Studies On Technologies Of Fermentation Of Gene Recombinant E.coli And Its Preparation And Extraction Of AHP

Hypertension has become the dominating disease which harms the human health and living quality owing to its high morbidity and serious syndrome. Antihypertensive peptides (AHPs) is the inhibitor of Angiotensin converting enzyme (ACE), which can control blood pressure through reducing the formation of angiotensinⅡ. AHP has many advantages deserving attentions, that is, low side effects and toxicity, and AHP could not reduce the normal blood pressure. Owing to these adventages as above, AHPs were expected to have a good prospect for an antihypertensive medicine. Nowadays, AHPs are mainly prepared from natural protein hydrolyzed by proteinase.But industrialization for product of AHPs will be faced with many difficulties, i. e. screening of specificity enzyme, purification and yield of product etc.. However, It is maybe a good choice to produce AHP by gene technology for the considerings above.The paper studied to carry out the high density fermentation of recombinant E. coli, use of domestic raw materials in fermentation and purification of end product on the basis of recombinant E. coli of AHP constructed successfully.The main results show as follows:1,The culture medium and fermentation technology in E. coli BL21 (pGEX-4T-2-AHP) were optimized through orthogonal experiment. The optimum fermentation culture medium was: yeast extract 3, tryptone 2.5, glucose 1%, sodium chloride 0.5%. The optimum cultivation and inducing conditions were: temperature 37℃, initial pH 7.0, medium loading ratio of 20%(v/v), inoculation quantity 2%(v/v), the initial OD600 of broth for inducing was 1.5, the final inducing concentration of IPTG was 0.5mmol/L and the inducing time was 4h.2,The exutory of E. coli BL21 (pGEX-4T-2-AHP) were optimized through orthogonal experiment. The paper find out use the culture medium without glucose the GST protein can be expressed induced by lactose. The optimum inducing conditions were: temperature 30℃, the initial OD600 of broth for inducing was 2.5, the final inducing concentration of lactose was 2% and the inducing time was 8h. The final expression of the GST protein was 38% higher than the exutory of IPTG.3,The high density fermentation technology of E. coli BL21 (pGEX-4T-2-AHP) were optimized through orthogonal experiment. The optimum cultivation conditions were: dissolved oxygen 40%, the culture medium was supplied at 2h after fermentation, the complementary speed was 1 mL/min, the complementary time was 1h. On this condition, the OD600 can reached 200, achieved the request of industrialization production.4, The purification technology of AHP was optimized through orthogonal experiment. The GST protein was purified by the ultrafiltration module(NMWL, 100kDa & 10kDa), the permeated rate was 47.4%. The GST protein can be digested with trypsinase which was cheaper than thrombin. The optimum conditions of trypsin cleavage were: added 15 U thrombin per mg GST, digested for 0.5h at 37℃. After the GST protein was digested with trypsinase and carboxypeptidase B, the AHP VLPVP can be detected with RP-HPLC, the content was 29.8ug/mL.