Study On Dehalogenase Conversion Of Specific Substrate

Dehalogenase dehalogenation enzyme also called to halide enzyme, it is canhydrolyze rift the corresponding catalytic halocarbon key and ion of theenzyme.Carbon-halogen bond is cleaved to generate the corresponding alcohol ionsenzyme, also known as enzyme to a halogen. Now the kind already knowdehalogenase mainly five kinds, namely dehalogenase alkyl, halo acid dehalogenase4-chlorobenzoyl-coenzyme a dehalogenase, halogenated alcohol dehalogenase,3-halogen acrylic acid dehalogenase. Dehalogenation enzymes are capable ofdegrading the contaminants in the environment many halide, and also a lot of organichalide can mediate dehalogenation reaction, making it an important pharmaceuticalintermediates. Therefore, dehalogenase has long been one of the hot research.This article is a specific substrate for screening existing laboratory strains, and thestrains were screened to identify, on the production dehalogenases optimizedfermentation conditions, the enzyme activity was measured in the dehalogenation,amplified experiment.First1,3-dichloro-propanol as substrate for laboratory strains were screened toone pair of substrate conversion rate is relatively high strains. Then the conversion rateof these strains were identified relatively high strains, identification results forAgrobacterium radiobacter. Then the transformation medium and fermentationconditions were optimized separately identified carbon, nitrogen, phosphate mixedvolume. Optimization of fermentation conditions from the liquid volume, seedcultivation time, inoculum size, fermentation temperature, initial pH value, shakingspeed, transforming time, several aspects of the amount of substrate added wereoptimized. Optimal conversion conditions are as follows:The best carbon source issucrose, the best nitrogen source is peptone mixture of diammonium phosphate andpotassium hydrogen phosphate, liquid volume was50mL/250mL, then all sorts of age16h, inoculum5%, the best substrate dosage40mg/mL, optimum temperature is30℃,shaking speed of160r/min, initial pH of7.0, the conversion time of48h; dissolvedthrough the substrate optimization methods, when dissolved in ethanol found using thehighest conversion rate, determining the way of dissolving ethanol dissolved. Takingall these optimized conditions, the total product of the unit cell conversion rate reached38%, compared to the previous optimization increased by17%. Next were20L fermenter into experimental observation as high after thecompletion of the fermentation conditions were optimized conversion rate whetheramplification experiment flask. Through several experiments comparing20Lfermenter was found in relatively good condition or control, conversion rate withessentially the same flask. Finally, we transformed Agrobacterium radiobacter1,3-dichloro propanol enzyme conditions studied. Conditions of enzyme is mainly theoptimum temperature and thermal stability; optimum stability of pH and pH; influenceon the activity of the buffer systems. And enzyme activity were determined.