Heterologous Expression And Application Of Fructosyltransferase From Aspergillus Niger

Fructooligosaccharides(FOS)is mainly produced by the substrate sucrose catalyzed by fructosyltransferase.Fructosyltransferase from the fungus Aspergillus niger have high FOS production capacity,and have been used in the industrial production of FOS.However,FOS was easily hydrolyzed by hydrolytic enzyme of A.niger,and its yield is not high.It is still not possible to explain the difference in FOS production based on the variations observed in the FOS synthesizing enzymes of A.niger.Nevertheless,the diversity of fructosyltransferases in A.niger needs to be explored,and this will be helpful for further understanding the molecular basis of differences in FOS synthesis,So as to better guide the production of FOS.A novel fructosyltransferase(FT-A)from A.niger TCCC41686 with high FOS synthesis ability was characterized in this study.The FT-A gene from A.niger TCCC41686 was obtained via RT-PCR and expressed in Pichia pastoris.Sequence analysis of the FT-A gene revealed that the ORF of FT-A was 1842 bp and encoded a protein of 613 amino acids with a apparent molecular mass of 116 kDa.The three-dimensional(3D)molecular model of FT-A from A.niger TCCC41686 was generated using the crystal structure of fructosyltransferase from A.japonicus CB05(PDB ID:3LF7)as template.Homology modeling was performed using the auto-model command.FT-A shared 96.50%(twenty-two different amino acids),97.06%(eighteen different amino acids)and 98.86%(seven different amino acid)sequence identity with A.niger SG610 fructosyltransferase(FruSG),A.niger YZ59 fructosyltransferase(fwt)and A.niger QU10 fructosyltransferase(ftsl),respectively.Moreover,the changes of residues in FT-A outside the conserved domains were identified and found to have an effect on its characteristics and its FOS-synthesis capacity.The optimal activity of the recombinant FT-A was observed at 50? and pH 6.0.Investigations of thermostability and pH stability using sucrose as substrate showed that FT-A was stable below 50? and pH 4.0-11.0.The Km,Cmax kat and kcat/Km values of FT-A were 151.13 g/L,6.55 g/(L·min)3.02 × 103 s-1 and 20.00 L/(g·s),respectively.The FT-A exhibit both hydrolytic and transfructosylating activities.When FT-A catalyzed the production of FOS,the control of the amount of enzyme and the time of catalysis was the key to the maximum yield of FOS.When the amount of enzyme is 40 U/g,the production of FOS remained above 60%during 50-80 min of synthesis based on sucrose as substrate and the maximum yield of FOS was 67%.The novel fructosyltransferase investigated in this study can potentially be applied for the efficient industrial production of FOS.The results are also provide more valuable information for explaining the relationship between the enzyme's characteristics and FOS synthesis,which will also be very useful for the rational design of fungal fructosyltransferases to improve the production of FOS.