| (1) The SAI was purified from mango by ammonium sulfate precipitation (0-85%saturation), DEAE-Sepharose Fast Flow (20column volume,0-0.27mol/L NaCl) and ultrafiltration (50kDa). The molecular weight of SAI was45kDa by SDS-PAGE, and isoelectric point (pI) is2.08by capillary electrophoresis. The CD spectra (Circular dichroism spectra) of SAI characterized a maximum at194nm and a minimum at222nm suggesting a large amount of (β-sheet structure. The amount of a-helix, antiparallel (3-sheet,(3-sheet,(3-corner and coil structure was8.3,49.2,3.7,16.6and29%respectively, calculated by the CDNN program. The fluorescence spectra excited at295nm shows a λmax:at331nm, suggesting the tryptophan residues of SAI partially exposed to solvent. Hydrodynamic radius of SAI was4-20nm by dynamic light scattering (DLS). The SAI was round granulate with diameter of20-50nm by atomic force microscope (AFM) and transmission electronic microscopy (TEM).(2) The optimal reaction temperature and pH of SAI was60℃and pH3.8, respectively. Km value was437.48、56.03and25.54μmol/L respectively, when stachyose, raffinose and sucrose were taken as substrate. SAI was stable with pH<7and temperature≤40℃, while lost the catalytic ability with alkaline (pH8,9) and temperature higher than50℃. The ellipticity value of CD spectra and intensity of fluorescence spectra were lowered by thermo (70℃/5min) and alkaline (pH8,9) treatment, but the secondary structure and environment of tryptophan residues stay unchanged after that.(3) The activity of purified SAI did not change after HPP at50-500MPa with temperature lower than40℃for10min, but decreased at600MP. The activity of SAI was inactivated significantly by HPP at45,50and55℃. Antagonistic effect of pressure and temperature was observed with pressure50-400MPa at45℃, and50-200MPa at50℃. No activity of purified SAI was observed after HPP treatment at55℃. Residual activity of purified SAI deceased with the increasing of treatment time at45℃/600MPa and50℃/50,600MPa. The activity of SAI in extract was stable after HPP at20-50℃/50-600MPa for0-30min, but decreased to60%after HPP at70℃and50MPa for5min. The activity of SAI in mango plup increased by0-20%after HPP. Low pH, bovine serum albumin (BSA) and pectins with different DE values (degree of easterified) could protect the SAI against the HPP. The protecition of pectins increase with the increasing of concention. The protection of pection with different DE values was natural pectin (DE:75%)> modified pectin (DE≥85%)> modified pectin (DE:20-35%). The residual activity of SAI with BSA (0.6%) was90%after HPP at50℃/600MPa for30min. The pressure resistence of SAI obviously increased at pH2,3and4. The trehalose and sucrose at0.6and6%exhibited no protection for SAI to HPP, while weak protection was oberved with sucrose at12%.(4) The fluorescence intensity of SAI excited at295nm decreased with the increasing of treatment time, pressure and temperature, but the λmax remained constant after HPP suggesting the environment of tryptophan residues remained unchanged. Pectins quenched the fluorescence intensity, which decreased with the increasing of pectin concentration. Fluorescence intensity of SAI with various concentration of pectin was lowed by HPP. Fluorescence intensity did not change with pH6, and increased lightly at2.1 and4. The intensity of SAI in different pH value after HPP was pH4>6>7.1>2.1. HPP lowed the ellipticity value of CD spectra, which decreased with the increasing of treatment time, pressure and temperature. The content of secondary structure did not change after HPP. Low pH affected the CD spectra. Ellipticity value at194nm was pH4>6>7.1>2.1, and pH7.1>6>4>2.1at222nm The result of DLS suggested the particle size of SAI stay unchanged after HPP. AFM and TEM showed that SAI shifted from granular to fibrillar after HPP and aggregation happened. |
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