| Fructooligosaccharides are a kind of important oligosaccharides. Fructooligosaccharides could be produced from sucrose by fructosyltransferase catalysis. Research on fructosyltransferases was mainly focused on the screening, purification and properties. The wild-type fructosyltransferases had some disadvantages(e.g., low thermal stability and low catalytic efficiency), these enzymatic characterizations should be improved by molecular engineering.In this work, a strain producing the fructosyltransferase with high fructooligosaccharides conversion efficiency was screened, and named Aspergillus niger YZ59(CICIM F0901). The fructosyltransferase was heterologously expressed in Pichia pastoris and Saccharomyces cerevisiae, respectively. The enzymatic properties of the fructosyltransferase purified were analyzed and discussed. Based on 3-D structure analysis, the thermal stability and catalytic efficiency of the fructosyltransferase had been improved by site-directed mutagenesis. This work plays an important role in efficient heterologous expression, molecular modification, and the industrial production of fructooligosaccharides by recombinant fructosyltransferases. The main research results are as follows: 1. One strain, YZ59, was obtained by secreening on fructosyltransferase expression and theconversion rate of fructooligosaccharides synthetized from sucrose. This strain belongedto A. niger through the analysis of morphology and 18 S r RNA sequence comparison. 2. The c DNA encoding the fructosyltransferase from A. niger YZ59 was obtained byRT-PCR, and heterologously expressed in P. pastoris GS115. The yield of recombinantfructosyltransferase in a 5-L fermentor reached 1020.0 U/m L after 96 h of induction. Theoptimum temperature and p H of the recombinant fructosyltransferase was 55°C and 5.5,respectively. The recombinant fructosyltransferase was stable at temperature below 50°Cand at p H from 3.0 to 10.0. The specific activity of recombinant fructosyltransferase was6.8×104 U/mg. The Km, Vmax, kcat, and kcat/Km values of recombinant fructosyltransferasewere 159.8 g/L, 0.7 g/(L·min), 1.1×104 min-1, and 68.8 L/(g·min), respectively. Therecombinant fructosyltransferase was slightly activated by 5 m M Ni2+, Mg2+, K+, Fe3+, orMn2+, but inhibited by all other metal ions. The highest yield of fructooligosaccharides forthe fructosyltransferase reached approximately 57.3%(w/w). 3. Fructosyltransferase was heterologously expressed in S. cerevisiae. The highest yield offructosyltransferase was 19.8 U/m L at 48 h. The optimum temperature offructosyltransferase was 55 oC and fructosyltransferase was stable below 50 oC. Theoptimum p H of fructosyltransferase was 5.5, and fructosyltransferase was stable at a p Hrange from 4.0 to 9.0. The Km and Vmax of fructosyltransferase were 170.0 g/L and 0.7g/(L·min). Ni2+ and Mg2+ could significantly activate fructosyltransferase. 4. The thermal instable amino acid residues of the fructosyltransferase were analyzed byPo PMu Si C Web Server software, and six kinds of mutation improving thethermal stability was obtained: Asp64 Leu, Gly80 Trp, Gly123 Leu, Glu377 Gly, Arg397 Ile,and Glu575 Tyr. The optimum temperature of mutants Glu377 Gly and Arg397 Ile wereincreased by 5oC than before. The half-life(t1/2) of Glu377 Gly was 26.9 min at 60 oC. Cationic-π interaction and β-turn of mutant Glu377 Gly were increased, and its hydrophobicity was enhanced. The affinity of that on sucrose was enhanced compared with that of the wild-type. Besides, the catalytic efficiency constant kcat value and kcat/Km value were respectively increased to 1.6 times and 2.1 times than that of the wild-type. After mutation, the inhibitory effect of glucose on Glu377 Gly was decreased compared with that of the wild-type. The highest yield of fructooligosaccharides for Glu377 Gly was increased by 2.7% than that of the wild-type. |
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