Cloning,expression And Characterization Of Fructosyl Hydrolases

Fructose and its derivatives have found wide applications in pharmaceutical,food,healthcare and feed industries because of their unique sweetness and health-promoting effects.Fructosyl hydrolases,including sucrase and inulinase,are employed industrially to produce fructose and its derivatives using sucrose and inulin as substrates.In this thesis research,two fructosyl hydrolase-coding genes in Aspergillus niger were cloned and heterologously expressed,and properties of the encoded enzymes were investigated,respectively,thus providing valuable data for their further molecular engineering to improve the process efficiency of production of fructose derivatives.Bioinformatics was employed to search for candidate genes encoding fructosyl hydrolases in the A.niger genome database.Based on multi-alignment analyses of the predicted amino acid sequence and homology-based modeling of the protein structure for each candidate gene,5 fructosyl hydrolase genes,designated fru1,fru2,fru3,fru4 and fru5,were selected for further study.Among these genes,fru1,fru2 and fru3 encode sucrase,and fru4 and fru5 encode inulinases,respectively.Total RNA was extracted from A.niger and cDNA synthesized by reverse transcription,which was then used as a template for cloning individual genes by PCR amplification,followed by construction of recombinant strains expressing individual fructosyl hydrolase genes.Enzymatic activity assays reveal that recombinant P.pastoris GS115/pPIC9K-fru2,P.pastoris GS115/pPIC9K-fru3 and P.pastoris GS115/pPIC9K-fru4 showed no enzyme activity,while P.pastoris GS115/pPIC9K-frul and P.pastoris GS115/pPIC9K-fru5 exhibited relatively high enzyme activities,which were determined to be 1360.06 U/ml and 1560.34 U/ml,respectively.The optimum pH and temperature of recombinant sucrase Frul were shown to be 5.5 and 45? respectively.Frul was stable in the pH range of 4.0?6.0 or at temperatures from 30? to 45?.Its enzymatic activity was enhanced by EDTA and inhibited by Fe2+,Na+,Co2+,Ca2+and Cu2+,Fru1 was shown to hydrolyze sucrose and inulin.The optimum pH and temperature of recombinant inulinase Fru5 were 5.0 and 50?,respectively.It was stable in the pH range of 5.0?6.0 or at temperatures from 40? to 50?.It was enhanced by EDTA,Na+and Li+,and strongly inhibited by Fe2+.Fru5 was shown to hydrolyze sucrose and inulin.