Trehalose Production By Enzyme With Starch As Raw Material

Trehalose,a non-reductive disaccharide,consists two pyran-rings of glucose with ?,?^-1,1-glycosidic linking,widely presents in nature.Trehalose can enhance the ability of micro-organisms to resist the bad environments,such as freezing,drying,high temperature,high permeability,etc.Trehalose plays important roles in protecting cells and bioactive substances.In this thesis,the trehalose-producing strain kept in the lab was treated by UV and atmospheric/room temperature plasma?ARTP?to obtain a mutant?Arthrobacter sp.SH-52?with high trehalose production ability.Then,the enzymes production conditions of this mutant were optimized and the environmental stress effects on the enzymes production were investigated.Enzymaticstarch-transformation process was optimized.The main results were summarized as follows:?1?A mutant?SH-52?with high trehalose production ability was obtained by UV and ARTP methods.The mutant converted the starch with polymerization degree more than 3 into trehalose via Tre Y-TreZ pathway.Trehalose hydrolase was also found in the mutant.Its catalysis ability reached 129.6 U·mL^-1,which was 46.1% higher than that of the original strain.Morphology and molecular biology indicated that the mutant was classified as Arthrobacter nicotinovorans,with a name of Arthrobacter sp.SH-52.?2?The enzymatic production of the mutant was optimized,including medium components and cells cultures/enzymes synthesis conditions.Under the optimal enzymatic production,the catalysis ability of the mutant reached 152.7 U·mL^-1.?3?Environmental stress was applied for the mutant.The enzymes catalytic capacity increased 31% by adding 0.2 mol·L^-1NaCl into the broth at 24 h.The enzymes catalytic capacity also increased 24.2% by heat-shocking under 40? for 60 min at 24 h.However,the combination of heat-shock and salt stress did not show obvious synergistic effect.?4?The starch-conversion process using the crude enzyme was optimized,and the enzymatic properties were studied.The optimal cells disruption method and enzymatic reaction conditions of the mutant were determined.Under the optimal reaction conditions,the conversion rate reached 37.6% and the concentration of trehalose was 37.6 g·L^-1.However,the enzyme activity was inhibited by 1 mmol·L^-1 Cu²⁺,Hg2 +,Zn²⁺,Fe²⁺ at some extent.?5?Trehalose was separated.Glucose and trehalose could be separated by activated carbon column with deionized water and 5% ethanol as the eluent.The purity of trehalose in the eluent reached 87.4%.