【摘要】：維生素K2是一種人類不可缺少的維生素。微生物發(fā)酵法生產維生素K2(MK-n)具有污染少、能耗低、產品生物活性高等特點,必然成為維生素K2生產的發(fā)展趨勢。異源表達了來源于雪白鏈霉菌的novQ異戊烯基轉移酶基因的重組畢赤酵母Gpn12,已被證實具有較大的MK-3生產潛力。為了使重組菌生產MK-3有更高的產量,本文對重組菌NovQ的表達和催化合成MK-3的條件進行優(yōu)化,以得到MK-3生產的較優(yōu)條件。本文探究了初始pH,誘導溫度,甲醇添加量和誘導時間對NovQ表達的影響。在起始pH7.5,誘導溫度25℃,每24h添加2%甲醇,誘導96h條件下,NovQ搖瓶表達量較優(yōu)化前提高了約10倍,30 L發(fā)酵罐流加發(fā)酵NovQ表達量較搖瓶優(yōu)化前提高了約15倍�？疾炝藫u瓶中初始pH、溫度、甲醇添加量、前體(甲萘醌、異戊烯醇)添加量、催化時間、十六烷基三甲基溴化銨(CTAB)添加量等七個因素對畢赤酵母全細胞催化合成MK-3的影響。對催化溫度、甲萘醌添加量和催化時間三個顯著因素進行響應面優(yōu)化得出催化條件為:催化溫度31.56℃,甲萘醌添加量295.54 mg/L,催化時間15.87 h。經(jīng)實驗驗證,優(yōu)化后的搖瓶MK-3產量達到98.47 mg/L,與響應面預測結果一致,較優(yōu)化前提高了 35%。基于搖瓶優(yōu)化條件,先后對30L發(fā)酵罐上MK-3催化的時間和細胞催化劑濃度進行了探究。發(fā)現(xiàn)在催化時間24 h和細胞催化劑濃度220 g/L干重時,30 L發(fā)酵罐中畢赤酵母合成MK-3產量達到189.67 mg/L。本文實現(xiàn)了對畢赤酵母NovQ的表達優(yōu)化和MK-3催化條件優(yōu)化,并對30L發(fā)酵罐中催化合成MK-3進行了初步探究,為Gpn12規(guī)�；aMK-3奠定了一定的基礎。為了提高維生素K2合成途徑中的側鏈供給量,本文還對維生素K2側鏈體外化學合成進行了探索性研究,制備的DMAPP和法尼基焦磷酸將直接用于MK-3的體外全細胞催化。
[Abstract]:Vitamin K 2 is an indispensable vitamin for human beings. The production of vitamin K _ 2 (MK-n) by microbial fermentation has the characteristics of low pollution, low energy consumption and high biological activity, which is bound to be the development trend of vitamin K _ 2 production. The recombinant Pichia pastoris Gpn12, which expressed the isoamyltransferase gene of novQ from Streptomyces albicans, has been proved to have great potential for MK-3 production. In order to increase the yield of MK-3 by recombinant bacteria, the expression of NovQ and the conditions of catalytic synthesis of MK-3 were optimized in this paper, and the optimum conditions for MK-3 production were obtained. The effects of initial pH, induction temperature, methanol addition and induction time on NovQ expression were investigated. Under the conditions of initial pH7.5, induction temperature 25 鈩，

本文編號：2280807