Immobilization Of Halodydrin Dehalogenase And Its Application For Biosynthesis Of Epichlorohydrin

Enantiopure epichlorohydrin(ECH)is recognized as a versatile chiral C3 platform compounds and has practical applications in the synthesis of atorvastatin,?-adrenergic blocking agents,L-carnitine,and trehalostatin.Although halohydrin dehalogenase-mediated biocatalysis pathway is a promising for biotranslation of ECH from 1,3-dichloro-2-propanol(1,3-DCP),it is hampered because of the high cost of biocatalyst,the poor operational stability and the difficulty of recyling product.To solve the problem,we will develop a strategy for immobilization of halohydrin dehalogenase(HHDH)to catalyse the production of epichlorohydrin.This paper was focused on the process of immobilization,the characteristics of immobilized halohydrin dehalogenase and the production of ECH in a recirulating packed bed reactor.Firstly,the preparation of immobilized HHDH was researched.HHDH was immobilized to a range of epoxy supports by multipoint covalent binding.The optimal conditions for immobilization of halohydrin dehalogenase were determined as follows: the enzyme was immobilized onto ES-103 B at 25 ? with shaking in 100 mM sodium phosphate buffer pH 7.0 for 10 h.Then the immobilized enzyme was shaking in 100 mM sodium phosphate buffer pH 9.0 at 30 ? for 24 h.At last the immobilized enzyme was incubated with the alkaline treatment for 24 h.In the optimal condition,The recovery of activity,immobilization yield and activity of immobilized enzyme were 72.4%,94.5% and 528 U/g,repectively.Then the characteristic of the immobilized halohydrin dehalogenase was investigated.The optimal reaction temperature was 70 ? and the optimal reaction pH was 9.0.The half-time of immobilized halohydrin dehalogenase at 40 ?,50 ? and 60 ? were 68.6 day,25.8 day,1.3 day,respectively.And the stability of immobilized halohydrin dehalogenase at pH 8.0-10.0 was very great and the activity of immobilized enzyme still remained 90 % while had been stored at a varied pH for 7 days.The good storage stability made immobilized enzyme retained 85 % of its activity after 30 days of incubation at 4 ?.When the concentration of substrate was 40 mM,the optimal concentration of immobilized enzyme was 40 g/L and the reaction time was 40 min.The immobilized halohydrin dehalogenase had a great operational stability in the stirrer,which was operated for 15 batches and the immobilized enzyme still remained more than 95 % of the original yield.Then a novel bioprocess to produce ECH in a recirculating packed bed reactor(RPBR)was investigated.The optimized operation parameters were examined as follows: the H/D of the reaction was 2.7 and the amount of immobilized enzyme was 60 g/L with a feeding rate of 8.0 mL/min.The immobilized enzyme was operated in a RPBR for 50 batches with 90 % of the original yield.In this work,in order to decrease the inhibition of enzyme from the production of ECH,the integrated bioprocess was performed in RPBR equipped with an external loop for ISPR using resin-based columns loaded with resin HZD-9.In the abrove optimal condition,the amount of macroporous resin of HZD-9 was about 20 g,which can remove a potentially inhibitory product from the reaction mixture as it is produced.Thus the volumetric productivity was varied from 4.20 g/L/h to 7.38 g/L/h.Finally,the process of stereoselective biotransformation 1,3-DCP to(S)-ECH by halohydrin dehalogenase(P175S/W249P)was studied.The reaction conditions were optimized,the results were as follows: The optimal reaction temperature and pH were 65 ? and 9.0,respectively.Considering the production of HCl in the reaction,so when the pH of reaction was controlled from 8.5 to 9.0,which could increase the concentration of substrate and the yield of ECH effectively.The immobilized enzyme had a great operational stability in the stirred tank reaction,which was operated for 25 batches and the immobilized enzyme still remained more than 80 % of the original yield.