微生物高效生产吡咯喹啉醌和聚苹果酸的研究

发布时间：2018-09-13 13:11

【摘要】：吡咯喹啉醌(Pyrroloquinoline quinone,PQQ)和β-聚苹果酸[poly(β-L-malic acid),PMLA]是两种具有多种不同用途的生物化学品,在食品、医药、化工等领域有广泛的应用。本文从多个角度较系统地开展了这二种重要生物化学品的高效合成机制研究及新工艺开发。论文首先对三种PQQ测定方法进行比较研究,明确了不同PQQ检测方法的特点和适用范围。利用GDH酶法检测高灵敏度特点,制备高效筛选PQQ产生菌的活性检测试纸,建立了从土壤中筛选PQQ高产菌的高通量筛选方法。在此基础上,筛选到了一株能以甲醇为唯一碳源生长的PQQ高产菌(Methlobbacillus sp.zju323),并保藏于中国典型培养物保藏中心(CCTCC),编号为M2016079,摇瓶培养结果表明PQQ产量达到25.8 mg/L。然后利用Plackett-Burman实验进行培养基优化研究。结合响应面、人工神经网络分析以及遗传算法优化,得到关键培养基组分和最佳浓度:CoC12·6H20、PABA 和 MgSO4·7H20,优化后浓度分别为 3.2 mg/L、418.7 μg/L 和 1.5 g/L。建立了 pH双阶段控制发酵策略,即发酵48 h前控制pH为6.8,之后pH为5.8,在补料发酵中前期补加甲醇,后期补加酵母粉。最后利用等离子体(ARTP)诱变和亚硝基胍(NTG)诱变以及两者复合诱变育种后,利用优化后的培养基和发酵工艺补料发酵,PQQ浓度接近450.0mg/L。利用本实验室早期筛选的聚苹果酸生产菌Aurobasidium pullulans ZD-3d(CGMCC4605),开展了高产聚苹果酸新策略的研究。首先比较了葡萄糖与木薯淀粉水解液对PMLA合成的影响,结果发现生木薯淀粉水解液更有利于PMLA合成。同时研究了膜微滤和离心分离回收细胞循环发酵技术,当离心分离回收细胞循环发酵时细胞活力提高,循环发酵次数增加至5次,聚苹果酸浓度为76.2～39.6 g/L,产率0.98～1.76 gL-1h-1,得率0.78～0.86 g/g,均高于膜微滤回收细胞循环发酵。利用蛋白质组学技术研究了钙离子对聚苹果酸合成的影响。结果发现钙离子组中有458种差异蛋白,对照组中含有726种差异蛋白。对差异蛋白GO富集分析发现钙离子组中有104种膜转运蛋白,这有利于苹果酸和聚苹果酸的胞内运输和胞外分泌。本论文一方面建立了高通量筛选高产PQQ菌的方法、优化了培养条件与发酵工艺、利用诱变育种技术提高了 PQQ产量,另一方面,发展了回收细胞循环发酵合成聚苹果酸工艺技术并通过蛋白质组学技术进一步研究了聚苹果酸的合成机理,本研究对这两类化合物的高效生物合成提供了指导意义。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ920.6

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