Screening And Utilization Of Sulfide Monooxygenase Producting Strains

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%.