Genes Heterologous Expression And Modification Of Cycloinulooligosaccharide Fructanotransferase

Cycloinulooligosaccharide fructanotransferase?CFTase?is a member of glycoside hydrolase family 32,which catalyses the intramolecular transfructosylation reaction converts inulin into cycloinulooligosaccharides?CFs?of?-?2,1?-linked D-fructofuranoses.Cycloinulohexaose?CF6?is major product of the CFTase reaction.At present,studies on CFTase only focus on the purification and characterization of enzymes,but catalytic mechanism of CFTase for inulin is rarely studied.CFTase106 derived from Paenibacillus sp.Lfos16 was heterologous expression by p.pastoris X-33,which was compared with E.coli BL21?DE3?constructed in the early stage of the laboratory.The results showed that the enzymatic properties of recombinant CFTase106expressed from different host sources were not significantly different.Considering that E.coli is easy to culture and has a short production period.Therefore,E.coli was used as the expression system to directed evolution and immobilization study of CFTase106 in the follow-up experiment.In order to study the catalytic mechanism of CFTase106 to better modification,directed evolution was used to introduce random mutations into cft106 gene to construct the mutant library by error-prone PCR.Two mutant strains with improved enzyme activity were screened.Meanwhile,considering the mutation site and the three functional regions of CFTase106?ERH,CR,R4?,it was found that The ERH region and the CR region have catalytic cyclic and hydrolytic functions,and the single CR region does not show any catalytic activity.However,the R4 region has an extremely important influence on the ability of enzymatic cyclic and hydrolytic reactions.CFs has potential application value in industrial production,in order to make the CFTase106 better application in industrial production,a new type of immobilization technology enzyme-inorganic hybrid nano flowers to immobilized mutant CFTase106.The results showed enzyme–inorganic hybrid nanoflower was assembled by using mutant CFTase106 as the organic component and Ca₃?PO₄?₂·3H₂O as the inorganic component,the hybrid nanoflowers have a hierarchical like flower morphology,the average diameter of a single nanoflower was 5?m.Meanwhile,on measuring stability and reusability of the hybrid nanoflower to inulin,it could maintain about 63%original activity after reuse of six times and more than 60%original activity after 10 days storage at room temperature,indicating the hybrid biocatalyst possesses an approving stability.