Cloning And Optimization Of Fermentation For Thermophilic Enzyme Gene In E.coli From Thermotoga Maritima

In the 21 st century,the development and utilization of marine microorganism has becomeone of the global hotspot,and the study of extreme microorganisms comes from marine environmental has also taken a new stage.Therefore,the study of extreme enzymes has also aroused considerale interest.in particular,thermophilic enzymes have been applicated in many area.Thermotoga maritime is a kind of extreme thermophilic bacteria that comes from the deep-sea volcano,and the growth environment is particularly strict.It is difficult to large-scale fermentation and use the enzyme resources for industrial production.The use of genetic engineering methods to clone and express the enzyme gene of extreme environmental microorganisms is the main method to utilize the resources of extreme enzymes.In this papers the focus was mainly on endo-1,4-?-glucanase(TM1525),arabinogalactan endo-1,4-?-galactosidase(TM1201)and phosphopentomutase(TM1067)of thermotoga maritima.The recombinant strains that express thermophilic enzyme was constructed by using gene cloning technology,and the protease was purified by affinty column.The phosphopentomutase(PPM)activity has been reported in the literature.The response surface methodology was used to optimize the fermentation conditions for PPM in recombinant strain.The main contents and results of this paper are as follows:1.According to the gene sequences of endo-1,4-?-glucanase,arabinogalactan endo-1,4-?-galactosidase and phosphopentomutase designed the primers,The thermotoga maritime genome as template,p ET-32a(+)as the expression plasmid and E.coli BL21(DE3)as the expression strain.Constructed to three kind of recombinant strain successfully.2.Studied the growth curves of the recombinant strains and induced by IPTG.The results that endo-1,4-?-glucanase and arabinogalactan endo-1,4-?-galactosidasewere failed to expression and phosphopentomutase successfully expressed,analyzed by polyacrylamide gel electrophoresis and Western blotting.3.Analysis of physical and chemical properties of target protein by bio-informatics software.Through the Ni-NTA resin column gained the pure phosphopentomutase.4.The regression equation was established by Plackett-Burman,the steepest ascent path and Box-Behnken tests.The results showed the optimum fermentation conditions were as followed:induction inoculum age 6.0,induction temperature 22.5 ?,induction time 13 h,IPTG0.45 m M,medium p H 7.6,inoculation quantity 2.25%.Under the conditions,the expression quantity of Phosphpentomutase increased 1.53 times more than that under initial conditions.