【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(chǎn)維生素K2(MK-n)具有污染少、能耗低、產(chǎn)品生物活性高等特點(diǎn),必然成為維生素K2生產(chǎn)的發(fā)展趨勢(shì)。異源表達(dá)了來(lái)源于雪白鏈霉菌的novQ異戊烯基轉(zhuǎn)移酶基因的重組畢赤酵母Gpn12,已被證實(shí)具有較大的MK-3生產(chǎn)潛力。為了使重組菌生產(chǎn)MK-3有更高的產(chǎn)量,本文對(duì)重組菌NovQ的表達(dá)和催化合成MK-3的條件進(jìn)行優(yōu)化,以得到MK-3生產(chǎn)的較優(yōu)條件。本文探究了初始pH,誘導(dǎo)溫度,甲醇添加量和誘導(dǎo)時(shí)間對(duì)NovQ表達(dá)的影響。在起始pH7.5,誘導(dǎo)溫度25℃,每24h添加2%甲醇,誘導(dǎo)96h條件下,NovQ搖瓶表達(dá)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(dá)量較搖瓶?jī)?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(shí)間、十六烷基三甲基溴化銨(CTAB)添加量等七個(gè)因素對(duì)畢赤酵母全細(xì)胞催化合成MK-3的影響。對(duì)催化溫度、甲萘醌添加量和催化時(shí)間三個(gè)顯著因素進(jìn)行響應(yīng)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(shí)間15.87 h。經(jīng)實(shí)驗(yàn)驗(yàn)證,優(yōu)化后的搖瓶MK-3產(chǎn)量達(dá)到98.47 mg/L,與響應(yīng)面預(yù)測(cè)結(jié)果一致,較優(yōu)化前提高了 35%�；趽u瓶?jī)?yōu)化條件,先后對(duì)30L發(fā)酵罐上MK-3催化的時(shí)間和細(xì)胞催化劑濃度進(jìn)行了探究。發(fā)現(xiàn)在催化時(shí)間24 h和細(xì)胞催化劑濃度220 g/L干重時(shí),30 L發(fā)酵罐中畢赤酵母合成MK-3產(chǎn)量達(dá)到189.67 mg/L。本文實(shí)現(xiàn)了對(duì)畢赤酵母NovQ的表達(dá)優(yōu)化和MK-3催化條件優(yōu)化,并對(duì)30L發(fā)酵罐中催化合成MK-3進(jìn)行了初步探究,為Gpn12規(guī)�；a(chǎn)MK-3奠定了一定的基礎(chǔ)。為了提高維生素K2合成途徑中的側(cè)鏈供給量,本文還對(duì)維生素K2側(cè)鏈體外化學(xué)合成進(jìn)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(xì)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(hào)：2280807