Biocatalysis Of Acrylic Acid By Nitrilase-Producing Strain Arthrobacter Nitroguajacolicus ZJUTB06-99

Considerable industrial interest in enzymatic conversion of nitrile into corresponding acid or amides was aroused in the recent years for the superiority of biotransformation in applications of synthesis of a wide range of high additional value chemicals.This environmental-friendly bioconversion is not only a much cleaner process than traditional chemical methods,but also a relatively mild synthesis with high selectivity and yield.And the product acrylic acid is one of bulk chemicals with widely application.The paper was focused on the bioconversion process of acrylonitrile to acrylic acid and investigated in terms of the screening,identification,characterization of the biocatalyst, the cultivation and application of the biocatalyst,preparation and application of immobilized catalyst and the clone of nitrilase gene and expression in E.coli. Firstly,isolates were obtained by enrichment using acrylonitrile as the sole nitrogen source.With this method,strain ZJUTB06-99 with the high nitrilase activity and stability was chosen as the best strain for further studies.Based on morphology,physiologic tests,and the 16S rDNA sequence,strain ZJUTB06-99 was identified as Arthrobacter nitroguajacolicus.It was the first to describe a nitrilase enzyme from A. nitroguajacolicus.The medium composition was optimized by single factors and response surface methodology（RSM）.The optimized medium composition was as follows（%w/v）:glucose 2.80,yeast extract 0.57,ε-caprolactam 0.42,K₂HPO₄ 0.05,KH₂PO₄ 0.05,MgSO₄ 0.05,and sodium glutamate 0.075.The activity of the cells after medium optimization was 2.86 folds of that with initial medium.The optimum conditions for cell growth and formation of nitrilase were as follows: inoculum volume 3%（v/v）,medium volumetric ratio 15-20%（v/v）,and initial pH value 7.0.The suitable culture time was about 72-78 h.Influences of reaction conditions on nitrilase activity were also evaluated.It was indicated that the nitrilase exhibited maximal activity in phosphate buffer（pH 6.5） at 40℃.It fit the Arrhenius equation between 25-40℃,the apparent activation energy Ea was 45.53 kJ/mol.The nitrilase of A.nitroguajacolicus ZJUTB06-99 had good thermostability with half-life(t_1/2) of 218.6,157.6 and 129.8 h at 4℃、30℃and 40℃, respectively.0.3 g cells in 10 mL reaction buffers could completely consumed 0.3%（v/v） after 165 min.Subsequently,the substrate spectrum test of ZJUTB06-99 exhibited that A.nitroguajacolicus ZJUTB06-99 had a relatively wide substrate spectrum which could convert series of nitrile to corresponding acid without any byproduct. Both acrylonitrile and acrylic acid inhibited the hydrolysis at concentrations above 0.7%（v/v） and 0.5%（v/v）.Moreover,kinetic studies of the nitrilase catalyzed reaction showed that the kinetic constants were as follows:K_m=11.71 mmol/L,V_m=0.26 mmol/（L·min）, K_s=133.05 mmol/L.To improve the reusability of the biocatalyst,chitosan and alginate were employed to immobilize the resting cells of A.nitroguajacolicus ZJUTB06-99.The chitosan immobilization preparation conditions were set as follows:2.0%（w/v） of chitosan,1.5%（w/v） of TPP,and 5%（w/v） of immobilized cells.The alginate immobilization preparation conditions were set as follows:1.5%（w/v） of sodium alginate,3.0%（w/v）of CaCl₂, 6 h of immobilization time,2 mm of bead diameter,and 4-6%（w/v） of immobilized cells.Reaction conditions of immobilized cell-mediated conversion were evaluated.The results showed that the suitable buffer of conversion were phosphate buffer with pH 6.2 and 7.4 at 45℃of chitosan and alginate beads.At 45℃,the half-lives(t_1/2) of the cells were 108.1 h and 120.7 h.Under these conditions,an initial acrylonitrile concentration of 0.3%（v/v） was completely converted to acrylic acid by alginate entrapped cells after 300 min.The reuse batches of chitosan and alginate beads were 8,and 11 times.The surface microstructures of the beads were observed by ESEM.The tolerance to substrate was enhanced from 0.7%（v/v） to 1.0%（v/v） after cells immobilization.Stabilization of the alginate immobilized cells with polyethyleneimine（PEI） and glutaraldehyde（GA） could alleviate the leakage of the cells.It was found that the optimum PEI,CaCl₂ and GA concentrations were 0.10%,30 mM and 0.75%,respectively,and the optimum cross-linkage time of GA was 30 s.Both the tolerance to high phosphate concentration and the bead strength of the PEI and GA stabilized cells were considerably enhanced. Moreover,reusability was significantly improved with 20 batches although the stabilized beads showed a little lower activity after PEI/GA treatment.ESEM observation implied that cells after PEI treatment were more densely distributed in the matrix.The cell debris was entrapped by chitosan and alginate.The chitosan immobilization preparation conditions were set as follows:2.5%（w/v） of chitosan,1.5%（w/v） of TPP,and 5%（w/v） of immobilized cells detris. The alginate immobilization preparation conditions were set as follows: 1.5%（w/v） of sodium alginate,3.0%（w/v） of CaCl₂,6 h of immobilization time,2.1 mm of bead diameter,and 5%（w/v） of immobilized cells debris.Reaction conditions of immobilized cells debris conversion were evaluated.The results showed that the suitable buffer of conversion were phosphate buffer with pH 6.2 and 7.4 at 30℃for chitosan and alginate carriers,respectively.The immobilized cells had relatively good stability under the optimum reaction pH.At 30℃,the half-lives(t_1/2) of the cells were 86.9,70.9 h for these two carriers.At 55℃,the half-lives(t_1/2) of the cells were 5.1 and 4.0 h.The convertion rate was improved after cells broken with an initial acrylonitrile concentration of 0.3%（v/v） completely converted to acrylic acid by alginate entrapped cells after 260 min.The reuse batches of chitosan and alginate beads were 6,and 8 times.The nitrilase gene of A.nitroguajacolicus ZJUTB06-99 was amplified by PCR method and then sequenced.The gene of A. nitroguajacolicus ZJUTB06-99 nitrilase was composed of 819 base pairs.The gene was ligated with pTrc99a and pET20b vectors and transformed into E.coli JM109 and BL21（DE3） to construct the recombinant strain.The nitrilase activities of the recombinant E.coli JM109/pTrc99a and E.coli BL21（DE3）/pET20b was 4.23 and 6.15 U/g DCW induced by IPTG,respectively.