| Alliinase (alliin lyase, EC 4.4.1.4) form garlic is the key enzyme, which catalyzes the conversion of alliin to allicin, the main biologically active component in garlic. Currently, there were many studies on factors of the alliinase activity, such as temperature, pH, E/S, however, for metal ions on the mechanism of alliinase activity rare reports. In this study, alliinase was purified from fresh bulbs using various steps, including Ammonium sulfate method, PEG6000 method, Sephadex G-200 column chromatography, the purity of alliinase was identified by SDS-PAGE, then got the optimal method of the alliinase extraction and purification from fresh garlic.We optimized the parameters of metal ions on the alliinase activity by orthogonal test and box-behnken test, studied the metal ions on kinetics and UV-Visible spectrum of alliinase, preliminary explored the activation and inhibition mechanism of the metal ions on alliinase. The quality of hot-air drying garlic slices is poor, while high-quality products such as freeze-drying garlic slices are costly in the domestic. Based on the method of single drying, develop high-quality dehydrated garlic slices by the technology of combined microwave-vacuum drying (MVD) with air-drying. The main conclusions stated as follows:1. Extraction of alliinaseContrast tests showed that, PEG6000 method has a higher extraction rate and alliinase activity recovery, using 20% PEG6000 to purify the crude enzyme, the purity was 2.96 times as much as crude enzyme and the recovery can reach to 72.75%. Then the crude enzyme was passed the Sepadex G-200 column. SDS-PAGE showed that no unpurposed protein was found, and one most intensive band at Mr=54.7 was detected, which is consistent with literature reports.2. Properties of alliinaseBased on the single-factor test, Orthogonal Test and Box-behnken test were used to optimize the parameters that activation effect of metal ions on allinase. The results showed that the optimal condition was:E/S value of 0.20, enzyme reaction temperature of 36℃, pH value of 6.4, Mn2+concentration of 8.15 mM. The allinase activity under this condition was 0.156±0.0053μmol/min.The kinetics of enzymatic activity results showed that, Mn2+as an activator increase the Km, Vmax and reaction speed, it is possible that Mn2+provides a suitable environment for the reaction alliinase with substrate, then improve the reaction speed. The role of L-cys as an inhibitor was opposite, it is possible that L-cys is an anti-competitive inhibitor in the reaction system, which affected the transformation ES complex to product. During the reaction, enzyme and substrate combined to form intermediate compound, which combined L-cys to form E-S-L-cys complex, not to transform product, then decrease the reaction speed.UV-Vis spectroscopy of alliinase showed that addition of activator did not move the absorption peak, just affected the strength of absorption peak. Mn2+as an activator increased the value of the absorption at 280nm,325nm and 430nm. L-cysteine as an inhibitor had the same effect at 280nm,325nm, however, decreased the value of the absorption at 430nm. It is possible that the activator and inhibitor affectd the structure of PLP and the combination of PLP as the cofactor to the main chain of alliinase, which affects the activity of alliinase.Preliminary explored of the activation and inhibition mechanism of the metal ions on alliinase, discussed the relationship between the alliinase activity and allicin formation. We found a significant positive correlation between the alliinase activity and allicin formation.3. Develop of odorless dehydrated garlic slicesBased on the color protection and deodorization, the technology of combined microwave-vacuum drying (MVD) with air-drying (AD) were optimized by orthogonal experiment. The results showed that the best processing was using microwave-vacuum drying for 20min (vacuum-90kpa, microwave power 375w), then air-drying 60min (60℃), the process obviously got higher quality garlic products than single method. |
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