Expression Of Trehalose Synthase Gene(AE015451.1) In B.Subtilis

Trehalose as a protective agent, it has stable nature and a non-specific role in theprotection of biological macromolecules and organisms organizations, has a broadapplication prospects in the field of molecular biology, medicine, food industry,cosmetics industry, agriculture. However, due to the difficulties of its preparation limitits widespread application. This experiment by genetic engineering techniques clonedtrehalose synthase gene from Pseudomonas putida KT2440,and transformed trehalosesynthase gene into E. coli BL21, named as P06.through enzyme activity validation,found it highly expressed to the trehalose synthase. The strains producing trehalosesynthas can transform maltose to trehalose by one step, but because E. coli waspathogens,it limited the trehalose produced by this strain in the food or pharmaceuticaluse.Trehalose, non-toxic and harmless, can be used for the preservation of manypharmaceutical products such as vaccines, anti-serum, cells and so on, or even surgicaldesired organ, blood, skin and so on.Trehalose can protect cells function in extracellulararoused great concern of scholars. Research found that trehalose was a very good role inthe protection of the antibody vaccine. It was recent reported that trehalose in thepreservation of organs for transplant had the remarkable effect. Reported in theliterature, preservation solution containing trehalose to save lungs can help patients withemphysema lung to transplant lung. In short, with the deepening of research on thepharmacological effects and its mechanism of trehalose, non-toxic trehalose will have awider application prospects in the medical field.However, based on the production of food grade and pharmaceutical gradetrehalose, the experimental use of B. subtilis as a recipient strain and pMA5shuttleplasmid successfully constructed recombinant B. subtilis producing trehalose synthasenamed as P08. The loss rate of recombinant vector pMA5-tres measured by pass-generation test from1generation to10generation, the loss rate of pMA5-tres is29.8%.The recombinant vecter can be stable to inherb.The recombinant vecter of B.subtilis P08with strong constitutive promoter HpaII did not need inducer to induce theexpression of enzyme, and ensure the enzyme safe. Through research, determinedenzymatic conversion conditions of recombinant B. subtilis P08. through the opticalconversion conditions,the enzyme activity increased to65.47u/g. And by contrast fermentation test between E. coli P06and recombinant B. subtilis P08,the experimentobtained differents in expression and enzyme activity.By shake flask experiments foundthat dry weight of E. coli P06and B. subtilis P08were2.14g/L and2.42g/L. Foundby enzyme contrast, activity of trehalose synthase of E. coli P06is much higher thanthat of Bacillus subtilis P08.But recombinant B. subtilis in the production of food gradeand pharmaceutical grade trehalose had unparalleled advantage than recombinant E. coli.