Gene Cloning ,Expression And Characterization Of β-fructofuranosidase Fromarthrobacter

Fructooligosaccharides (FOS) have efficient propagation of bifidobacteria, intestinal detoxification clean, prevention of dental caries ,improve lipid metabolism (fat and cholesterol) and other physiological functions,people give more attention to them . At the current,industry mainly useβ-fructofuranosidase to produce FOS.β-fructofuranosidase use sucrose as substrate to the catalyze FOS.The active mechanism ofβ-fructofuranosidase is that it breaks the bond ofβ-(1-4) of the surcose,which generates fructose and glucose.Then many fruetoses polymerize the fructoolignsaecharide with the enzyme.β-fructofuranosidase is abroad in the biological world, the character of the enzyme from different microorganisms were different. Aspergillus niger, Arthrobacter, Aspergillus and other microorganisms could produceβ-fructofuranosidase. This experiment cloned the genome from Arthrobacter 10137, and inserted into cloning vector pMD19-T.The gene sequence showed that the gene fragment was 1488bp, the protein consist of 496 amino acids. A expression plasmid pFL-B13cl carrying the gene was transformed into E.coli BL21,andβ-fructofuranosidase gene was successfully expressed in the recombinant strains by double digestion identification, and named E. coli pFL-B13cl-bff, the target protein was about 67KDa.The research design a series of single factor experimenst about the best culture condition of pFL-B13cl-bff. The results showed t that the enzyme activity was the maxium when induce temperature remained at 22℃and induce time lasted for 12h.IPTG and lactose could both induce pFL-B13cl-bff produce recombinant protein, the activity reached the maximum 5.67U/mL when lactose 12g / L, and IPTG concentration 0.5mol/L the activity 6.14U/mL. Lactose was the better inducer because of safety and price cheap. Cultivation E. coli pFL-B13cl-bff in the fermenor,the results showed that the activity was 9.69 U / mL, activity increased to 10.39U/mL by pH control and 11.27U/mL by continuous flowing of glucose, but acetate metabolism products would lead to decline phase coming early.The crude enzyme was purified by metal chelate chromatography, SDS-PAGE analyses showed that 300mmol/L imidazole buffer contain the target protein of 67 KDa.β-fructofuranosidase had the following characteristics: the optimum temperature of 30 ~ 40 ℃,β-fructofuranosidase was stable below 40℃; theβ-fructofuranosidase was stable at pH6.0～7.5, theβ-fructofuranosidase was activated by Mg2+,Ca2+ and inactivated by Cu2+,Ag+ .