| Microbial side-chain cleavage of phytosterols is a key point in steroid transformation,and the product of androstenedione(AD),as an important intermediate,can be converted into hundreds of hormone drugs.However,the catalytic process is complicated and mechanism is not clear,so it can only be conducted by whole cells so far.Herein,the poor solubility of substrate and inhibition of hydrophobic product are two major problems,which are becoming research hotspots in steroid synthesis.Ionic liquids,as novel green solvents in 21st century,have been used to enhance steroid bioconversion efficiency in recent years,but main researches concentrated on hydroxylation and 1-dehydrogenation of,steroids.In this work,ionic liquids were applied to construct favorable solvent systems for the production of AD via side-chain cleavage of phytosterols by Mycobacterium sp.MB 3683 cells.Moreover,several novel ionic liquids were designed and synthesized with superior dissolving power and biocompatibility,in order to further enhance the bioconversion efficiency and promote the development of sustainable process engineering.First,13 kinds of representative hydrophobic/hydrophilic ionic liquids were selected to systematically evaluate the solvation properties relevant to phytosterols bioconversion.The relationships among ionic liquid structures,physicochemical properties,dissolving capacity and biocompatibility were established.It was found that with the extension of cationic alkyl chain length,the density of ionic liquids decreased,but viscosity increased.When the hydrophobicity value of log P was greater than-2,ionic liquid could form biphasic system with water.The hydrophobicity sequence of different cations was quaternary phosphonium/ammonium>dialkyl pyrrolidinium>dialkyl imidazolium,and sequence of anions was[NTf2]’>[PF6]->[BF4]->[Lac]-.Besides,the log P increased with the elongation of cationic alkyl chain.The equilibrium phase compositions of ionic liquid/water(mutual solubility between ionic liquid and water)were always inversely related to log P.The solubility of phytosterols in ionic liquids was improved more or less compared with that in water,and positively correlated with H-bond basicity(β)of ionic liquids.Surprising,the solubility of AD was enhanced approximately 1000 times with log D>2,which would facilitate in situ product removal(ISPR),and the values also increased with extension of cationic alkyl chain.The membrane integrity of Mycobacterium sp.cells was inversely related to ionic liquid concentration and anionic hydrophobicity,whereas glucose metabolic activity was positively related to anionic hydrophobicity.The systematical evaluation of solvent properties provided basic data for the prospective application of ionic liquids in bioconversion.Then,9 of water-immiscible ionic liquids were used to construct biphasic systems for the extractive fermentation of phytosterols side-chain cleavage by Mycobacterium sp.,in which[PrMIM][PF6]was determined as a suitable candidate to establish a biphasic ionic liquid/aqueous system.Under optimal conditions,i.e.inoculum size of 12%with 48 h age,IL addition at 84 h,and phase ratio of 1:20(ionic liquid/aqueous,v/v),AD production reached 2.23 g L-1 after 5 day biotransformation with substrate concentration of 5 g L-1 in a scaled-up biphasic system of 200 mL.Compared with monophasic aqueous system,this bioconversion conducted in biphasic ionic liquid/aqueous system was more efficient,and AD production could be further enhanced at high substrate concentration.The mass transfer mechanism of two-phase biotransformation was tentatively discussed to determine the key role of ISPR implemented by ionic liquids.Furthermore,11 of water-immiscible and 2 of water-miscible ionic liquids were utilized for phytosterols bioconversion catalyzed by Mycobacterium sp.resting cells.The cell culture conditions were investigated,and at glucose concentration of 8 g L’1,with 1 g L-1 of phytosterols microdispersed in tween 80 solution as inducer,cells with relatively high catalytic activity were collected when harvested at late logarithmic growth phase(60 h).After bioconversion catalyzed by 50 g L’1 wet cells with the feeding of 3 g L’1 substrate in biphasic systems or cosolvent systems x composed of 0.1 M Tris-HCl(pH 7.5)and ionic liquids for 12 h,[PrMIM][PF6]generated the highest AD production due to the proper cell membrane permeability(MI≈ 40%),which facilitated the transport of substrate/product across cell membrane,thus enhancing bioconversion efficiency.After bioconversion,AD was extracted from ionic liquid phase by ethanol and the ionic liquid recovered was reused for batches.Moreover,the immobilization of Mycobacterium sp.cells on dry fruit of Luffa cylindrica was attempted in the bioconversion of phytosterols.Finally,24 kinds of novel ionic liquids consisting of biomolecule lactic acid and long-chain fatty acid as anion donors and quaternary phosphonium/ammonium as cation donors were designed and synthesized,considering the insufficient substrate solubility and cell biocompatibility in commercial ionic liquids.Their structures and purities were characterized by 1H NMR and 13C NMR.The solvation properties were evaluated,and these ionic liquids were determined with relatively low polarity and extremely high H-bond basicity,thus performing excellent dissolving power to phytosterols,which was approximately 100 times higher than commercial ionic liquids.The 8 of water-immiscible ionic liquids were used to construct biphasic systems for phytosterols bioconversion by resting cells.The conversion efficiency was improved more than 40%with 0.1 vol.%[P6,6,6,14][C12]or[N10,10,10,10][Lac];however,the result was not desirable at higher ionic liquid concentrations due to the agglomeration of ionic liquids,substrate and cells.Correspondingly,the other 16 of water-miscible ionic liquids were utilized as cosolvents in aqueous systems.Their aqueous solutions at low concentration possessed superior dissolving capacity to substrate/product than the organic solvents or even surfactant involved due to their amphiphilicity.What’s more,in the cosolvent systems,the viscosities were less than 1.6 cP,with cells and substrate well dispersed,and the cell membrane integrity maintained to keep the viability.The bioconversion efficiency was investigated in detail by varying both ionic liquid species and concentrations,in view of their correlation with glucose metabolic activity.At a very low ionic liquid concentration of 0.1 vol.%,the system with[N4,4,4,4][C10]as cosolvent generated an AD productivity as high as 136 mg L-1 h-1.In summary,biphasic ionic liquid/aqueous systems could be applied to phytosterols bioconversion,accompanied by the recovery of ionic liquids.The customized novel ionic liquid cosolvent systems,with less dosage of ionic liquids,saved cost and ionic liquid recycle process practically.The combination of ionic liquids and bioconversion greatly conforms to the desire for green and sustainable process,and presents a potential application prospect. |
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