微生物制备β-聚-L-苹果酸的研究
文献类型：学位论文
| 作者 | 曹伟锋 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-05-22 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 万印华 |
| 关键词 | β-聚-L-苹果酸 出芽短梗霉 补料发酵 膜细胞循环 氧化还原电位 |
| 其他题名 | Study on the production of β-poly (L-malic acid) by microorganism |
| 学位专业 | 生物化工 |
| 中文摘要 | β-聚-L-苹果酸（PMLA）是一种具有极佳水溶性的阴离子聚酯，在医学及其它工业中受到越来越多的重视。本文系统地研究了菌株出芽短梗霉ipe-1生产PMLA的发酵条件及培养基组成、PMLA的代谢途径、细胞生长与PMLA合成的关系、在对数生长后期提高PMLA产量的策略和PMLA的结构特性。 pH 6.0和溶解氧浓度（DO）大于70%时PMLA产量最高，同时pH对细胞的形态有明显的影响，当pH大于等于6.0时，细胞主要呈酵母形态，此形态细胞是主要的PMLA生产者。高DO、高转速将促进细胞生长，但却降低了PMLA对葡萄糖的得率，而在对数生长前期高细胞浓度有利于PMLA的合成，因此采用三段DO和搅拌控制策略有利于促进PMLA合成且提高PMLA对葡萄糖的得率，第一阶段恒定高转速（800 rpm）维持DO大于70%；第二阶段当DO自然降至70%时，通过搅拌与DO偶联控制DO 70%促进PMLA的合成；第三阶段低转速（400 rpm）高DO（70%）降低细胞生长速率，进而提高PMLA对细胞的得率。 PMLA的合成与葡萄糖的消耗速率及细胞的生长速率成正比。采用氧化态氮源硝酸钠时PMLA产量最高，而随着还原态铵浓度的增加PMLA产量降低；当硝酸钠浓度大于6 g/L时，将显著降低黑色素的合成，最大PMLA产量达到37 g/L。初始 5 g/L酵母粉和190 g/L葡萄糖对PMLA的合成较适宜，进一步增加酵母粉浓度对细胞生长无明显促进作用，葡萄糖浓度大于190 g/L将抑制细胞的生长。当在培养基中添加锌离子和磷酸二氢离子时将提高PMLA的生产强度，尽管锰离子对PMLA的生产强度也有促进作用，但是同时促进了黑色素的合成，铁、镁、铜及钾离子对PMLA的产生无明显促进作用，而添加0.1 g/L氯化钙却使PMLA产量提高11.38%。氢氧化钠和碳酸钠做中和剂对PMLA合成无明显影响，而采用氨水做中和剂导致生物量增加23.41%，PMLA产量降低19.96%。 PMLA的合成与细胞的生长在对数生长前期相偶联，而在对数生长后期出现部分解离，原因是高浓度的PMLA（大于40 g/L）不仅抑制PMLA的合成而且对细胞的生长有害，另外，在对数生长后期培养基中氧化还原电位（CRP）从57 mV升高到100 mV，缺乏还原力也将降低PMLA的生产强度。因此，通过半连续发酵维持细胞处于对数生长阶段或通过膜-细胞循环移除新产生的PMLA将提高PMLA的生产强度，同时无论在哪种发酵模式下在对数生长后期控制CRP小于70 mV都将促进PMLA的产生。在超过240 h的5个半连续发酵周期内PMLA的平均浓度60.19 g/L、生产强度达到1.15 g/L·h。 当葡萄糖作为补料溶液的碳源，发酵结束时分批发酵与分批补料发酵中PMLA产量无明显差别。初糖为蔗糖时PMLA产量最高，而为果糖时PMLA对菌体的得率最高，自36 h开始连续补充葡萄糖与蔗糖（10:1）混合液可维持CRP小于70 mV，PMLA产量达到63.2 g/L，同时发酵体积增加25%。另外，补充外源L-苹果酸、丁二酸、三氟乙酸和亲和素对PMLA的合成无明显影响，而生物素、丙酮酸及高的CRP将抑制PMLA的合成，这说明PMLA的合成途径主要涉及磷酸烯醇式丙酮酸（PEP）在PEP羧化酶催化下经由草酰乙酸合成PMLA的还原途径。 从菌株ipe-1发酵液提取获得的PMLA重均分子量为10.58 KDa，分散系数1.13，而在发酵过程中PMLA重均分子量变化范围为12.7-18.6 KDa，在190-260 nm 具有正向的CD谱带。 |
| 英文摘要 | β-poly (L-malic acid) (PMLA) is a highly water-soluble anionic polyester of L-malate, which has attracted industrial interest for its potential applications in medicine and other industries. In this dissertation, with Aureobasidium pullulans ipe-1 as the PMLA producer, fermentation conditions and medium compositions, metabolic pathway for PMLA production, relations between cell growth and PMLA production, intensification of PMLA production in the late exponential growth phase and PMLA structural features were systematically investigated. The strain produced a high concentration of PMLA when pH and DO remained about 6.0 and above 70%, respectively. The cells morphogenesis, affected significantly by pH values, mainly remained yeast-like form when pH was above 6.0, which was the main PMLA producer. High stirring speed would increase cell growth at high DO values with decreased PMLA production on per gram glucose (Yp) as a cost, while high cell concentration would be beneficial to PMLA production at early logarithmic growth phase. To enchance PMLA production, the cultivation was divided into three control phases. In phase I, cell growth was accelerated by maintaining DO more than 70% with constant high stirring speed (800 rpm). In phase II, PMLA production was increased by controlling DO at constant value of 70% by adjusting automatically the stirring speed. In phase Ⅲ, Yp was enhanced by keeping DO at about 70% with low stirring speed to decrease cell growth. This control strategy increased the production of PMLA and Yp by 15% and 18%, respectively. The production of PMLA was related positively to glucose consumption and biomass increase. The maximum amounts of PMLA were synthesized in the presence of an oxidized nitrogen source (NO3-) in the medium, while increasing the reduced nitrogen source (NH4+) concentration would decrease the yield of PMLA. With high sodium nitrate concentrations (above 6 g/L), the strain produced PMLA without melanin synthesis, maximum PMLA production was up to 37.0 g/L. Initial 5 g/L yeast extract and 190 g/L glucose were preferred for PMLA production. Further increasing yeast extract concentration above 5 g/L could not promote cell growth, and glucose concentration higher than 190 g/L would inhibit PMLA production. When the medium was supplemented with Zn2+ and H2PO4-, PMLA productivity was promoted. Though the productivity could also be enhanced by Mn2+, it promoted the strain to secrete much melanin to the broth. The addition of Fe2+, Mg2+, Cu2+ and K+ showed no significant enhancement on the yield of PMLA. While adding exogenous Ca2+ (0.1 g/L CaCl2) to the medium led to a significant increase in PMLA production by 11.38%. Furthermore, whatever NaOH or Na2CO3 was used, there were no obvious differences in PMLA production and cells growth, while neutralization using NH3∙H2O led to increased cells growth and decreased PMLA production by 23.41% and 19.96%, respectively. PMLA production was associated with cell growth in early logarithmic growth phase, while the dissociation of the cell growth and PMLA production appeared in the late exponential growth phase. It was found that the high concentration of produced PMLA (above 40 g/L) not only inhibited its production, but also was detrimental to cell growth. Moreover, when culture redox potential (CRP) increased from 57 mV to100 mV in the late growth phase, lack of reducing power in the broth also decreased PMLA productivity. Therefore, PMLA productivity could be enhanced by repeated-batch culture to maintain cell growth in exponential growth phase, or by membrane cell-recycle culture to remove the produced PMLA, and by controlling CRP below 70 mV no matter which kind of fermentation mode was adopted. During 5 cycles repeated batch culture over 240 h, the PMLA concentration of 60.19 g/L on average with a productivity up to 1.15 g/L·hwas obtained. When glucose was used as carbon source in fed-solution, there was no significant difference between fed-batch and batch culture in the final PMLA and biomass concentration. Sucrose was most suitable for PMLA production, while fructose gave a higher yield of PMLA in terms of the amount of PMLA produced per dry cell weight. Furthermore, exogenous addition of L-malic acid, succinic acid, trifluoroacetic acid and avidin did not significantly affect PMLA production, while pyruvic acid, biotin and CRP inhibited PMLA production, indicating that PMLA biosynthesis was probably related to phosphoenolpyruvate via oxaloacetate catalyzed by PEP carboxylase. Fed-batch fermentation with continulously feeding solution of glucose and sucrose from 36 h could maintain CRP below 70 mV, and led to 20% increase in PMLA concentration, reaching 63.2 g/L, meanwhile the end fermentation volume increased by 25%. The weight-average molecular weight of PMLA obtained from the broth of the strain ipe-1 was 10.58 KDa with a polydispersity index of 1.13. While in the fermentation process, the weight-average molecular weight of PMLA varied between 12.7 KDa and 18.6 KDa. PMLA had a positive CD band in the 190-260 nm. |
| 语种 | 中文 |
| 公开日期 | 2013-09-25 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1843]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 曹伟锋. 微生物制备β-聚-L-苹果酸的研究[D]. 中国科学院研究生院. 2012. |
入库方式： OAI收割
来源：过程工程研究所
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