Research On Trehalose Synthase Gene Engineering Bacteria

Trehalose is known as a new class of carbohydrates in the 21st century.It consists of two molecules of glucose by?,?-1-1 key linkages from the non-reducing disaccharides,and has many excellent properties,so can be widely uesed in food,medicine,agriculture,biological products and cosmetics and other industries.That making rice starch to maltose by amylase at high temperature,then turning maltose into trehalose by trehalose synthase,greatly simplifies the traditional process,and is a hot topic of current research.The vitality of trehalose synthase easily affected by external environment.Therefore,by increasing the enzyme activity to improve the utilization of trehalose synthase to produce trehalose that have a great prospect research.Yeast display technology enables Microbial surface dispaly technique also further develop and improve,and therefore have a wide range of application in many fields.Yeast has the specific protein modification effect of eukaryotic cells display system?such as glycosylation and disulfide bond isomerization,etc.?.So that the involuted eukaryotic proteins with functional activity can be displayed,especially for mammalian proteins even some human-derived protein.In contrast to prokaryotes surface display system,the new system retains the advanrages of ease of selection and amplification,etc.It can make up for the lack of it unable to display complex eukaryotic protein.Previous recombinant E.coli most existing some problems such as wall-breaking take enzymes,inclusions,immobilization,etc.Yeast surface display system is expected to solve these problems.So the new technology is expected to greatly reduce the cost of production.Yeast surface display technology will have great prospects for development with the theory and technology gradually improved in the coming years.In order to improve the trehalose synthase activity,At the early of the reeseaech,Zhang Qiong has successfully constructed the recombinant E.coli?TSG2013?.In this study we use the single factor experiment and the response surface experiment selected the main ingredient in fermentation culture medium?such as glucose,peptone,yeast extract and magnesium sulfate?,and obtained the optimal solution:8.56 g/L glucose,peptone,20.44 g/L,yeast extract,20.8 g/L,Mg²⁺:13.9 mmol/L.We cultivated the recombinant E.coli in the optimized medium and found the bacteria wet weight can reach 6.135 g/L,raised by 30 percent than before optimization.The TSG2013 growth was reduced and easy formation of inclusions,so we need to study a more effective way to improve the trehalose synthase activity.We combined the GPI-modified wall proteins with the sky blue Streptomyces trehalose synthase?TS?,then clone it into the pPIC9k and transformed it into GS115,so we successfully constructed the carrier?pPIC9k GCW14-TS?.The TS is successfully showed in the surface of pichia GS115.The enzymology properties were studied,and found in 30⁴5?temperature range it can play a role,the optimum pH of the TS is 8.5,K⁺,Mg ²⁺and other metal ions has a promoting effect on it and reused after 5 times it can still remain more than75%of the enzyme activity.The trehalose synthase can be immobilized,so it can be used to industrialized production of convert maltose into trehalose.Success show the DNA?GCW14-TS?that which from the coelicolor trehalose synthase in pichia GS115 surface.Comparing the cell growth and enzyme activity of the whole cell catalyst in different medium,we found it can grow in BMGY,potato medium,bean sprout medium,malt medium and the largest growth exsit in the BMGY,and no growth in the examine's medium.By studying the components of BMGY?glycerint and methanol?and culture conditions?inoculation quantity,capacity and initial pH?.Preliminary study of the growth and enzyme activity under different fermentation conditions of the whole cell catalyst,we found that the optimum condition is:glycerol 2%,methanol 2%,inoculation amount 3%,capacity 75 ml/250 ml,initial pH6.5.After the optimized medium culture bacteria growth up to 27.56 g/L.Compared with TSG2013 the wet weight proportion can be up to 5 times.The enzymatic activity of whole-cell catalyst can reach 490.72 U/g.