Biocatalysis Of Dichloropropanol By Recombinant Halohydrin Dehalogenase

Epichlorohydrin(ECH)or chiral isomer is an important C3 compound mainly used as raw material for the production of fine chemicals and organic chemicals.DCP(1,3-dichloro-2-propanol,2,3-dichloro-1-propanol)are mass-produced in the production of industrial ECH.In this study,the biotransformation DCP to ECH by using the strain of E.coli BL21(DE3)/pET28b-HheC expressing halohydrin dehalogenase were investigated in detail.Firstly,in order to obtain the(S)-2,3-dichloro-l-propanol,E.coli BL21(DE3)/pET28b-HheC bioresolution(R,S)-2,3-dichloro-1-propanol(2,3-DCP)were studied,the results showed that the whole cells of recombinant E.coli BL21(DE3)expressing halohydrin dehalogenase was limited by sbustrate inhibition when the concentration was above 150 mM.The further study showed that the low concentration of epichlorohydrin(ECH)could cause inhibition of resting cells.The toxicity of ECH towards cells was the major rate limiting factor for the low efficiency of kinetic resolutions.When substrate concentration was 100 mM,could obtain e.e.>99%of the(S)-2,3-DCP after 49 h,yield 31.7%.An aqueous-organic biphasic system was developed in this work.The influential operational parameters such as phase volumetric ratio,buffer pH and reaction temperature were determined and optimized.The results showed that n-heptane was found to be the best organic solvent and the optimum volume ratio of the organic to aqueous phases was 1 to 4.The aqueous phases pH was 9.0.Other optimum conditions for biocatalysis were 20 mg DCW/mL for cell concentration,45? for temperature.Under the optimized reaction conditions,the substrate concentration significantly increased from 150 mM to 375 mM(2.5 times),and the biocatalyst productivity remarkably improved from 2.97 mmol/g to 7.64 mmol/g(2.57 times).Subsequently,the scale-up bio-resolution of DCP was successfully performed in a stirred reactor(2-L scale),affording 128.8 mM(S)-2,3-DCP in 99.0%e.e.(enantiomeric excess)and a volumetric productivity of 16.1 mmol/L/h.Biotransformation 1,3-dichloro-2-propanol(DCP)to ECH by resting cells was studied.The reaction conditions of recombinant E.coli BL21(DE3)expressing halohydrin dehalogenase were optimized,the ruslts showed that the optimal reaction conditions were as follows:pH 8.3,45? for temperature,Cell concentration was 10 mg DCW/mL.Under the optimized reaction conditions,30 mM of 1,3-DCP could afford 21 mM ECH after 10 min reaction(conversion 63.3%).The result showed that halohydrin dehalogenase was limited by product inhibition and value of the Ki was 9.13 mM.Obviously,ECH inhibits biotransformation activity of recombinant E.coli BL21 cells already at low concentration(<20 mM).In order to remove the product inhibition issue along with low net productivity,a biotransformation strategy using resin was investigated.Several macroporous resins were chosen to adsorb ECH in situ during the bioconversion.Resin HZD-9 was found to be the best,which adsorbed the most ECH and the least DCP.In batch biotransformation,When 10%(w/v)HZD-9 resin was added to the biotransformation system,53.3 mM ECH was obtained within 20 min.The supplement of the HZD-9 increased molar yield from 49.7%to 88.3%,the ECH volumetric productivity up to 2.77 mmol/L/min and this was 4.57 times enhancement compared to without addition of the HZD-9.In fed-batch biotransformation,this approach increased the ECH concentration from 31.2 mM to 87.2 mM(2.79 times).