毕赤酵母异戊烯基转移酶NovQ的表达及催化合成MK-3条件优化

发布时间：2018-10-19 10:05

【摘要】：维生素K2是一种人类不可缺少的维生素。微生物发酵法生产维生素K2(MK-n)具有污染少、能耗低、产品生物活性高等特点,必然成为维生素K2生产的发展趋势。异源表达了来源于雪白链霉菌的novQ异戊烯基转移酶基因的重组毕赤酵母Gpn12,已被证实具有较大的MK-3生产潜力。为了使重组菌生产MK-3有更高的产量,本文对重组菌NovQ的表达和催化合成MK-3的条件进行优化,以得到MK-3生产的较优条件。本文探究了初始pH,诱导温度,甲醇添加量和诱导时间对NovQ表达的影响。在起始pH7.5,诱导温度25℃,每24h添加2%甲醇,诱导96h条件下,NovQ摇瓶表达量较优化前提高了约10倍,30 L发酵罐流加发酵NovQ表达量较摇瓶优化前提高了约15倍。考察了摇瓶中初始pH、温度、甲醇添加量、前体(甲萘醌、异戊烯醇)添加量、催化时间、十六烷基三甲基溴化铵(CTAB)添加量等七个因素对毕赤酵母全细胞催化合成MK-3的影响。对催化温度、甲萘醌添加量和催化时间三个显著因素进行响应面优化得出催化条件为:催化温度31.56℃,甲萘醌添加量295.54 mg/L,催化时间15.87 h。经实验验证,优化后的摇瓶MK-3产量达到98.47 mg/L,与响应面预测结果一致,较优化前提高了 35%。基于摇瓶优化条件,先后对30L发酵罐上MK-3催化的时间和细胞催化剂浓度进行了探究。发现在催化时间24 h和细胞催化剂浓度220 g/L干重时,30 L发酵罐中毕赤酵母合成MK-3产量达到189.67 mg/L。本文实现了对毕赤酵母NovQ的表达优化和MK-3催化条件优化,并对30L发酵罐中催化合成MK-3进行了初步探究,为Gpn12规模化生产MK-3奠定了一定的基础。为了提高维生素K2合成途径中的侧链供给量,本文还对维生素K2侧链体外化学合成进行了探索性研究,制备的DMAPP和法尼基焦磷酸将直接用于MK-3的体外全细胞催化。
[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，

本文编号：2280807