Screening Of Producing Thehalose Synthase Enzyme Strain And Studies On Its Enzymatic Properties

Trehalose(α-D-glucopyranosyl-α-D-glucopyranoside) exists in nature widely, especially lower plants, alga, bacteria, fungi, yeasts, insects, and invertebrates. Because trehalose exhibit non specialized protective action against damage of biologic molecule, it could be widely used in molecular biology, pharmaceuticals, foodstuff, cosmetic and agriculture etc.. Trehalose's uses are restricted due to the difficulty in production. At present, there are many ways to produce trehalose, but the enzyme method is regarded as the most promising one. This study mainly discussed the synthesis of trehalose by trehalose synthase. The main research contents include the screen of a strain producing trehalose synthase, the investigation of culture condition, the separation and purification of trehalose synthase enzyme and separation and purification of trehalose.A strain, No.62, producing trehalose synthase highly was screened using improved method according to the interaction of trehalose with high temperature and infiltration. Based on No.62, we screened and got the P-23-37 strain that was treated with ultraviolet inducement. The strain′s modality and biochemical character are researched. Using orthogonal experiment and Respond Surface Analysis(RSA)to optimize the constitute and culture condition of inducement culture medium which is most adapt to produce enzyme, then we culture Bacillus under the optimal condition .We can find that the harvest of the enzyme come up to 48.82U/g wet thalli. The activity of the enzyme is 32.93% more than that under originality condition (36.75U/g wet thalli).Trehalose synthase is a kind of intercellular enzyme, so cell disruption is important for releasing it. Toluene disruption was chosen as our experiment method. The optimal disruption conditions were as follows: toluene dosage 2%, thalli?toluene2% 1:20, 35℃and 150r/min for 2.5h. The crude enzyme was purified by using gel filtration chromatography of Sephadex-G75 and Sephadex-G200. The enzyme appeared to be a dimmer when determined by SDS Polyacrylamide gel electrophoresis (SDS-PAGE).Their molecular weight were 55kDa and 50kDa respectively. And then work over the characteristic of the enzyme.Following Michaelis-Menten kinetics we get the inherent parameter of trehalose synthase enzyme, Km is 8×10^-4mol/L,Vm is 3.24×10^-5 mol/(L.S).The separation and purification of trehalose was also researched. The maltose that existed in enzymatic reaction liquid was hydrolyzed into glucose by maltase, with the trehalose remained 91.9%. And then glucose and trehalose can be separated by successive displacement with H2O and 5% CH3CH2OH through a charcoal column. Trehalose was purified by ion-exchange etc and purity is 97.5%. At last we canaffirmed that trehalose synthase enzyme cantransform maltose to a,a-trehalose by infrared spectrometry etc determined means.