Biocatalytic Production Of Chiral Epichlorohydrin From 1,3-Dichloropropanol

Chiral epichlorohydrin is an important intermediate for organic synthesis. It has been widely used as chiral buildingblocks for optial pure drugs such as metoprolol, alprenolol, and atenolol. Chemical synthysis employing Salen-Co as catalyst is currently the main method for industrial production of chiral epichlorohydrin, but this approach has the drawback of high cost and severe environmental pollution. Whereas enzymatic hydrolysis of racemic epichlorohydrin to （S）-epichlorohydrin carrys out under mild conditions and is much more ecofriendly. A new biotechnological method for production of chiral epichlorohydrin was developed in the dissertation which employed halohydrin dehalogenase and epoxide hydrolase as biocatalysts with 1,3-dichloropropanol as substrate.Firstly, two screening models were established employing 1,3-dichloropropanol and epichlorohydrin as carbon source through conventional enrichment culture and GC and GC-MS. With these methods, 11 strains with halohydrin dehalogenases and 5 microorganisms bearing epoxide hydrolase activity were isolated which were capable of converting 1,3-dichloropropanol to epichlorohydrin and epichlorohydrin to （S）-epichlorohydrin. Strain 09102, with the maximum halohydrin dehalogenases activity, was chosen as the best strain for further studies. Based on morphology , physiological and biochemical characteristics, the 16S rDNA sequence and phylogeny, it was identified as Pseudomonas putida ZJB-09102. Strain 09103, with the maximum epoxide hydrolase activity, was chosen as the best strain for further studies. Based on morphology, the ITS sequence and phylogeny, it was identified as Aspergillus niger ZJB-09103.The medium composition was optimized by single factors to enhance halohydrin dehalogenase production by P. putida ZJB-09102. The optimized medium composition was as follows （g/L）: starch 14.0, yeast powder 5.0, peptone 5.0, K₂HPO₄ 0.8, KH₂PO₄ 0.4, NH₄Cl 0.6, MgSO₄ 0.4. The optimum conditions for cell growth and formation of halohydrin dehalogenases were as follows: initial pH value, 7.0; 30℃; inoculum volume, 2 % （v/v）; medium volumetric ratio, 60ml/250ml. Under these conditions, concertration of epichlorohydrin was achieved 1.62 g/L after cultivation for 48h , which was 1.25 times higher than before optimization.The medium composition was optimized by single factors and response surface methodology （RSM） for A. niger ZJB-09103. The optimized medium composition was as follows （g/l）: starch 17.56, soybean cake 3.89, peptone 4.03, K₂HPO₄ 0.8, KH₂PO₄ 0.4, MgSO₄·7H₂O 0.4. The optimum conditions for cell growth and formation of epoxide hydrolase were as follows: initial pH value, 6.5; 30℃; inoculum volume, 1 % （v/v）; medium volumetric ratio, 20 % （v/v）. Under these conditions, a specific epoxide hydrolase activity of 149.98 U l^-1 was achieved after cultivation for 4 d, which was 2.33 times higher than before optimization. Influences of reaction conditions on e.e. and yield were also investigated. Results indicated that e.e≥99% and 18.6% yield were achieved after biotransformation for 10 h.