| Lentinus edodes is welcomed for its delicious taste, strong and pleasant flavour and valuble nutrients, but in recent years, the issue of its endogenous formaldehyde is highly controversial. So it was high time to analyze the formation mechanism of endogenous formaldehyde. In order to reach that aim, the key enzyme in formaldehyde formation pathway, y-Glutamyltranspeptidase (GGT), was isolated and purified, and its properties were also determined during post-harvest Lentinus edodes. The major findings are as follows:Compared with ordinary extraction by stiring, the yield by ultrasound-assisted extraction could get 8.24 U/g, which was 30%more than ordinary extraction by stiring. The optimum conditions of ultrasound-assisted extraction were obtained as follows:extraction power 200 W, the dry/water weight ratio 1:50, extraction time 30 min. Then the response surface methodology (RSM) was applied to optimize the ultrasonic-assisted extraction of y-Glutamyltranspeptidase.Box-Benhnken experimental design was used for experimental design and analysis of the results to obtain the optimum extraction conditions. Based on the RSM analysis, optimum conditions were:material/water ratio 1:52.29, extraction time 28.19 min and extraction power 204.84 W. Under the optimized conditions, the experimental value was 11.07 U/g, in close agreement with values predicted by the mathematical model.The ammonium sulfate precipitation of 30% and 60% on the broth pretreatment could get target enzyme. the optimum conditions of Phenyl-Sepharose were:cp 2.6 cm×20 cm Column, fluid 2 mL/min, concentrated buff 0.05 mol/L Tris-HCl, elution 1.0 mol/L(NH4)2SO4 (pH 7.6),0.05 mol/L Tris-HCl (pH 7.6). through 30%-70% of ammonium sulfate precipitation, Phenyl-Sepharose, sephadex G-25, the singled GGT was got. The yield was 23.7%, and the concentration multiple was 178.1.SDS-PAGE method indicated that the molecular weight of GGT is 88 KDa, and it had two subunits. The relative molecular weight of two subunits was estimated to be about 28 KDa and 60 KDa, respectively. By amino acid compositions analysis, Leu and Thr were found in relatively large amounts, whereas, Met and Cys are low in the purifed enzyme. The optimum temperature and pH of enzyme reaction were 37℃and 7.6, respectively. GGT is very stable when the temperature is under 40℃, but its activity was almost lost when the temperature was over 60℃. The GGT convertedγ-glutamyl-P-nitroanilide to P-nitroanilide with the Km of 2.601mg/ml and the Vmax of 0.0287μmol/min. The GGT activity could be activated by Na+, K+, Ca2+ however, inhibited by Ag+, Cu2+, Zn2+, Fe3+. IEF-PAGE method indicated that its pI was 6.4. FT-IR method indicated that the percentage ofβ-sheet, a-helix, random coil andβ-bend were 40.94%,18.26%, 15.90%and 24.90%respectively. CD method indicated that the percentage ofβ-sheet, a-helix was 47.7%and 18.2%respectively, which was similar with FT-IR.The effects of various factors on GGT in Lentinus edodes were examined. Among 14 compounds in 5 various groups (basic nutriment, amino acid, inorganic substances containing sulfur group, signal-related compound and antioxidant) used in this study, The GGT activity could be activated by much of amino acid such as cysteine, glutamine, glycine, lysine, methionine and arginine. Norganic substances containing sulfur group such as Na2SO4 and Na2SO3 can exhibit the activity of GGT. The GGT activity could be activated by Vc, however, was effected less by NO.With cysteine, methionine, lysine, arginine, Vc, sodium sulfate to stimulate the bodies of Lentinus edodes, finding that formaldehyde content and GGT activity was correlative directly.
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