Biocatalyzation Routine Development Of Key Chiral Intermediate Of Pregab Alin And Its Process Design At 10 Tons Per Year Scale

(3S)-2-carboxyethyl-3-cyano-5-methyl-exanoic acid is a valuable synthetic intermediate for Pregabalin,which was developed for the treatment of neuropathic pain and epilepsy.Thus,the production of the chiral intermediate through biocatalyzation has been a hot issue.The E.coli BL21(DE3)/pET28b-TLL,a lipase produced by recombinant bacteria,can transform racemic 2-carboxyethyl-3-cyano-5-methylhex-anoic acid ethyl ester(CNDE)to(3S)-cyano-2-earboxyethyl-5-methyl-hexanoie-acid.In this study,the expression conditions of recombinant bacteria was optimized,and the transformation of CNDE to(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid using recombinant E.coli as whole cell biocatalyst was studied.Additionally,a preliminary design of technological process for pregabalin production at the scale of 10 tons per year was created.These results provided a basis for industrial production of the intermediate of Pregabalin.Firstly,the batch fermentation conditions of E.coli BL21(DE3)/pET28b-TLL in the 5 L fermenter was obtained by single factor experiment.The fermentation parameters was determined as follows:Cultured temperatue was 37?,stirring speed of 500 r/min and ventilation of 1.5 L/min.10 g·L-1 lactose was used as the inducer and added to the culture when OD600=7,then cooled to 28 ? to induce for expression.After 22 h cultured at 28?,the biomass reached 8.3 g DCW/L,and the highest lipase production reached 252.4 U·L-1.These results provided a basis for fed-batch fermentation.Afterwards,the fed-batch fermentation conditions of E.coli BL21(DE3)/pET28b-TLL was optimized in a 5 L bioreactor.The optimized parameters of the fed-batch fermentation determined as follows:The temperature of growth stages was controlled at 37? for 12h,then cooled to 28 ? and induced for expression.The pH value was kept at pH 7.2 by automatic feeding with 20%ammonia solution.The flow rate of air was 1.5L/min to keep DO 50%with feedback control of the growth process,increased the stirring rate through computer software control when the dissolved oxygen was lower than the set value.Feeding strategy:feeding pump operation was directly controlled by the dissolved oxygen value.When the dissolved oxygen was higher than 50%,the feeding pump fed the culture at 1s/20s,10mL/min rate.Induction:after 12 h fermentation,cooled to 28 ?,adding lactose 45 g,after 18 h induction,adding 45 g lactose again,so that the final concentration was 30 g/L.After 26 h cultured,the lipase activity and biomass reached 798.4 U·L-1 and 38.4 g dcw·L-1,respectively.In addition,the biotransformation of racemic 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl estered to(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid using recombinant cell as biocatalysts was investigated.The results indicated that optimal pH was 8.0,and the lipase showed highest activity at 50?.In a 2 L reaction system,150 mM calcium acetate and 75g/L cell was added,and the substrate was added 4 times,600 mM for each time.After 24 h,the final substrate conversion was 28.93%,e.e.value of products was 92.59%,space-time yield reached 8.18g/(L·h).Finally,the technological design and material balance of the production process of fermentation section,biological catalysis section and decarboxylation section were established.The results provided a basis for production of key chiral intermediates of Pregabalin at the scale of 10 tons per year.