Study On The Technology Of Trehalose Synthase On The Surface Of Bacillus Subtilis Spore Capsid Protein CotC

Trehalose synthase?TreS?belongs to the 13th family of glycoside hydrolase,capable of catalyzing the maltose directly into trehalose,and is a kind of enzyme conversing maltose into trehalose by one step.Its substrate specificity is high,the process of using the enzyme for trehalose production is simple and not affected by the substrate concentration of maltose,it is the first choice for industrial producting trehalose.To obtain trehalose synthase having good catalytic surface,which is displayed in a highly efficient and stable surface of Bacillus subtilis.At the same experiments were selected enhanced green fluorescent protein?EGFP?and trehalose synthase?TreS?as a model protein,to come from Bacillus subtilis spore coat protein Cot C?Cot B?Cot G?Cot X as Bacillus subtilis anchored proteins displayed on the surface.Flow cytometry analysis of the situationthat EGFP could be immobilized on the surface of spore.Then replace fluorescent protein gene egfp and trehalose synthase gene tres.The recombinant strains was hang up using pH 7.5 buffer suspension and the concentration of substrate for30%of the maltose in 40?water bath roling 2h.Reaction products were analyzed by HPLC and the trehalose peak can be detected,indicating that the trehalose synthase gene can be displayed on the surface of the spore by fusion with the sporozoite Cot C.This suggests that Cot C is associated with the outer part of the coat.Cot C can therefore be used as a molecular vehicle for spore surface display of exogenous proteins.The results showed that the addition of 2%wheat bran,2%soybean peptone,0.1%CaCl₂ and 0.002%MnSO₄·H₂O,using pH 7.5,and 40?were optimized by optimizing the spore culture medium of the recombinant bacteria in carbon source,nitrogen source and inorganic salt.The highest rate of spore formation was higher.In the optimized conditions of enzymatic reaction after pH 7.5,40?,the surface of trehalose synthase activity was 27.2U/mL.The enzyme activity was stable between 40?and 45?,the relative activity of the residual enzyme activity was up to 83%or more after heat preservation 6 h,and in the range of pH 7.0-7.5 after heat preservation 6 h,its enzyme activity is still maintained at 70%.