| Fructooligosaccharides(FOS) have been widely used in the food industry because of their physiological functions(e.g., regulating the intestinal flora, low sweetness and low calorie). Fructosyltransferases can catalyze sucrose to produce FOS. In this study, the fructosyltransferase gene from Aspergillus niger, was successfully cloned and heterologously expressed in Pichia pastoris host. Moreover, protein engineering and process optimization were performed to improve the catalytic efficiency, thermostability and FOS yield of the recombinant enzyme. The main important results are listed below:(1) The cDNA sequence encoding fructosyltransferase was cloned from A. niger SG610 by RT-PCR, and heterologously expressed in P. pastoris GS115. The optimum temperature and pH of recombinant FruSG were 50℃ and 5.5, respectively. The recombinant protein could perform high stability below 40℃ and from pH 3.0 to 8.0. The Km and kcat values of the recombinant FruSG were 214.13 g·L-1 and 63.33 s-1, respectively. On addition of 50 g·L-1 and 100 g·L-1 glucose, the Ki values were 130.60 and 170.00 mmol·L-1, respectively. 1 mmol·L-1 Fe2+ or 5 mmol·L-1 Mg2+ could activate the recombinant FruSG.(2) The optimal conditions in flasks were investigated. Results showed that medium volume 30 mL/250 m L, glycerol:methanol 1:20(v/v), the initial pH 5.5, inducing temperature 30℃, the initial methanol concentration 1.0%, the subsequent methanol concentration 1.5% and ammonium sulfate concentration 10 g·L-1 were optimal conditions for recombinant enzyme expression. After induction for 120 h, the yield reached 218.32 U·m L-1, which was 4.63 fold higher than that before optimization. In 5 L fermenter, the maximal yield(2294.7 U·m L-1) of recombinant FruSG was obtained at DO 30%, which was 10.51 fold of the highest level in flasks.(3) The 3-D structure of recombinant FruSG was obtained by Swiss Model. Four amino acid residuses(C66, P65, I62, Q61) close to the active sites were selected for site-directed mutagenesis(C66A, C66 T, C66 N, C66 S, P65 A, P65 S, I62 A, I62 S, I62 M, Q61 A, Q61 Y, Q61W) after 3-D structure analysis. The catalytic efficiency constant kcat values of mutants were enhanced than that of wild-type, of which, the mutant C66 N was increased significantly. The kcat value increased to 1402.95 s-1 from 63.33 s-1. Also, the specific activity of C66 N increased by 5.86 fold, which was increased from 2284.89 U·mg-1 to 15671.15 U·mg-1.(4) The structure of recombinant FruSG was analyzed by PoPMuSiC Web Server software, and nine mutants were obtained to improve the thermostability: E617 F, S585 F, D570 Y, E543 I, E508 F, D336 W, D311 I, Q229 I, Q159 I. The thermal stability of mutants Q159 I, E617 F and D336 W were improved compared with that of wild-type. The mutants Q159 I, E617 F and D336 W retained 95.65%, 79.52% and 80.86% of its initial activity after incubation 2 h at 40℃, which were 1.34, 1.12, 1.14 fold of that of wild enzyme, respectively. Residual activity of mutant Q159 I was increased by 249.47 fold at 50℃ and the half-life(t1/2) of it was from 30.20 min of wild type to 166.78 min at 45℃.(5) After fusion with fructosyltransferase gene and glucose oxidase gene using six different linkers, the fusion enzymes were expressed in P. pastoris GS115. FOS yield was analysised in this work. The highest yield of FOS by Fru-GOD-D was 48.06%, which was 1.28 fold of that by wild type. After adding catalase, the highest yield of FOS by Fru-GOD-D reached 89.96%, which was 1.97 fold of that by the wild-type. |
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