| Pregabalin(Lyrica)is one of the y-aminobutyric acid(GABA)analogs.It was the 3-isobutyl substituted analogue of the neurotransmitter inhibitor and widely applied in the treatment of neuropathic pain and epilepsy.In this paper,the main purpose is to establish the fermentation process of E.coli for production of lipase from Thermomyces lanuginose and prepare cross-linked aggregates.Definite research content is as follows:The batch fermentation conditions of E.coli BL21(DE3)/pET28b-TLL in the 5 L fermenter were obtained by single factor experiment.The fermentation parameters were determined as follows:Cultured temperatue was 37℃,stirring speed of 500 r/min and ventilation of 2.0 L/min.15 g·L-1 lactose was used as the inducer and added to the culture when OD600=8,then cultured at 28 ℃ for 20 h,the biomass reached 20.2 g DCW/L and the highest lipase production reached 800.3 U/L.These results provided a basis for fed-batch fermentation.The fed-batch fermentation conditions of E.coli BL21(DE3)/pET28b-TLL were optimized in a 5 L bioreactor.The optimized parameters of the fed-batch fermentation as follows:The temperature of growth stages was controlled at 37 ℃ for 16h,then cultured at 28 ℃ and induced for expression.The pH value was kept at 7.0 by feeding with ammonia solution.The flow rate of air was 2.0 L/min to keep DO 30%with feedback control of the growth process,increased the stirring rate through computer 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 30%,the feeding pump fed the culture at 2s/30s,20 mL/min rate.Induction:after 16 h fermentation,cooled to 28 ℃,adding lactose 90 g.After 42 h,the lipase activity and biomass reached 4030.2 U/L and 71.8 g DCW/L,respectively.E.coli BL21(DE3)/pET28b-TLL which was got by the fed-batch fermentation was ultrasonic broken,in order to collect cell extract.The optimal CLEAs preparation conditions was determined as follows:using 60%ammonium sulfate co-precipitated with additive of 0.75%(w/v)SDS for 1 h,then cross-linking with 2%(v/v)glutaraldehyde at 0℃for 2 h.The activity of CLEAs-TLL reached 159%compared to the free enzyme.The activity of CLEAs-TLL for pH 8.0 was higher than others.The best stability for pH is 9.0 and the remaining activity of CLEAs-TLL remained 60%after 26 days.The optimal reaction temperature was 45℃.The best stability for tempe-rature is 45℃.The half-life of CLEAs-TLL at 35℃ and 45℃ were 19 days and 10 days respectively.Reusing for 8 consecutive batches,the remaining activity of CLEAs-TLL has been 90%.CLEAs-TLL was stored at 0℃for 60 days,the remaining activity has been 89%. |
