本文選題：苷酸　切入點(diǎn)：生產(chǎn)　出處：《天津科技大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：5’-肌苷酸作為新一代增味劑的重要組成成份,在調(diào)味品行業(yè)占據(jù)了十分重要的地位,此外,在奶粉、飼料和保健品行業(yè)中,其作為功能型添加劑的應(yīng)用也十分廣泛。20世紀(jì)初,日本科學(xué)家開始了發(fā)酵生產(chǎn)5’-肌苷酸的研究,但目前為止,5’-肌苷酸發(fā)酵生產(chǎn)仍存在發(fā)酵周期過長,工藝控制過程十分復(fù)雜等缺點(diǎn)。本文以酸性磷酸酶(Acid phosphotranferase, AP/PTase)為研究對象,通過構(gòu)建大腸桿菌及枯草芽孢桿菌重組菌,對5’-肌苷酸二步發(fā)酵工藝進(jìn)行研究；并以構(gòu)建的大腸桿菌重組菌為出發(fā)菌,對其發(fā)酵工藝,催化工藝進(jìn)行了初步探究,為5’-肌苷酸新生產(chǎn)方法的應(yīng)用奠定了基礎(chǔ)。本文研究了以基因序列phoCY及突變序列phoCYM分別編碼的AP/PTase及AP/PTaseM催化生產(chǎn)5’-肌苷酸的最適條件,并驗(yàn)證了二者催化能力,發(fā)現(xiàn)二者最適pH分別為pH 4.5和pH 5.2,最適溫度均為35℃。AP/PTaseM催化能力為AP/PTase的3.05倍。在此條件下,探究了多種金屬離子及表面活性劑對AP/PTaseM的影響,結(jié)果表明在5-10mmol/L范圍內(nèi),Mg2+、Mn2+、Fe2+、Zn2+對AP/PTaseM酶活有促進(jìn)作用,但在高濃度環(huán)境中,酶活受到強(qiáng)烈抑制；此外,表面活性劑對酶促反應(yīng)有明顯促進(jìn)作用,當(dāng)添加8.0%Tween60或5.0%Triton X-100時(shí),轉(zhuǎn)化率分別提高49.98%和40.57%。接著,以大腸桿菌重組菌XL1-Blue+pQE30-phoCYM為出發(fā)菌株,利用5L發(fā)酵罐確定了一種獲得高酶活的細(xì)胞發(fā)酵工藝。誘導(dǎo)劑最適添加時(shí)機(jī)為對數(shù)前期(OD600=2.0～2.5),32℃持續(xù)誘導(dǎo)培養(yǎng)8 h,酶活達(dá)到最高,為1980.07 U/L。在此條件下,催化8h底物最適濃度分別為肌苷120 mmol/L,十水合焦磷酸鈉200 mmol/L,轉(zhuǎn)化率達(dá)99.32%.由于目前肌苷發(fā)酵生產(chǎn)技術(shù)已十分成熟,本文進(jìn)一步提出了二步法生產(chǎn)5’-肌苷酸,即與肌苷發(fā)酵相偶聯(lián),在肌苷發(fā)酵液中催化生產(chǎn)5’-肌苷酸,設(shè)計(jì)出兩種方案：1、以肌苷生產(chǎn)菌B. subtilis JG為出發(fā)菌株,利用穿梭質(zhì)粒pBE43和pBSA43,構(gòu)建了兩株能夠表達(dá)AP/PTaseM的重組菌B. subtilis JAB及B. subtilis JAF,進(jìn)行二步法搖瓶發(fā)酵培養(yǎng)和催化反應(yīng),二者5’-肌苷酸產(chǎn)量分別為2.35g/L、2.96g/L。2、以XL1-全細(xì)胞催化B. subtilis JG肌苷發(fā)酵液。初步探究了發(fā)酵液處理方式及EDTA添加量,Tween 60對轉(zhuǎn)化率的影響。結(jié)果表明對發(fā)酵液進(jìn)行離心或煮沸處理可使轉(zhuǎn)化率提高7.2%,達(dá)48.0%。加入60 mmol/L EDTA和8.0% Tween60可使轉(zhuǎn)化率達(dá)61.62%,5’-肌苷酸含量為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，

本文編號：1566354