Process Study Of Tert-butyl-6-cyano-(3R,5R)-dihydroxyhexanoate Ate Using Whole Cells And Its Process Design On A 200 Tons Per Year Scale

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate((3R,5R)-CDHHB)is the key chiral intermediate of atorvastatin,the best selling drug for hypercholesterolemia treatment.Therefore,improving synthesis process of(3R,5R)-CDHHB is always desired.Thanks to its mild reaction conditions,high conversion rate and outstanding sterochemical specificity,biocatalysis has become the most promising and preferred method in the phameaceutical synthesis.In this thesis,process of(3R,5R)-CDHHB was studied by using whole cells of E.coli BL21/p ET28a-cr carrying carbonyl reductase gene in conjunction with E.coli BL21/pET28a-gdh carrying glucose dehydrogenase gene.Firstly,the fermentation process was scaled up successfully from 5 L to 50 L,500 L and up to 5000 L sacle by using the scale-up principle of constant air input rate per unit volume.Then,the cultivation conditions of E.coli BL21/pET28a-cr were optimized and given as follows: first order seed(shake flask)7~9 h,second order seed(50 L fermentor)3~5 h,third order seed(500 L fermentor)2~3 h,tap water chosen as the fermentation of water,commericial glycerol used as the material,inoculum size of 5.0%,media loading size 0.6(v/v),lactose 15.0 g/L.Under these optimized cultivation conditions,volumetric activity peaked at 17628.2 U/L,with biomass of 12.7 g DCW/L and specific activity 1387.9 U/g.(3R,5R)-CDHHB was synthesized via asymmetric bioreduction of t-butyl 6-cyano-(5R)-hydroxy-3-dihydroxyhexanoate((5R)-CHOHB)catalyzed by E.coli BL21/pET28a-cr coupled with E.coli BL21/pET28a-gdh in the whole cell form.The biocatalytic conditions were studied,and the ideal biocatalytic conditions were given as follows: triethanolamine buffer at 6.5 g/L,mass ratio of(5R)-CHOHB to glucose of 1 : 0.75(w/w),pH maintained at 6.0~7.0,temperature between 30~32 °C,E.coli BL21/pET28a-cr cell loading size 10.0 g/L,E.coli BL21/pET28a-gdh cell loading size 5.0 g/L.Under the optimized conditions,300 g/L of(5R)-CHOHB was converted within 7h,with(3R,5R)-CDHHB d.e.value over 99.5%.In addition,the forementioned bioconversion can be performed using the broths of E.coli BL21/pET28a-cr together with E.coli BL21/pET28a-gdh.Fed-batch bioconversion was evaluated to weaken the substrate inhibition.The optimized fed-batch process was obtained: the initial(5R)-CHOHB concentration was 100 g/L plus 100 g/L glucose,first feeding of 100 g/L(5R)-CHOHB and 100 g/L glucose added after 40 min conversion,second feeding at 2 h,third feeding at 4 h.The accumulated concentration of(5R)-CHOHB reached 400 g/L,which was completely converted in 6 h.Also,continuous bioconversion was tested.The optimal continuous bioconversion conditions were given as follows: the initial(5R)-CHOHB concentration was 100 g/L.(5R)-CHOHB was continuous feeding by peristaltic pump at a speed of 75 g/L·h,with(5R)-CHOHB conversion reaching 99% in 6h.The accumulated concentration of(3R,5R)-CDHHB was 400 g/L.Finally,process design at 200 ton/y(3R,5R)-CDHHB was preformed based on the above experimental results.