5’-肌苷酸生产菌构建及发酵工艺研究

发布时间：2018-03-04 16:08

  本文选题：苷酸　切入点：生产　出处：《天津科技大学》2015年硕士论文　论文类型：学位论文

【摘要】：5’-肌苷酸作为新一代增味剂的重要组成成份,在调味品行业占据了十分重要的地位,此外,在奶粉、饲料和保健品行业中,其作为功能型添加剂的应用也十分广泛。20世纪初,日本科学家开始了发酵生产5’-肌苷酸的研究,但目前为止,5’-肌苷酸发酵生产仍存在发酵周期过长,工艺控制过程十分复杂等缺点。本文以酸性磷酸酶(Acid phosphotranferase, AP/PTase)为研究对象,通过构建大肠杆菌及枯草芽孢杆菌重组菌,对5’-肌苷酸二步发酵工艺进行研究；并以构建的大肠杆菌重组菌为出发菌,对其发酵工艺,催化工艺进行了初步探究,为5’-肌苷酸新生产方法的应用奠定了基础。本文研究了以基因序列phoCY及突变序列phoCYM分别编码的AP/PTase及AP/PTaseM催化生产5’-肌苷酸的最适条件,并验证了二者催化能力,发现二者最适pH分别为pH 4.5和pH 5.2,最适温度均为35℃。AP/PTaseM催化能力为AP/PTase的3.05倍。在此条件下,探究了多种金属离子及表面活性剂对AP/PTaseM的影响,结果表明在5-10mmol/L范围内,Mg2+、Mn2+、Fe2+、Zn2+对AP/PTaseM酶活有促进作用,但在高浓度环境中,酶活受到强烈抑制；此外,表面活性剂对酶促反应有明显促进作用,当添加8.0%Tween60或5.0%Triton X-100时,转化率分别提高49.98%和40.57%。接着,以大肠杆菌重组菌XL1-Blue+pQE30-phoCYM为出发菌株,利用5L发酵罐确定了一种获得高酶活的细胞发酵工艺。诱导剂最适添加时机为对数前期(OD600=2.0～2.5),32℃持续诱导培养8 h,酶活达到最高,为1980.07 U/L。在此条件下,催化8h底物最适浓度分别为肌苷120 mmol/L,十水合焦磷酸钠200 mmol/L,转化率达99.32%.由于目前肌苷发酵生产技术已十分成熟,本文进一步提出了二步法生产5’-肌苷酸,即与肌苷发酵相偶联,在肌苷发酵液中催化生产5’-肌苷酸,设计出两种方案：1、以肌苷生产菌B. subtilis JG为出发菌株,利用穿梭质粒pBE43和pBSA43,构建了两株能够表达AP/PTaseM的重组菌B. subtilis JAB及B. subtilis JAF,进行二步法摇瓶发酵培养和催化反应,二者5’-肌苷酸产量分别为2.35g/L、2.96g/L。2、以XL1-全细胞催化B. subtilis JG肌苷发酵液。初步探究了发酵液处理方式及EDTA添加量,Tween 60对转化率的影响。结果表明对发酵液进行离心或煮沸处理可使转化率提高7.2%,达48.0%。加入60 mmol/L EDTA和8.0% Tween60可使转化率达61.62%,5’-肌苷酸含量为16.03g/L,提高了33.3%。
[Abstract]:As an important component of a new generation of flavor enhancers, 5- inosinic acid occupies a very important position in condiment industry. In addition, it is widely used as functional additive in milk powder, feed and health products industry in the early 20th century. Japanese scientists have begun to study the production of 5- inosine monophosphate by fermentation, but so far, the fermentation cycle is too long and the process control process is very complicated. In this paper, acid phosphatase acid phosphotranferase (APP / PTase) was used as the research object. By constructing Escherichia coli and Bacillus subtilis recombinant bacteria, the two-step fermentation process of 5-inosinic acid was studied, and the fermentation process and catalytic technology of the recombinant Escherichia coli strain were studied. In this paper, we studied the optimum conditions for the production of 5- inosinic acid by AP/PTase and AP/PTaseM encoded by gene sequence phoCY and mutated sequence phoCYM, respectively, and verified their catalytic ability. It was found that the optimum pH was 4.5 and 5.2, respectively, and the optimum temperature was 35 鈩，

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