Synthesis Of Lactic Acid By Thermophilic Dehalogenase ST2570

Dehalogenase is a series of enzyme that can hydrolysis the halogen fromhalogen-containing organic compounds including stable carbon-halogen bond. It ismainly classified into four classes: haloacid dehalogenase, fluoroacetatedehalogenase, haloalkane dehalogenase and reduction dehalogenase which screenedfrom microorganisms that lived in the environment with organic halides. Above all,L-2-halogenated acid dehalogenase is known widely than other kinds. ST2570derived from thermophilic archaea Sulfolobus tokodaii, which belong to L-2-HADsuperfamily. ST2570catalyze the cleavage of carbon-halogen bond of L-2-haloacidin the reaction type of SN2. After reaction, the L-2-alkyl halide acid is changed intoD-2-hydroxy acid. Because of efficient catalytic activity, stereoselectivity, highthermal stability and wide pH tolerance range (2~12), ST2570was applied toproduce D-lactic acid. Details are as follows:1. The expression and purification of the ST2570. The gene fragement of ST2570was constructed in pET28b. The recombination protein with His tag wasoverexpressed in BL21(DE3) pLysS and purified by Ni-NTA chromatography. Afterpurification, above90%purity protein was obtained.2. Activity detection and identification stereoselectivity of ST2570. Through thedetection the enzymatic parameters of ST2570, we obtained specific activity,optimum temperature and optimum pH respectively. The specific activity of ST2570is2963.33U/mg, optimum temperature is70°C and optimum pH is9.5. Theseparameters will provide the reference for the subsequent optimization of the catalyticreaction conditions. The main purpose of this essay is by using ST2570as catalyst tohydrolyze racemic2-CPA for high-purity D-lactic acid produce.2-CPA and lacticacid that contained in the reaction solution were analyzed qualitative by usingreverse HPLC. At the same time, detection of substrate stereoselectivity of ST2570 found that only S-2-CPA can be dechlorinated by this enzyme, but never happened toR-2-CPA.3. Optimal catalytic reaction conditions of ST2570. Racemic2-CPA is used assubstrate for D-lactic acid produce and the catalytic reaction is optimized. For lacticacid is a heat-sensitive substance, it will decompose at high-temperature. So thereaction temperature was reduced to60°C and the reaction pH was constant at9.5.Under the condition that was mentioned above, optimized processes include threeaspects: buffer concentration, substrate concentration and enzyme concentration.From the results, we found that: the reaction was effective in pH (9.5), temperature(60°C), the buffer concentration (3mol/L), initial substrate concentration (0.5mol/L)and the initial enzyme concentration (30000U/L) of reaction mixture. After reactionof36h, the optimal reaction is obtained under these conditions. According to theresults, the factors that influence reaction rate are concentration of substrate, bufferand enzyme. The concentration of substrate contribute main effect to conversionsignificantly.