【作者】 李爱涛；

【导师】 许建和；

【作者基本信息】 华东理工大学 ， 发酵工程， 2011， 博士

【摘要】 手性化合物的不对称合成或拆分已成为药物化工的研究热点和关键技术。手性亚砜由于其高度的光学稳定性,作为手性辅剂、手性催化剂配体以及起始原料,在不对称合成中具有广泛的用途。它的功能还进一步体现在一些含硫原子手性中心的手性药物中。本研究通过筛选获得一株红球菌Rhodococcus sp. ECU0066,它不仅能够高对映选择性地将硫醚氧化成手性亚砜,而且还可以对外消旋的手性亚砜进行对映体拆分。本论文研究了以目标反应为导向的筛选方法,并以芳香手性亚砜为目标产物,对来自土壤中的微生物进行了广泛的筛选；对筛选得到的红球菌ECU0066的产酶和反应特性进行了考察,并拓展了其底物谱；针对疏水性硫醚底物氧化过程中的瓶颈问题,寻找合适的解决方法,提高了细胞的利用率；对硫醚氧化和亚砜拆分两种生产手性亚砜的途径进行了比较,发现在单一的水相体系中,外消旋亚砜的拆分更具有优势。本论文主要分为四个部分。在第一部分中,以手性亚砜为目标产物,根据目标反应建立了以目标产物为导向的筛选模型,对土壤中的微生物进行了广泛筛选。通过初筛得到了100株具有硫醚单加氧酶活性的菌株,其中能生成R构型产物的为57株；能生成S构型产物的为43株；转化率在20%以上的占60%；综合考虑活性和立体选择性因素,选定了菌株ECU0066为最终的研究对象。后续研究发现它可以通过两步对映选择性氧化,使产物(S)-苯甲亚砜的ee值从80%左右提高到99%以上。菌株ECU0066经16S rDNA鉴定为红球菌属,命名为Rhodococcus sp. ECU0066。该菌株已交由中国普通微生物保藏中心保藏,编号为CGMCC 2547。在第二部分中,在摇瓶中对红球菌ECU0066细胞生长及产酶进程进行了考察,最佳产酶培养时间为30 h；在对数生长前期加入底物苯甲硫醚或者苯甲亚砜可以对酶活进行诱导,诱导后的酶活力提高了约10倍多(比活力由0.064 U/g提高至0.76 U/g)；红球菌ECU0066中硫醚单加氧酶存在于细胞膜上,利用辅酶NADPH对硫醚进行不对称氧化。优化了红球菌ECU0066催化苯甲硫醚不对称氧化的反应条件,最适温度为30℃,pH为8.0。考察了红球菌ECU0066对底物苯甲硫醚的耐受性,发现ECU0066可以耐受的底物浓度较低,能完全转化底物的最高浓度为10 mM,葡萄糖可以作为辅底物帮助辅酶再生,葡萄糖的加入可以使20 mM的底物几乎完全转化。利用红球菌ECU0066整细胞作为催化剂,对其底物谱进行了拓展,成功地实现了一系列手性亚砜的生物制备。在第三部分中,采用了细胞固定化和两相反应体系两种方法来缓解苯甲硫醚对细胞的抑制作用。实验结果表明：海藻酸钙固定化细胞对细胞有保护作用,细胞固定化后其半衰期提高为游离细胞的2.1倍,固定化小球内较高的含水量可以对底物起到稀释作用,缓解了底物的抑制。以固定化细胞作为催化剂,反应可以重复进行10次,底物总量达50 mM,与游离细胞所能完全转化的10 mM底物浓度相比提高了4倍。在水-异辛烷两相体系中,利用红球菌整细胞催化苯甲硫醚不对称氧化,当有机相中底物浓度为150 mM时,得到的水相中产物浓度最高,达18.3 mM,远远高于相同反应条件下单一水相体系中2.17 mM的产物浓度,并且ee值也从单一水相的59.9%提高到了99.0%以上。利用生长细胞作为催化剂,对在水-异辛烷两相体系中催化苯甲硫醚的氧化反应进行了上罐放大,反应总体系3.0 L,反应结束后水相产物的终浓度高达26.0 mM,ee>99.0%,得率为86.7%,相对于静息细胞催化的反应又有了进一步的提高。第四部分中,对硫醚氧化和亚砜拆分两种生产手性亚砜的途径进行了比较,苯甲硫醚对细胞的毒性较大,对红球菌细胞抑制明显,最大反应速率Vmax=0.063 mmol h-1 g-1 wet cell,米氏常数Km=0.26 mM；而底物苯甲亚砜对细胞几乎没有毒性,抑制作用不明显,最大反应速率Vmax=0.27 mmol h-1 g-1 wet cell,米氏常数Km=0.69 mM。优化了红球菌细胞催化拆分苯甲亚砜的条件；反应通过采用分批补料的方式,得到产物(S)-苯甲亚砜的浓度高达37.8 mM,ee值为93.7%,远远高于苯甲硫醚氧化反应得到的结果(10 mM,80% ee)；表明在单一的水相体系中,亚砜的拆分更具有优势。拓展了红球菌催化亚砜拆分的底物谱,成功地实现了四种手性亚砜和相应砜的生物制备。最后,利用生长细胞实现了苯甲亚砜拆分反应的放大制备,反应采取分批补料的方式,反应体积2.5 L,反应液中产物终浓度为21.8 mM,ee>99.0%,最后对反应的产物进行了分离纯化,得到了(S)-苯甲亚砜6.35 g,分离得率为36.2%。更多还原

【Abstract】 Nowadays, asymmetric synthesis and resolution of chiral compounds have been a hot topic of research and key technology in the chemical and pharmaceutical industries. Because of the high configurational stability of sulfinyl group, chiral sulfoxides as valuable starting materials or chiral auxiliaries are widely used in asymmetric synthesis. The value.of its functionality is further illustrated by their diverse biological activities and pharmaceutical uses. A new biocatalyst-Rhodococcus sp. ECU0066, had been discovered in this study, which could not only oxidize sulfides to sulfoxides with high enantioselectivity, but also enantioselectively transform racemic sulfoxides to sulfone to produce enantiopure sulfoxides. The main purpose of this work was to design the strageties of target reaction-oriented screening, to screen for new biocatalysts from soil using chiral sulfoxide as the target product, to optimize the culture and reaction conditions of Rhodococcus sp. ECU0066, to extend its use for asymmetric oxidation of some other prochiral sulfides, to investigate the causes of this biocatalyst deactivation, to present some methods to improve the yield, and to compare the two methods of asymmetric oxidation and kinetic resolution for the preparation of chiral sulfoxides.The study aimed to search for new biocatalyst with excellent enantioselectivity and high activity. The target reaction-oriented screening model was established using chiral sulfoxide as the target product, and 100 strains with obvious sulfide monooxygenase activity were obtained after primary screening, of which 57 could oxidize phenyl methyl sulfide (PMS) to (R)-phenyl methyl sulfoxide (PMSO),43 could produce (S)-PMSO, and 60%had>20% conversion. According to the overall performances including conversion and product ee, a bacterial strain marked as ECU0066 was selected for further study. This strain could transform PMS to (S)-PMSO with 99% ee via two steps of enantioselective oxidations. The 16S rDNA sequencing and taxonomic analyses revealed that this strain belongs to the genus Rhodococcus. Therefore, the strain ECU0066 was marked as Rhodococcus sp. ECU0066, which was presently deposited in China General Microbiological Cultures Center, with an accession number of CGMCC No.2547.The time courses of cell growth and enzyme production were investigated, the optimum cultivation time was 30 h, its enzyme activity could be effectively induced by adding PMS or racemic PMSO directly to the medium at the early log phase of fermentation, resulting in over 10 times higher production of the enzyme. The sulfide monooxygenase of Rhodococcus sp. ECU0066 is located on the cell membrane, which only use NADPH for asymmetric oxidation of sulfide. The reaction conditions of Rhodococcus sp. ECU0066 catalyzed asymmetric oxidation of PMS were optimized, the pH and temperature optima were shown to be pH 8.0 and 30℃, respectively. The substrate tolerance of Rhodococcus sp. ECU0066 was examined, the highest substrate concentration that the bacterium could transform was only 10 mM, but such a concentration (10 mM) still represents the highest level. Glucose was helpful for the regeneration of coenzyme. The addition of glucose could enhance the substrate concentration that the bacterium could transform from 10 to 20 mM. The substrate spectrum of Rhodococcus sp. ECU0066 was investigated, a series of chiral sulfoxides were successfully prepared with resting cells of Rhodococcus sp. ECU0066.The immobilized cells in alginate beads and water-organic solvent biphasic system were used to alleviate the substrate inhibition. The results indicated that the half-life of the immobilized cells is 2.1 times as much as that of free cells, and also the amount of water in alginate beads could dilute the substrate and make substrate concentration around cells decreased, which prevents the toxicity to cells and inhibition. The asymmetric oxidation of PMS proceeded for 10 batches using immobilized cells as the catalyst, the total PMS concentration was increased up to 50 mM, which was 4 times higher than that of the result obtained from the reaction catalyzed by free cells. The asymmetric oxidation of PMS in water/isooctane biphasic system was investigated by the resting cells of Rhodococcus sp. ECU0066, when the PMS concentration of 150 mM in isooctane was employed, the final product concentration reached as high as 18.3 mM with>99.0% ee, which were much higher than that of the results (2.17 mM,59.9% ee) in aqueous system under the same reaction conditions. Scale-up reaction for asymmetric oxidation of PMS during the cultivation of Rhodococcus sp. ECU0066 in water/isooctane biphasic system was studied, reaction volume was amplified to 3.0 L, after reaction, higher product concentration (26.0 mM) and yield (86.7%) were achieved than before.Comparison of the substrate toxicity and kinetic characteristics was made between the whole-cell-catalyzed asymmetric oxidation of sulfides and kinetic resolution of sulfoxides. The toxicity of PMS to the resting cells of Rhodococcus sp. ECU0066 was higher than that of rac-PMSO. Determination of apparent kinetic parameters indicated that severe substrate inhibition was observed when PMS was used as substrate, but vice verse for substrate rac-PMSO. The apparent maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were 0.063 mmol h-1 g"1 wet cell and 0.26 mM for PMS, and 0.27 mmol h-1 g-1 wet cell and 0.69 mM for rac-PMSO, respectively. The reaction conditions for kinetic resolution of rac-PMSO were optimized in a fed-batch reaction, where the product (S)-PMSO was formed in a high concentration of 37.8 mM and good selelctivity of 93.7% ee, which are much better than those of the product from asymmetric oxidation of PMS (10 mM,80%ee). The substrate spectrum of kinetic resolution of rac-sulfoxides by the resting cell of Rhodococcus sp. ECU0066 was investigated. Four kinds of enantiopure sulfoxides and corresponding sulfones were successfully prepared. The kinetic resolution of rac-PMSO using growing cells of Rhodococcus sp. ECU0066 was scale-up to 2.5 L, resulting in a final product concentration of 21.8 mM with>99.0%ee, and the product was isolated and purified, giving 6.35 g of (S)-PMSO with an isolated yield of 36.2%.更多还原