Application And Mechanism Of Ionic Liquid Cosolvent Systems In The Side-chain Cleavage Of Phytosterols By Mycobacterium Sp.

Androstenedione(AD)obtained by microbial side-chain cleavage of phytosterols is an important intermediate,which can be further converted into different steroid hormone drugs.However,some technical problems existed in this process,such as the poor solubility of substrate and the inhibition of product,limit the reaction efficiency of this bioconversion.In recent years,ionic liquids as novel green solvents have a broad application prospect in biocatalysis due to their unique physical and chemical properties,but the side-chain cleavage of phytosterols was rarely referred to.The biocompatibility and biodegradability of ionic liquids are two crucial factors that affect the bioconversion efficiency and AD industrial production.Therefore,novel ionic liquids composed of natural and non-toxic materials such as choline,amino acids and carboxylic acids are suitable alternatives.In this work,cholinium amino acid ionic liquids were introduced as cosolvents for the side-chain cleavage of phytosterols to produce AD by Mycobacterium sp.MB 3683 cells,which was combined with other efficient strategies to improve AD production.Moreover,the mechanism of ionic liquids as cosolvents for the whole cell biotransformation was explored to promote the development of green process engineering of ionic liquids.First,18 kinds of novel ionic liquids composed of choline cations and amino acid anions were synthesized and their solvation properties was systematically evaluated.The densities of ionic liquids were slightly higher than that of water,and the relatively high viscosities decreased rapidly in the presence of a little water.The hydrophobicity values log P of most ionic liquids were less than-2,indicating ionic liquids could act as cosolvents in the medium.The hydrophobicity sequence of ionic liquids with different side-chain groups of amino acids was as follows:long-chain alkyl>short-chain alkyl>polar group.The solubilities of hydrophobic substrate phytosterols and product AD were significantly improved by the addition of ionic liquids,which were inversely proportional to their polarities.Most of ionic liquids had good biocompatibility to Mycobacterium sp.cells,and[Ch][Asp],[Ch][Glu]and[Ch][Gln]even exhibited considerably low toxicity at quite high concentrations.The production rate of AD was both affected by the solubility of phytosterols in ionic liquid aqueous solution and the biocompatibility of ionic liquid to cells.Generally,in the system containing of 1 vol.%ionic liquid,cells demonstrated the best catalytic activity to conduct the phytosterols bioconversion.Considering the long-term phytosterols bioconversion,the biocompatibility of ionic liquids to cells was more important compared with physterols solubility.These ILs of[Ch][Asp],[Ch][Glu]and[Ch][Gln]with good biocompatibility could effectively alleviate the degradation pathway of product and improve AD production.Overall,the production of AD in 1 vol.%[Ch][Asp]for 30 h was about twice as high as that in the buffer.Then,the side-chain cleavage of phytosterols to AD by Mycobacterium sp.cells immobilized on cryogel beads in cholinium amino acid ionic liquid cosolvent systems was investigated.By adjusting the ratio of hydrophilic monomer(2-hydroxyethyl methacrylate,HEMA)and hydrophobic monomer(butyl methacrylate,BMA),seven kinds of cryogel beads with different hydrophobicities were prepared using dropping and freezing method.With the increase of hydrophobicities of cryogel beads,the Mycobacterium sp.cells tended to distribute on the surface of beads,and the remaining cells in the interior of beads were uniformly dispersed.The cells immobilized on the semi-hydrophobic(HEMA/BMA=1:1)cryogel beads distributed well in a relatively dispersed form,and a nearly 13-fold higher AD production per unit of cells than that of suspension cells was achieved due to the effective avoidance of cell agglomeration.Biocompatible ionic liquids[Ch][Asp]and[Ch][Glu]also had the alleviating effect on the degradation of AD produced by immobilized cells,and the highest AD production(0.37 g/L)was obtained by immobilized cells in 1 vol.%[Ch][Asp]cosolvent system for 48 h,which was 1.5 times as high as that by immobilized cells in buffer solution.Although the biocatalytic activity of immobilized cells decreased rapidly after multiple cycles,they demonstrated good storage stability.After storage for 20 days,the residual biocatalytic activity was still more than 80%of the initial value.Furthermore,macroporous resins were employed as in-situ product adsorbents for the side-chain cleavage of phytosterols to AD by Mycobacterium sp.cells in cholinium amino acid ionic liquid cosolvent systems,which played an important role in promoting substrate dissolution and relieving product inhibition/degradation.Seven representative macroporous adsorption resins were selected,and HZ-818 resin exhibited big AD adsorption capacity and good biocompatibility;meanwhile,it also had high selectivity for AD.After adding 20 g/L HZ-818 resin,AD production by Mycobacterium sp.resting cells in buffer for 24 h reached 1.01 g/L,which was 181%higher than that of resin-free system.The optimal conditions of phytosterols bioconversion with HZ-818 resin as the adsorbent in ionic liquid cosolvent were determined.After adding 20 g/L HZ-818 resin,AD production by resting cells in 1 vol.%[Ch][Glu]for 36 h reached 1.86 g/L.Under optimal conditions of fermentation,i.e.resin addition at 60 h,resin amount of 20 g/L and[Ch][Glu]concentration of 5 vol.%,AD production reached as high as 2.08 g/L after 120 h biotransformation.Besides,the recycle of HZ-818 resin for 5 times proved the good reusability for phytosterols bioconversion.Finally,the effect of cholinium amino acid ionic liquid on some key enzymes of the phytosterols side-chain cleavage by Mycobacterium sp.cells was revealed via experimental methods and molecular dynamics simulation.Two genes chO1 and chO2 encoding cholesterol oxidase(ChO),one gene hsd encoding 3β-hydroxysteroid dehydrogenase(3β-HSD),three genes kshA1,kshA2,kshB encoding 3-ketosteroid-9α-hydroxylase(KSH),and one gene kstD encoding 3-ketosteroid-Δ1-dehydrogenase(KstD)were located from the whole genome of Mycobacterium sp.Due to the flexible hydrophobic ring structure above their active sites,biocatalytic activities of ChO1 and ChO2 were almost not affected in a system containing low concentration(0-1 vol.%)ionic liquid,but the exposed catalytic region of another sterol dehydrogenase 3β-HSD made it more vulnerable to ionic liquids than ChO.Cholinium cations of ionic liquids could be attracted by the negatively charged region near the active pockets of KshA1 and KshA2,thereby destroying the substrate binding channel structure of proteins.On the contrary,the positively charged region near the active pockets of KstD tended to attract amino acid anions of ionic liquids,which led to the change of the relative position between the substrate and the active sites,thus the biocatalytic activity reduced.After the addition of ionic liquids,someα-helixes of AD degradation enzymes were gradually converted into random coil structure,and the fluorescence spectra showed that ionic liquids interacted with Trp residues of AD degradation enzymes and their stability diminished.Therefore,AD degradation enzymes KshAlB,KshA2B and KstD had the poor tolerance to ionic liquids,and their biocatalytic activities were significantly inhibited in systems containing low concentration(0-1 vol.%)ionic liquid.Compared with[Ch][Glu],[Ch][Asp]caused relatively great damages to the activities and structures of KshA1,KshA2 and KstD,which may be related to more interactions with protein residues resulted from its higher ionization degree.The inhibitory effects of[Ch][Glu]and[Ch][Asp]on the degradation pathway of AD were verified by the analysis of the activities and structures of key enzymes involved in Mycobacterium sp.cells using ionic liquid cosolvent system.In conclusion,cholinium amino acid ionic liquids with good biocompatibility could be applied as cosolvents to the side-chain cleavage of phytosterols.Combined with other intensified strategies,the bioconversion efficiency could be greately improved,which presented a potential application prospect in the production of steroid drugs.Meanwhile,the interaction mechanism of ionic liquid on whole-cell biotransformation provided more theoretical references for the design of ionic liquids and the sustainable development of biotransformation process could be expected.