【摘要】：吡咯喹啉醌(Pyrroloquinoline quinone,PQQ)和β-聚蘋果酸[poly(β-L-malic acid),PMLA]是兩種具有多種不同用途的生物化學(xué)品,在食品、醫(yī)藥、化工等領(lǐng)域有廣泛的應(yīng)用。本文從多個角度較系統(tǒng)地開展了這二種重要生物化學(xué)品的高效合成機制研究及新工藝開發(fā)。論文首先對三種PQQ測定方法進行比較研究,明確了不同PQQ檢測方法的特點和適用范圍。利用GDH酶法檢測高靈敏度特點,制備高效篩選PQQ產(chǎn)生菌的活性檢測試紙,建立了從土壤中篩選PQQ高產(chǎn)菌的高通量篩選方法。在此基礎(chǔ)上,篩選到了一株能以甲醇為唯一碳源生長的PQQ高產(chǎn)菌(Methlobbacillus sp.zju323),并保藏于中國典型培養(yǎng)物保藏中心(CCTCC),編號為M2016079,搖瓶培養(yǎng)結(jié)果表明PQQ產(chǎn)量達到25.8 mg/L。然后利用Plackett-Burman實驗進行培養(yǎng)基優(yōu)化研究。結(jié)合響應(yīng)面、人工神經(jīng)網(wǎng)絡(luò)分析以及遺傳算法優(yōu)化,得到關(guān)鍵培養(yǎng)基組分和最佳濃度:CoC12·6H20、PABA 和 MgSO4·7H20,優(yōu)化后濃度分別為 3.2 mg/L、418.7 μg/L 和 1.5 g/L。建立了 pH雙階段控制發(fā)酵策略,即發(fā)酵48 h前控制pH為6.8,之后pH為5.8,在補料發(fā)酵中前期補加甲醇,后期補加酵母粉。最后利用等離子體(ARTP)誘變和亞硝基胍(NTG)誘變以及兩者復(fù)合誘變育種后,利用優(yōu)化后的培養(yǎng)基和發(fā)酵工藝補料發(fā)酵,PQQ濃度接近450.0mg/L。利用本實驗室早期篩選的聚蘋果酸生產(chǎn)菌Aurobasidium pullulans ZD-3d(CGMCC4605),開展了高產(chǎn)聚蘋果酸新策略的研究。首先比較了葡萄糖與木薯淀粉水解液對PMLA合成的影響,結(jié)果發(fā)現(xiàn)生木薯淀粉水解液更有利于PMLA合成。同時研究了膜微濾和離心分離回收細胞循環(huán)發(fā)酵技術(shù),當離心分離回收細胞循環(huán)發(fā)酵時細胞活力提高,循環(huán)發(fā)酵次數(shù)增加至5次,聚蘋果酸濃度為76.2～39.6 g/L,產(chǎn)率0.98～1.76 gL-1h-1,得率0.78～0.86 g/g,均高于膜微濾回收細胞循環(huán)發(fā)酵。利用蛋白質(zhì)組學(xué)技術(shù)研究了鈣離子對聚蘋果酸合成的影響。結(jié)果發(fā)現(xiàn)鈣離子組中有458種差異蛋白,對照組中含有726種差異蛋白。對差異蛋白GO富集分析發(fā)現(xiàn)鈣離子組中有104種膜轉(zhuǎn)運蛋白,這有利于蘋果酸和聚蘋果酸的胞內(nèi)運輸和胞外分泌。本論文一方面建立了高通量篩選高產(chǎn)PQQ菌的方法、優(yōu)化了培養(yǎng)條件與發(fā)酵工藝、利用誘變育種技術(shù)提高了 PQQ產(chǎn)量,另一方面,發(fā)展了回收細胞循環(huán)發(fā)酵合成聚蘋果酸工藝技術(shù)并通過蛋白質(zhì)組學(xué)技術(shù)進一步研究了聚蘋果酸的合成機理,本研究對這兩類化合物的高效生物合成提供了指導(dǎo)意義。
[Abstract]:Pyrroloquinoline quinone (Pyrroloquinoline quinone,PQQ) and 尾 -L-malic acid (poly) are two kinds of biological chemicals with many different uses. They are widely used in food, medicine, chemical industry and so on. In this paper, the efficient synthesis mechanism and new process development of these two important biological chemicals have been studied systematically from many aspects. In this paper, three PQQ detection methods are compared and studied firstly, and the characteristics and applicable range of different PQQ detection methods are clarified. Based on the characteristics of high sensitivity of GDH enzymatic assay, a high throughput screening method for screening high yield strains of PQQ from soil was established by preparing a high efficiency test paper for screening PQQ producing bacteria. On this basis, a strain of PQQ high yield strain (Methlobbacillus sp.zju323), which could grow with methanol as the sole carbon source, was screened and stored in Chinese typical culture center (CCTCC), number M2016079. The result of shaking flask culture showed that the yield of PQQ reached 25.8 mg/L.. Then the Plackett-Burman experiment was used to optimize the culture medium. Combined with response surface, artificial neural network analysis and genetic algorithm optimization, the key media components and optimal concentration of MgSO4 7H20 and PABA were obtained. The optimized concentrations were 3.2 mg/L,418.7 渭 g / L and 1.5 g / L, respectively. A two-stage control strategy for pH fermentation was established. The control of pH was 6.8 before 48 h fermentation, and pH was 5.8 after 48 h fermentation. Methanol was added in the early stage of feedstock fermentation and yeast powder was added in the later stage of fermentation. At last, plasma (ARTP) mutagenesis, nitrosoguanidine (NTG) mutagenesis and their compound mutagenesis were used. The concentration of PQQ was close to 450.0mg / L using optimized medium and fermentation technology. A new strategy of high yield polymalic acid (Aurobasidium pullulans ZD-3d) was studied by using Aurobasidium pullulans ZD-3d (CGMCC4605), which was screened early in our laboratory. Firstly, the effects of glucose and cassava starch hydrolysate on PMLA synthesis were compared. The results showed that raw cassava starch hydrolysate was more favorable for PMLA synthesis. At the same time, the cell cycle fermentation technology of membrane microfiltration and centrifugal separation and recovery was studied. When the centrifuge separated and recovered cells were circularly fermented, the cell viability was increased, and the number of circulatory fermentation increased to 5 times. The concentration of poly (malic acid) was 76.2 ~ 39.6 g / L, and the yield of 0.98 ~ 1.76 gL-1h-1, was 0.78 ~ 0.86 g / g, which was higher than that of membrane microfiltration. The effect of calcium ion on the synthesis of polymalic acid was studied by proteomics. The results showed that there were 458 differentially expressed proteins in calcium group and 726 differential proteins in control group. GO enrichment analysis of differential proteins revealed that there were 104 membrane transporter proteins in calcium group, which was beneficial to the intracellular transport and extracellular secretion of malic acid and polymalic acid. On the one hand, the method of high-throughput screening of high-yield PQQ bacteria was established, the culture conditions and fermentation process were optimized, and the yield of PQQ was improved by mutagenesis and breeding. The polymalic acid synthesis technology was developed and the synthesis mechanism of polymalic acid was further studied by proteomics. This study provided guidance for the efficient biosynthesis of these two kinds of compounds.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TQ920.6

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