| The microbial transformation of sterols is a low-efficient,low-productivity,low-benefit process.The reasons are as below:low solubility of sterols,inhibition of substrate and product to microbial cells,product degradation,branch metabolic pathway producing by-products,and so on.To reach the highest productivity and the industrialization application request,in our study,highly effective,economical,appropriate transformation systems were established based on the metabolism analysis.1.Metabolism process analysis of phytosterols biotransformationThe strains of Mycobacterium neoaurum NwIB-R10choM2,M.neoaurum NwIB-01MSchoM2,M.neoaurum NwIB-yV,M.neoaurum NwIB-XIV1,M.neoaurum NwIB-BNA,can convert phytosterols to produce AD,ADD,9-OH-AD,1,4-HBC and 4-HBC respectively.The metabolism process pathway was studied by TLC,HPLC and LC-MS.The two factors,reducible by-product and branch metabolism pathway,are the reason of low product purity.The two aspects of low sterol availability and product degradation are the reason of low product yield.The model process is the phytosterols biotransformation producing 9-OH-AD by M.neoaurum NwIB-yV.2.Optimization of the culture mediumAll the optimization process of culture medium was based on the original medium MYC/02.After the investigation of the carbon source,nitrogen source,pH,we designed an orthogonal array L9(34)and obtained the optimized culture medium MYC/03(1 L)which consist of glucose 30 g,soluble starch 2 g,corn steep 1 g,K2HPO4·3H2O 0.5 g,MgSO4·7H2O 0.5 g,(NH4)2HPO4 3.5 g,citric acid 2 g,ferric ammonium citrate 0.05 g,initial pH 8.When the phytostterols concentration was 5 g/L,the 9-OH-AD production reached to 2.24 g/L from 1.69 g/L and the space-time yield was enhanced from 0.28 g/(L d)to 0.37 g/(L d).The molar yield reached to 64.02%from 48.3%.The different pH caused by different carbon source is the reason for the transforming efficiency.Sodium gluconate was a good pH conditioner.When the glucose and the sodium gluconate were 25 g/L and 5 g/L respectively,the space-time yield reached to 0.44 g/(L·d).3.The oil-water emulsion system applied for biotransformationA simple oil-water emulsion system was developed for highly efficient biotransformation of phytosterols to 9-OH-AD.The results indicated that mechanical shearing force generating shear force and addition of tween80 as dispersant were the essential factors to form a stable emulsion system.And the ratio of cereal oil to water was related to the phytosterols concentration.In the emulsion system,a higher biomass was got at an evenly dispersed state.Application of the emulsion system accelerated biotransformation process at higher phyosterols concentration of 20 g/L phytosterols with a 64.06%molar yield and a 1.28 g/(L·d)productivity of 9-OH-AD.After biotransformation,the major by-products partitioned in the oil phase would be removed with oil,the content of relative percentage of 9-OH-AD by the chromatography area normalizing increased from 66.3%to 90%which simplified the downstream purification process.Besides of purification by means of separation of oil and water,a simple and effective isolation and purification progress was introduced.The product broth can be oil-water emulsion system fermentation broth or resting cell system transformation broth.Through ethyl acetate extraction three times,evaporated and recycled the ethyl acetate,methyl alcohol extraction,the majority of oils would be removed.Addition of water(in a ratio of 1:9-2:8 to methyl alcohol)to methyl alcohol extraction liquor,residual oil was eliminated.And the residual substrate could be removed by addition of petroleum ether or n-hexane.After these processes,the 9-OH-AD crude product was obtained and almost no 9-OH-AD loss.After column chromatographic or crystallization,the 9-OH-AD purity reached 92-95%by HPLC.But a 19.8%product loss was found by methods of crystallization.4.The effect of cosolvent and coenzyme on the phytosterols biotransformationThe key point was focused on the application of different cosolvents in aqueous system such as tween80 and cyclodextrins(CDs).The effect of cosolvents on the transformation efficiency and product proportion was discussed.Both tween80 and CDs can increase the productivity,and CDs was superior to tween80.CDs increase the product concentration more and decrease the transformation time less than Tween80.The optimized molar ratio of HP-β-CD to phytosterols was 2:1.And the space-time yield was 0.79 g/(L·d),which was better the no cosolvents addition system 0.47 g/(L·d)within 3 d.HP-β-CD was superior toβ-CD.The main reasons were as belows,better substrate solubility and inhibition product degradation.Tween80 was a toxic reagent to the microbial cells and can promote the reduction reaction(increasing C17-hydroxyl-reduction and decreasing C1,2-dehydrogenation),while HP-β-CD can promote the oxidation reaction(Cl,2-dehydrogenation).The incomplete of side chain degradation by-product such as 1,4-HBC would be increased in the presence of HP-β-CD.The process of biotransformation of phytosterols producing 9-OH-AD or AD was studiedin the presence of agent of coenzyme or coenzyme regeneration.The preliminary conclusion was that the importance of coenzyme does not lie in the absolute amount,but lies in the coenzyme regeneration.5.Steroid metabolites production of resting cells biotransformation containing HP-P-CDHydrophobic phytosterols could be efficiently solubilized by the formation of inclusion complex with HP-β-CD.Using resting cells as biocatalyst could separate the biotransformation from cell growth which has been found to be an effective way to improve the cell concentration and eliminate the impurities from cell metabolites.The above two strategies have been well integrated and an enhanced process of microbial biotransformation of phytosterols to 9-OH-AD was achieved.After optimization,the best resting cell was culture broth at 3 d;0.1 g/L phytosterols was added in the culture medium as the inducer;the best molar ratio of HP-P-CD to phytosterols was 1:1.We demonstrate that resting cell biotransformation can be run at 70 g/L phytosterol in a bioreactor,where 9-OH-AD yield and space-time yield reached up to 36.4 g/L and 9.1 g/(L·d)respectively,using 200 g/L resting cell.Biotransformation costs were greatly reduced through the development of simple recycling procedures of cells and HP-β-CD.Using of appropriate antifoaming strategy,this transformation system succeed in the small fermentation equipment.And this combination strategy could be widely suitable for other steroid metabolites(AD,ADD,1,4-HBC and 4-HBC)production process.The production of AD,ADD,1.4-HBC and 4-HBC reached 23.2、23.6、32.3、33.6 g/L respectively.6.The effect of dissolved oxygen level on the 9-OH-AD production form phytosterols biotransformationIn the typical laboratory rocker condition,the dissolved oxygen level was varied by adjusting the reaction volume and rotational speed.The reaction rate and assignment of 9-OH-AD pathway and AD pathway were studied.Dissolved oxygen level was a critical condition that can influence the speed of reaction at high concentration of resting cells and phytosterols.The 9-OH-AD pathway and AD pathway were relatively independent.With the increased of dissolved oxygen level,the reaction rate increased and the ratio of 9-OH-AD/AD also increased.The provided high oxygen level at the earlier period(1-2 d)is critical on the phytosterols biotransformation. |
