The Property Of Cutinase In Ethanol Solution And Application In Extraction Of Apple Phenolics

Cutianse is a mutilfunctional enzyme and can hydrolyze cuin of plants. The catalytic behavior of cutinase has been studied in various types of non-aqueous media, such as reversed micelles. Ethanol usually is used to produce apple polyphenols. In order to apply cutinase in different systems, first of all, the properties of cutinase were evaluated in ethanol solutions, and the effect of ethanol on conformation was also researched. And at the same time, the optimum technological parameters of cutinase-assiatant extradtion conditions of phenolics were optimized. The component of polyphenols which were obtained by different extracting methods was identified by HPLC-MS. The results were as follows:Cutinase was stable at 37 oC in 20% ~ 50% ethanol solutions. Cutinase were incubated in 30% ethanol solution at pH 6.5 ~ 10.5 for 12 hours, the remaining activityies hardly changed. The activity of cutinase in 30% ethanol solutions was twice as high as in aqueous phase at 55 oC.The optimum temperatures of cutinase were 65 oC, 55 oC and 50 oC respectively in 20%, 30% and 40% (V/V) ethanol solutions. The activity of cutinase was much lower in 50% ethanol solution. The optimum pHs of cutinase were 9.5 and 9.0 in 30% and 50% ethanol solutions, respectively.The inactivation kinetics of cutinase was tested in ethanol solutions at 50 oC. The results showed that the inactivation kinetic of cutinase in ethanol solutions followed the first order reaction (R~2>0.98).The conformation of cutinase was investigated by CD and fluorescence spectra. The secondary of cutinase was not correlated with the activity, but the tertiary structure conformations greatly changed in ethanol solutions. The fluorescence signal of cutinase was lower but the activity was higher.The extraction rate of phenolics was improved by cutinase. The maximum extraction rates of polyphenols were 99.16% and 86.43% in 30% ethanol solution and aqueous phase with cutinase. The optimum conditions were as follows: temperature was 37 oC, the rate of solid to solution was 1:50, extraction time was 15 min, and the rate of enzyme to pomace was 1250:1 (U/g) in 30% ethanol solution; and temperature was 60 oC, the rate of solid to solution was 1:75, extraction time was 20 min, and the rate of enzyme to pomace was 2000:1 (U/g) in aqueous phase at pH 7.5.As the result DA201-C was the best in separating of polyphenols. The purity of polyphenols which was approximately 30 times higher than the previous was 58.33%. The yield of polyphenols could reach 73.09%. The optimum conditions were that the concentration of polyphenols was 3.0 mg/mL, pH was 5.0, the eluent was 50% ethanol, the speed of eluent was 1.0 mL/min.The puried polyphenols determined by HPLC-MS, the component of polyphenols obtained by different extracting method were the same. The component of polyphenols were caffeic acid derivatives, chlorogenic acid, procyanidia dimmer, quercetin-glucoside, quercetin- galactoside, quercetin- arabinopyranoside, quercetin-rhamnoside, phloridzin. And the content of quercetin-glucoside was highest.