摘要
光学活性仲醇是重要的医药和精细化学品中间体,光学纯苯基乙二醇(PED)是其中的重要代表,广泛用于手性药物、农药和液晶材料的合成。与化学合成法合成手性化合物相比,生物催化剂在位置、化学和立体选择性上具有独特优势。其中,微生物去消旋和不对称还原法由于其理论最大得率100%和操作简便已经成为当今生物催化中前沿领域和热点内容。但这两类反经常遇到的几个共性问题是底物浓度低,辅酶循环效率低和催化剂不稳定。本论文以酵母细胞催化不对称氧化还原反应高效合成光学纯苯基乙二醇为目标,针对近平滑假丝酵母(Candida parapsilosis)催化PED去消旋和酿酒酵母(Saccharomyces cerevisiae)不对称还原α-羟基苯乙酮反应的瓶颈,采取过程控制和介质工程手段明显提高了其反应效率。此外,还揭示了C. parapsilosis细胞代谢D-木糖(戊糖)偶联去消旋苯基乙二醇过程的作用机理。这些发现不仅进一步丰富了还原酶手性生物催化理论,而且有助于合成其他精细化学品。主要结果如下：
(1)以C. parapsilosis细胞催化PED去消旋为模式反应,对可能限制细胞催化活性的因素进行了系统考察。结果发现底物浓度高于25 g/l或产物浓度高于17.5 g/l对该反应产生强烈的抑制作用。较高的底物或产物浓度(如超过30 g/1)导致细胞死亡率大于18.3%。此外,在高底物浓度下(如30 g/1)NADPH再生也是限制该反应效率的因素之一。
(2)运用树脂原位底物补料和产物移除“二合一”策略提高去消旋反应的效率。大孔树脂H103由于吸附容量高和对反应促进最明显被选作吸附材料。最佳反应条件如下：pH 8，温度30℃,细胞120 g/l,树脂72 g/l,转速150 r/min。在该条件下30g/l PED反应48 h后,产物(S)-PED光学纯度和得率分别达到99.2%和93.6%。基于通过异构反应实现生物催化去消旋的特点,提出一种合理运用树脂原位底物补料和产物移除“二合一”策略的方法。该新方法可使C. parapsilosis催化50g/l底物反应90 h后,产物e.e值和得率分别高达99.3%和92.0%。
(3)C. parapsilosis催化苯基乙二醇去消旋分批连续反应中,添加D-木糖有利于细胞代谢活性的恢复,但对催化去消旋关键反应的(S)-羰基还原酶活力无影响。在证实细胞内存在木糖还原酶和木糖醇脱氢酶的基础上,通过磷酸戊糖途径抑制试验和直接测定胞内NADPH含量相结合的方法,揭示了C. parapsilosis细胞代谢D-木糖偶联去消旋苯基乙二醇过程的作用机理：D-木糖先后经过木糖还原酶和木糖醇脱氢酶的还原和氧化反应生成木酮糖,再经木酮糖激酶生成5-磷酸木酮糖并由此进入磷酸戊糖途径。通过磷酸戊糖途径中间代谢产物6-磷酸葡萄糖、6-磷酸葡萄糖酸的两步脱氢反应产生大量NADPH用于去消旋PED,提高反应效率和催化系统的稳定性。此外,萃取转化(树脂/缓冲液体系)和辅酶再生(添加D-木糖)相结合的策略,不但可以解除底物/产物抑制,还能提高生物催化剂的可持续性。
(4)通过靶向反应定向筛选成功获得一株高立体选择性(e.e>99.9%)不对称还原α-羟基苯乙酮合成(R)-PED的酵母S. cerevisiae JUC15。当底物浓度为2g/l时游离细胞重复使用40次,产物得率和光学纯度无明显降低。该催化剂使用廉价的蔗糖作为辅助底物用于辅酶再生,可在相当宽的pH范围内(4-9)催化α-羟基苯乙酮。在所有考察条件下,产物e.e值始终保持大于99.9%。当α-羟基苯乙酮浓度低于其溶解度(11.4g/1)时,产物抑制是不对称还原反应的主要瓶颈。当底物浓度高于其溶解度,尤其是超过20g/l,产物抑制和催化剂失活是引起(R)-苯基乙二醇得率降低的主要原因。此外,S.cerevisiae细胞催化部分产物降解。为克服这些缺点,采取缓冲液和水不溶性有机溶剂组成的原位分离的策略。通过测定不同有机溶剂对底物/产物分配系数和对不对称还原反应的影响,筛选出对此反应具有较好促进作用的有机溶剂邻苯二甲酸二丁酯。在合适的相比例下,产物浓度达到20.7 g/l，明显高于目前已报道的其他生物法水平。
Optically active secondary alcohols are important intermediates of pharmaceuticals and fine chemicals, and enantiomerically pure phenyl-1,2-ethanediol (PED) is an important representative for optically active secondary alcohols, which is a versatile chiral building block for the synthesis of pharmaceuticals, agrochemicals, pheromones, and liquid crystals. Compared with chemical synthetic methods, biocatalysts offer distinct advantages in terms of of regio-, chemo- and enantioselectivity under benign conditions. Amonies, microbial deracemization and asymmetric reduction have been the front and topic research of present biocatalysis thanks to the maximum theroretical yield of 100% and easy-to-handle conditions. Nevertheless, some weaknesses frequently met in both microbial deracemization and asymmetric reduction are small substrate concentrations, the inefficient regeneration of cofactors and the poor stability of biocatalysts. In order to achieve highly effective synthesis of chiral PED by yeast-mediated asymmetric oxidoreduction, the bottleneck of Candida parapsilosis-catalyzed deracemization of PED to (S)-PED and S. cerevisiae-mediated asymmetric reduction of 2-hydroxyacetophenone to (R)-PED were investigated. The efficiency of above-mentioned reactions was significantly enhanced by process engineering and media engineering. Furthermore, the mechanism of coupling involving D-xylose metabolism and C. parapsilosis-catalyzed deracemization of PED was also uncovered. Those findings not only further enriched the theory of oxidoreductase-catalyzed biocatalysis, but also helped to systhesis of other fine chemicals.The main results were shown as follows:
(1) C. parapsilosis catalyzing deracemization of PED was used as a model reaction to systemically investigate the factors that might limit the reactivity of such cells. It was found that there was a marked inhibition of reaction when the substrate and product concentration exceeded 25 g/1 and 17.5 g/1, respectively. Besides, excessive substrate or product concentration (such as>30 g/1) led to a high cell mortality rate of more thanl8.3%. Furthermore, NADPH regeneration was one of the factors limiting the reaction efficiency under higher substrate concentration (such as 30 g/1).
(2) A highly efficient process for C. parapsilosis-mediated deracemization of PED was described, based on a "two-in-one" resin-based in situ substrate feeding and product removal methodology. The macroporous resin H103 was selected as the adsorbent material thanks to its high adsorbent capacity and greatest promoting effect on the reaction. The optimal conditions were selected as follows:pH 8,30℃,120 g/1 cells,72 g/1 resin H103 and 150 r/min. Under the optimal conditions,30 g/1 of racemic substrate was converted to (5)-PED with 99.2% enantiomeric excess (e.e) in 93.6% yield after 48 h. Based on the characteristic of biocatalytic deracemization by stereoinversion, a more rational method was proposed to make use of a "two-in-one" resin-based in situ substrate feeding and product removal strategy. Using this new method, when substrate concentration was 50 g/1, a reaction yield of 92.0% and e.e of 99.3% were obtained for (S)-PED within 90 h.
(3) D-xylose added to multi-batch reactions had no influence on the activity of (S)-carbonyl reductase catalyzing the key step in deracemization, but performed a promoting effect on the recovery of the metabolic activity of the cells in each batch. Based on the fact that the activities of xylose reductase and xylitol dehydrogenase from cell-free extract of C. parapsilosis were detected, the depression of the pentose phosphate pathway (PPP) and the investigation of the cofactor pool were performed to uncover the mechanism of coupling between D-xylose metabolism and the deracemization of PED. The proposed mechanism was as follows:D-xylose was converted to xylulose via a two-step reduction and oxidation mediated by xylose reductase and xylitol dehydrogenase, respectively. Xylulose was phosphorylated to xylulose 5-phosphate, which then enters into PPP. When glucose-6-phosphate and 6-phpspho-gluconate, the intermediate metabolites of PPP, were catalyzed by glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively, they can afford more NADPH for deracemization of PED to enhance the reaction efficiency and the stability of biocatalytic system. Furthermore, a strategy involving extractive biocatalysis (resin/buffer system) coupled with cofactor regeneration (adding D-xylose) not only alleviated the substrate/product inhibition, but also enhanced the sustainability of the biocatalyst.
(4) S. cerevisiae JUC15 was successfully obtained by target reaction-oriented screening, which asymmetrically reduced 2-hydroxyacetophenone to (R)-PED of excellent e.e (>99.9%). There was no significant decrease in the yield and optical purity of (R)-PED when the free cells were reused for 40 repeated cycles at 2 g/1 substrate concentration. The biocatalyst used cheap sucrose for cofactor regeneration and worked over a considerably wider range of pH (4-9). The product e.e. kept above 99.9% in all examined conditions. When 2-hydroxyacetophenone concentration was below solubility limit (11.4 g/1), product inhibition was the primary bottleneck of the asymmetric reduction. However, when the substrate concentration exceeded its solubility, especially more than 20 g/1, the observed decrease in the yield of (R)-PED can be mainly attributed to a combination of product inhibition and the inactivation of the biocatalyst. Moreover, the catalytic decomposition of partial product by S. cerevisiae occurred. Biphasic systems consisting of buffer and a water-immiscible organic solvent were applied to circumvent those limitations. Dibutyl phthalate was selected as the suitable organic solvent thanks to its higher organic phase-buffer partition coefficent for the substrate and product, and the largest promoting effect on the reduction. Use the appropriate volume ratio could make the product concentration reach 20.7 g/1, which was remarked higher than that of other bio-methods reported so far.
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