| Nitrile hydratase?NHase,EC 4.2.1.84?,a kind of metalloenzyme,is able to catalyze nitrile to the corresponding amide and mainly used for production of acrylamide and nicotinamide.Nowadays,the strain applied in industrial production is Rhodococcus rhodochrous J1 which produces high-and low-molecular mass NHases containing cobalt ion?H-NHase and L-NHase,respectively?.R.rhodochrous J1 has a few limits such as the long cultivation time,rigorous cultivation conditions and low quantity of protein expressionThis study focused on two recombinant Escherichia coli:BAG which expresses H-NHase and BAE which expresses L-NHase.The bioprocess using genetically engineered strain to synthesize nicotinamide was established by adopting methods of substrate fed-batch reaction,high cell-density cultivation and addition of protective agent.The main contents of this study were summarized as followed:?1?The shake flask medium of recombinant strains,E.coli BL21?DE3?/p ET-24a?+?-nhh Brbs Arbs G?BAG?and E.coli BL21?DE3?/p ET-24a?+?-nhh Brbs Arbs E?BAE?,was optimized based on the elements in 2YT.The carbon and nitrogen sources and cobalt ions were firstly evaluated and then the orthogonal test was applied.The final component of medium was decided:tryptone 12 g·L-1,yeast extract 6 g·L-1,glycerol 10 g·L-1,Co Cl2·6H2O 0.1 g·L-1.After the optimization,the OD600 of BAG and BAE reached to 12±1.5 and 8.3±1.9 from 10±1.2and 4.2±0.4,respectively;The cell activities of BAG and BAE reached to 90.1±4.2 U·m L-1and 331.4±10.2 U·m L-1 from 29.7±3.2 U·m L-1 and 84.3±4.7 U·m L-1,respectively.?2?The whole-cell catalysis to catalyze 3-cyanopyridine was studied.The processes of substrate flow reaction and fed-batch reaction were compared and the latter method performed better.BAG was more suitable to be applied in industrial production because of its better tolerance to 3-cyanopyridine and nicotinamide.After the evaluation of effect of substrate concentration on reaction rate,we decided to add 0.4 mol·L-1 of 3-cyanopyridine into reaction system each time and the final concentration of nicotinamide reached to 39%(w·v-1)in 105min.?3?High cell-density cultivation using BAG was applied in 5 L bioreactor.Fed-batch cultivation was performed and the exponential feeding strategy which allows cells to grow at a constant specific growth rate of 0.2 h-1 was constructed.After the optimization of concentration of lactose and Co2+,mixed solution containing 20%(w·v-1)lactose and 0.4 g·L-1 Co Cl2·6H2O was added as inducer.The maximal OD600 can reached to 200±9.8(73.6±3.6 g DCW·L-1)and cell activity was about 4 000±58 U·m L-1.Different high densities of cells were used to produced nicotinamide and the final concentration reached to 50.8%(w·v-1)in 1 h.?4?The effects of protectant and metal ions to enzyme's catalytic stability were studied.The relative activities can maintain 75%with the addition of 1%(w·v-1)magnesium ion and0.1%(w·v-1)calcium ion.These two kinds of ions were added into reaction system catalyzing3-cyanopyridine and the final concentration of nicotinamide reached to 63%(w·v-1). |
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