| Halohydrin dehalogenases catalyze the conversion of vicinal halohydrin into itscorresponding epoxide. The enzymes paticipate in the biodegradation processes ofhalogenated compounds, which have been introducted into the environment as aconsequence of industrial waste disposal and widespread open use of synthetichalogenated compounds in agriculture. Until now, six different halohydrindehalogenases, which can be grouped into three subtypes A, B, and C on the basis ofamino acid sequence similarities, have been isolated. So far many researches andapplications on halohydrin deahlogenases are related to the enzyme obtained fromAgrobacterium radiobacter AD1(HheC), the knowledge concerning A and B subtypesis still in shortage. The3D structure of halohydrin dehalogenase HheAAD2has beensolved recently, which could facilitate the further researches on HheA. The enzymedisplays a different substrate range and enantioselectivity compared with HheC. Thus,the studies on HheA will enlarge the application range of halohydrin dehalogenases inpharmaceutical and fine chemical synthesis.The expression level of HheA is very low which hinders the further research ofthe HheA. the use of halohydrin dehalogenase to produce β-replaced optical purecompounds has important applications, but till now there is no any screen methodsavailable to detetermine the epoxide ring-openning reaction activity of halohydrindehalogenases. In this work, the expression level of HheA has been optimized bychanging the+2codon usage. The resulted variant displays a4fold improvedexpression level and the purified yield of HheA is8fold to the wild type. Besides, ahigh-throughput screening method based on the adrenaline assay was constructed toscreen halohydrin dehalogenases variants with improved epoxide ring-opening activity.After that, directed evolution to HheA was carried out combining random mutation withDNA shuffling method to obtain variants with higher activity and enantioselectivity. |
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