| Nitrilase can directly convert nitriles into carboxylic acids.It exhibits broad application prospects in production of medical intermediates,food additives,and bulk chemicals.Therefore,the biosynthesis of nicotinic acid via hydrolysis of 3-cyanopyridine with nitrilase is a promising and green approach with superior application potential.In our previous study,a high nitrilase producing strain Pseudomonas putida was isolated from environmental samples.However,the biomass of Pseudomonas putida in the fermentation process is relatively low and the level of enzyme production is thus limited,and conventional recombinant strains which produced nitrilase generally need to be induced by IPTG,which increased the operation procedures and production costs.In this study,the constitutive expression system of nitrilase was successfully constructed based on cloning of P.putida nitrilase gene,which avoided the use of toxic IPTG and the increase of production costs.Furhtermore,the strategy of high cell density cultivation?HCDC?was adopted in order to improve the biomass and nitrilase productivity.Moreover,the continuous process for bioproduction of nicotinic acid was investigated.The details were as follows:?1?This study used the nitrilase gene from Pseudomonas putida CGMCC3830 as the research objective.Firstly,the constitutive expression plasmids of nitrilase were successfully constructed with pET-3b and pMA5,and named as pET-3b-NIT and pMA5-NIT,respectively.After that,recombinant plasmids pET-3b-NIT and pMA5-NIT are transformed into Escherichia coli and Bacillus subtilis WB600,respectively.The recombinant strain E.coli BL21?DE3??pET-3b-NIT?reached the highest nitrilase activity(25.18 U·mL-1)in LB medium at 8 h.The nitrilase from B.subtilis WB600?pMA5-NIT?reached the highest activity of 4.55 U·mL-1 at 9.5 h.Because of the higher amount of nitrilase production,shorter fermentation cycle,and feasibility of performing high-density cultivation,E.coli BL21?DE3??pET-3b-NIT?was selected as the target strain for further study.?2?In order to improve the biomass of recombinant strain E.coli BL21?DE3??pET-3b-NIT?and nitrilase production,the technology of high cell density cultivation was employed in this study.The basic fermentation conditions and nitrilase production level were observed by batch fermentation.In the batch fermentation,the highest enzyme activity and OD600 were 43.98 U·m L-1and 12.92,respectively.Meanwhile,constant-rate feeding strategy and pH-stat feeding strategy were utilized.In the constant-rate feeding fermentation,pH was maintained at 6.8 by feeding medium and ammonia;results showed that the highest enzyme activity and OD600 were 111.48 U·mL-1and 43.50,respectively.In the pH-stat feeding fermentation,pH was maintained at 6.8 by feeding ammonia;at each pH increase by 0.01,0.03,and 0.05,the feeding medium was fed back to fermentation system at the early,middle,and late stage of the cultivation process,respectively.And the highest enzyme activity reached up to 653.84 U·m L-1;the highest OD600 value was 86.40.To our knowledge,the nitrilase production of this study is the highest level reported in literature.?3?In order to fully explore the catalytic potential of recombinant strain E.coli BL21?DE3??pET-3b-NIT?,the catalytic properties of recombinant strain were investigated.The optimum catalytic conditions were pH 7.2 and 50?.It also displayed the best stability at 4? and pH 7.2.The suitable substrate concentration was 50 mM.The enzyme activity was obviously inhibited when the nicotinic acid concentration was more than 200 mM.The enzyme activity was strongly inhibited by Ag+;whereas Li+?Ni2+?Sr2+?Mg2+?Sn2+ and Co2+ promoted the enzyme activity.5% and 20% propylene glycol could improve the enzyme activity by 14% and 38%,respectively.In order to explore the bioconversion potential of E.coli BL21?DE3??pET-3b-NIT?,the biotransformation of 3-cyanopyridine was carried out with resting cells as the catalysts.We studied the effects of substrate feeding concentration and resting cell concentration on production of nicotinic acid.The results showed that,the average transformation rate per gram cells to substrate reached the highest value(22.90 g·h-1)at 200 mM(20.8 g·L-1)feeding substrate concentration.418 g·L-1of nicotinic acid was obtained within 410 min after 17 batches of feeding with 200 mM 3-cyanopyridine.The results of biotransformation with different cell concentration showed that it was disproportional between the average transformation rate and resting cell concentration,where the low cell concentration supported a relatively higher conversion rate.200 mM 3-cyanopyridine coud be completely hydrolyzed by using 3.51 g·L-1 resting cell within 290 min after 22 batches of feeding with 200 mM 3-cyanopyridine,and finally 541 g·L-1 of nicotinic acid was obtained.Besides,the conversion rate reached 100% without generation of byproducts.This study paves the way for the biosynthesis of nicotinic acid in industrial scale. |
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