Construction Of Engineering Bacteria And Application Of Thermophilic β-fructosidase

Thermophilic bacteria is a kind of extremophiles,it has extremely high requirements for growing up and the common cultures can not supply with the optimal growth conditions.Thermophilic enzyme are from thermophilic bacteria,with good heat resistance and property broader activity conditions,they are often used in food production and industrial production,the thermophilic enzyme has considerable application prospects.In this study,the gene of thermophilic β-fructosidase from Thermotoga neapolitana DMS 4359 was cloned,pET-28a(+)as the carrier and expressed in Escherichia coli BL21(DE3),we successfully constructed the thermophilic β-fructosidase engineering bacteria,and obtained the thermophilic glycosidase after culturing the engineering bacteria.After purifying the molecular weight detected by SDS-PAGE is about 60.0 kDa.The results of substrate specificity show that the sucrose is the best substrate for the thermophilic β-fructosidase.Characteristics of the thermophilic β-fructosidase: the optimal reaction temperature is 85 ℃;the optimal reaction pH is 5.0;10 mmol/L Mg2+ play the best role to promote the thermophilic β-fructosidase activity,it can make the enzyme activity increase nearly 2.6 times,while 1 mmol/L Cu2+ play as strong inhibitor,it can almost completely inhibit the activity;the thermophilic β-fructosidase has good thermostability,after insulation treatment 40 h at 95 the ℃enzyme activity retains 50%,the results of optimal temperature and the thermostability can be clearly identified again for the thermophilic β-fructosidase.In order to further improve the expression of thermophilic β-fructosidase,we combine the single factor tests and response surface method and optimize the thermophilic β-fructosidase culture medium,finally the optimal culture medium as follows: sucrose 10.11 g/L,NH4NO3 12.30 g/L,angel yeast powder FM905 8.24 g/L,MgSO4·7H2O 5.61 mmol/L,NaCl 10 g/L,the new culture medium can improve the enzyme activity nearly 2.1 times than before,it not only raised the expression quantity of thermophilic β-fructosidase but also save the materials costs of culture medium at the same time.In order to determine the degradation properties of thermophilic β-fructosidase in the applied research,the results show that the thermophilic β-fructosidase have hydrolysis ability and transglycosylation,it can hydrolyze sucrose and receive glucose and fructose,combined with fructooligosaccharide,and the main products are fructose and fructooligosaccharide after hydrolysing inulin.The results of tests can open up a new path for the high fructose syrup and fructooligosaccharide production.