Study On The Structure And Biochemical Reaction Of Wax Synthase

Wax ester synthase is a key enzyme in the synthesis of wax esters in various organisms.It exists in a variety of animals,plants and microorganisms.It can catalyze the production of a series of ester compounds with different structures and different functions.With the development of the research in wax ester synthase,it was found that the wax ester synthases in different sources varied in function and activity,which led to the diversity of their products and applications.At present,the most widely studied Wax Synthases are those from the Marinobacter aquaeolei VT 8 and Acinetobacter baylyi ADP1.These enzymes have been used by several laboratories for the production of biodiesel and other wax esters in vivo.In this paper,the protein WS2 expressed by the MhWS2 gene in Marinobacter aquaeolei VT 8 was studied.The main findings are as follows:1.We constructed a new expression vector with pET11a and transferred it into E.coil BL21 competent cells,and the soluble expression of the protein increased.2.The optimal induction condition for WS2 was determined by using 0.8 mM IPTG at 30? and 170 rpm for 16 h.After purification of each liter of E.coli,a maximum of 10 mg of WS2 monomer protein and 5 mg of WS dimer protein were obtained.3.By conducting crystallization experiments on WS2 protein,we have preliminarily determined the crystallization conditions of WS2 dimer,and carried out some optimizations to pave the way for further optimization to obtain crystals.4.We studied the enzyme kinetics of the WS2 dimer and determined that the optimal conditions,for which the ideal pH is 8.5 and the optimal temperature is 50?.Then we obtained the better mutant PLPQ protein by rational design and mutant,indicated that reaction efficiency and substrate affinity of WS2 to different chain alcohols are elevated remarkably,50 times increased of catalytic efficiency for ethanol.