发酵-膜分离耦合制备生物材料β-聚苹果酸的规律及机理研究

β-poly(malic acid) (PMLA) is an anionic polyester of L-malate, which has attracted industrial interest for its potential applications in medicine and other industries. However, the productivity of PMLA produced by Aureobasidium pullulans is low and the molecular weight distribution is wide. It was also found that the high concentration of produced PMLA not only inhibited PMLA synthesis and cell growth, but also resulted in the degradation of high molecular weight PMLA. Therefore, PMLA will be synthesized and separated in situ by microfiltration-ultrafiltration membrane technology coupled with PMLA fermentation process in this project. We plan to increase PMLA productivity and achieve different degree of polymerization of PMLA products by using this new coupling process. Through studying the mechanism of matter-energy conversion and transfer in the process, a mathematical model will be established to calculate parameters after coupling membrane device into the fermentation process of A. pullulans. Furthermore, system control and optimization of the coupling process will be developed. The mechanism for the variation of PMLA molecular weight will also be investigated in the process, and the strategies of the regulation to synthesize different molecular of PMLA will be established. After investigating and analyzing the filtration performance and membrane fouling behavior, the mathematical models will be developed to fit the experimental flux data to identify the predominant membrane fouling mechanisms during microfiltration and ultrafiltration. Then, the cost-effective fouling control and cleaning strategies will be proposed. The process in this project will not only provide reliable theory and experimental evidences together with technical conditions for scale production of PMLA, but also provide a platform for other biopolymers.

β-聚苹果酸（PMLA）是一种在医学及其它工业中受到越来越多重视的阴离子聚酯，然而Aureobasidium pullulans合成PMLA的生产强度较低且分子量分布宽，高浓度PMLA既抑制其合成过程也抑制细胞生长，同时也致使高分子量PMLA降解。因此，本项目拟采用微滤-超滤膜集成技术与PMLA发酵工艺相耦合，实现PMLA的原位分离与生产并解除其抑制作用，提高PMLA的生产强度，制备不同聚合度的PMLA产品。研究发酵-膜分离耦合过程中物质与能量转化和传递规律，PMLA分子量变化的机理及调控合成不同分子量PMLA的策略，并建立数学模型模拟计算膜耦合装置引入A. pullulans发酵过程的发酵参数，进而对耦合过程进行系统控制与优化。结合膜面污染物特性分析，考察膜过滤过程的特征和膜污染行为，建立相关数学模型，提出经济有效的膜污染控制技术和膜再生清洗技术，为制备PMLA提供理论依据和技术支持。

β-聚苹果酸（PMLA）是一种在医学及其它工业中受到越来越多重视的阴离子聚酯，然而Aureobasidium pullulans合成PMLA的生产强度较低且分子量分布宽。因此，本项目采用微滤-超滤膜集成技术与PMLA发酵工艺相耦合，实现PMLA的原位分离与生产并解除其抑制作用，提高PMLA的生产强度，制备不同聚合度的PMLA产品。. 首先，在相同条件下，分别利用孔径1.4 μm、0.2 μm和0.14 μm的微滤膜及截留分子量300 kDa、150 kDa和50 kDa的超滤膜过滤料液。结果表明，300 kDa和150 kDa超滤膜的细胞去除率和PMLA透过率都较高，膜污染相近，但300 kDa的平均通量是91.91 L•m-2h-1而150 kDa的平均通量是82.64 L•m-2h-1，因此截留分子量300 kDa的超滤膜更适合用于发酵液除菌过程。在确定了去除菌体合适膜的基础上，对膜过滤的操作条件进行了优化，获得的优化条件如下：进料体积1300 mL，浓缩倍数7.2，错流速度6 m/s，跨膜压力0.2 MPa，温度40℃。同时发现膜污染主要由一些大分子（细胞碎片，多糖，蛋白等）造成，而且污染机理主要是膜孔堵塞。. 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，缺乏还原力也将降低PMLA的生产强度。因此，通过膜-细胞循环移除新产生的PMLA将提高PMLA的生产强度，生产强度达到1.15 g/L•h。. 进一步利用膜技术对发酵所得PMLA进行二次分离纯化，并验证了用双膜夹心的方法可以逐级分离获得不同分子量的PMLA，并且得到的PMLA分散系数很接近1，达到了很好的效果；采用离子交换树脂吸附的方法进一步除去PMLA里面的小分子糖，其中发现树脂D296的吸附量最大，达到近160 mg/g，可去除70.86%的糖；通过核磁H谱的分析得出，发酵所得PMLA侧链中含有糖基；并通过单独添加普鲁兰酶、淀粉酶、糖化酶进行水解得出这些糖基大多以a-1,6糖苷键相连接，加入三种酶同时进行酶解，发现糖基侧链分子量大概为3534.5 Da；红外光谱结果表明所提取纯化的PMLA都是β位聚合而成。