been used as an important intermediate for the production of pharmaceuticals such as semi-synthetic penicillins,cephalosporins,antitumor agents,antiobesity agents and chiral resolving agent for the resolution of racemic alcohols and amines.R-MA can be produced by two routes including chemical and biosynthetic methods.Biocatalytic synthesis of R-MA has been widespread because of its industrial development and application prospects.In this study,screening,molecular modification,industrial application and immobilization of stereoselective nitrilase were investigated in details.Firstly,all the strains expressing nitrilase in our lab were screened,and mandelonitrile was mainly researched.A strain called WtNit was obtained with high activity and stereoselectivity.In this study,the recombinant nitrilase from WtNit was selected to improve activity by using gene site saturation mutagenesis method(GSSM).Based on the secondary and three-dimensional structures,interactions between substrate and the nitrilase,and previous reports,five sites were selected for further mutagenesis.After mutagenesis,the positive mutants were selected using a convenient two-step high-throughput screening method based on the product formation and pH indicator combined with HPLC method.After three rounds of GSSM,a best mutant Mut3,with 200% relative activity than WtNit in pure water system,was selected for further studies.After loaded to a nickel-NTA superflow column,the characteristics of purified WtNit and Mut3 nitrilases were studied and compared.Both of nitrilase activity of WtNit and Mut3 shared almost the same trends under different pH conditions or different temperature conditions,and the optimum pH and temperature were 8.0 and 45 oC,at the same time,the specific activity of Mut3 nitrilase was 154% higher than WtNit nitrilase at pH 8.0.Kinetic analysis showed that the values of Km,Vmax and kcat were calculated to be 20.64 mM,33.74 ?mol mg-1 min-1 and 24.45 s-1 for WtNit nitrilase,while 9.24 mM,47.68 ?mol mg-1 min-1 and 34.55 s-1 for Mut3 nitrilase,respectively.In the second part,the application of nitrilase was studied.100 mM R,S-MN was converted to R-MA by 10 g/L wet cells,and the yield of Mut3 could reach 100% in 0.5 h which was higher than WtNit.In a fed-batch reaction,800 mM R,S-MN was converted to R-MA by whole-cell catalysis of 50 g/L Mut3,and the accumulative concentration of R-MA reached 693 mM at 7.5 h in this work.Mut3 was produced in the fermentor,and specific activities in 700 L,7000 L and 20000 L fermentors were 114.19,145.11 and 138.60 U/g(DCW),respectively.Fermentation broth from the 700 L,7000 L and 20000 L fermentors was used for R-MA production in a 500 L reaction tank,respectively.R,S-MN was added by fed-batch,and the R-MA concentration could reach 57.39,68.71 and 64.85 g/L,respectively.At the same time,the separation characteristics of R-MA were also studied.The optimum alkaline p H,alkaline extraction ratio,the optimum acidic pH,acidic extraction ratio and the amount of activated carbon were 10.0,1:0.5(once),2.50,1:2(twice)and 1% for the separation of R-MA,respectively.The recovery of R-MA with >99% purity and 99.9% ee was 78.8%.In the third part,five kinds of immobilization methods were used and the direct cross-linking Mut3 using DA/GA/PEI was selected,and the relative activity of DA/GA/PEI was 135.95%.After investigating the operation stability of immobilized cell at 35 °C,the results showed that the operational efficiency of immobilized cells remained stable,and approximately 100% of activity remained after 28 reuse cycles,whereas free cells was 8.79% after 10 batches.In a fed-batch reaction,832 mM R,S-MN was converted to R-MA by 50 g/L immobilized cells DA/GA/PEI,and the cumulative concentration of R-MA in the reaction mixture reached 805.3 mM.The optimum temperatures of immobilized cells and free cells for the efficient hydrolysis of R,S-MN were 50 °C and 45 °C,respectively.The study of storage stability indicated that the immobilized cells can be used in long-term storage at 4 °C,however,the free cells showed better stability than immobilized cells at-20 °C.To achieve a RPBR reaction,the immobilized cells need to be air dried to a constant weight at 25 oC,and the shapes and sizes of the dry immobilized cells were studied.The granular shape of dry immobilized cells with 10~18 mesh were the best.And the kinetic resolution process showed higher initial reaction rate and yield by recycling 100 mL of substrate solution(70 mM)through RPBR packed with 6.0 g immobilized cells with a feeding rate of 19 mL/min while the H/D ratio was 2.8.The immobilized cells kinetic resolution of R,S-MN was studied in the RPBR for 50 batches,and the relative activity was >78%.Finally,to resolve the problems of coupling with glutaraldehyde above,THP instead of glutaraldehyde as a coupling reagent was used for the immobilization of nitrilase for the first time.The efficient hydrolysis of R,S-MN was observed using immobilized cells DA/PEI/THP when the temperature was 55 °C and pH was 8.0,and the half-life periods about thermal stabilities of immobilized cells measured at 35,40 and 50 °C were 1800,965,and 163 h,respectively.The study of substrate tolerability showed that the specific activity of immobilized cells DA/PEI/THP was 6.08-fold higher than that of immobilized cells DA/GA/PEI at the concentration of R,S-MN 300 mM.The concentration of R-MA reached 358 mM after 1 h conversion with 500 mM R,S-MN by 250 g/L immobilized cells DA/GA/PEI.In a fed-batch reaction,the accumulative concentration of R-MA of immobilized cells DA/PEI/THP(1.49 M after 15 h conversion)was higher than that of DA/GA/PEI(1.27 M after 13 h conversion),affording the highest productivity 363.70 g L-1d-1.The operational efficiency of immobilized cells remained stable,and 64% of relative activity remained after 60 cycles with the successive production of 5.69 M R-MA for the dried immobilized cells with granular shape.