Study On Enzymatic Characteristics Of Polyphenol Oxidase And β-amylase From Purple Sweet Potato

Purple sweet potato is a new breed that was introduced from foreign countries in recent years and widely used for industrial processing.With the further study,purple sweet potatoes will be more and more widely exploited and utilized.In this paper,the basic study on polyphenol oxidase(PPO)and P-amylase of purple sweet potato enzyme system is mainly carried out.These two kinds of enzymes may have a great influence on the quality and benefit of purple sweet potato products.This paper is to provide basic data for deep-processing,comprehensive utilizing and large-scale production of purple sweet potatoes;and to increase the income of peasants and strengthen the competition of sweet potato in the international market.In this paper,PPO andβ-amylase in purple sweet potato are extracted,purified and determined through modern separation techniques and analysis methods by first studied.At the same time enzymatic characteristics of PPO andβ-amylase are studied and kinetics of enzyme-catalyzed reactions of PPO is also analyzed.The main results are as follows:The polyphenol oxidase is extracted from fresh purple sweet potato by phosphate buffer solution,and spectrophotometry method is applied in the experiment to study the process of the reaction of PPO from purple sweet potato.The result shows that the maximum absorption wavelength of the product is 370nm with catechol as a substrate.PPO activity is determined under the circumstances.The speed of the reaction catalyzed by PPO is linear with reaction time within 3 minutes.And it is stable after the reaction started 12 minutes.The activity of PPO from purple sweet potato peel is 1.17 times than that of inner-tissue, 1.14 times than that of intact purple sweet potato;Browning degree(BD)of purple sweet potato peel is 1.99 times than that of inner-tissue,1.55 times than that of intact purple sweet potato.The relativity between PPO activity and browning degree of purple sweet potato is positive.Relativity coefficient is 0.9895.This result shows that the browning of purple sweet potato is mainly due to polyphenol oxidase in it.Browning degree of purple sweet potato peel is more serious than that of inner-tissue,because the activity of PPO from peel is stronger than that of inner-tissue.The effects of pH and temperature on activity of polyphenol oxidase are assayed.PPO from purple sweet potato is very sensitive to the changes of pH value.The optimum pH of PPO is 4.1 with the second optimum pH of 6.0 and PPO activity dropped rapidly when it is below 3.0 or above 7.5.The optimum temperature of PPO from purple sweet potato is 16℃with the second optimum temperature at.30℃.The enzyme is more stable at 16℃than that at any other temperatures.Maybe PPO from purple sweet potato has its isoenzymes because of its two optimum pH values and two optimum temperatures.We are waiting for this problem to be solved in the near future.The heat stability is determined and it shows that PPO is sensitive to temperature and it becomes very unstable above 70℃.The PPO activity and its stability decreases rapidly with the increase of temperature when it is above 45℃.The half-lives of PPO at different temperatures are 450,240,66,50,21 and 13s.It can be referred that the higher the treating temperatures,the shorter the half-lives of PPO.Browning intensity of fresh-cut purple sweet potato slices during storage is found to be correlated with polyphenol oxidase activity.During storage,the activity of PPO extracted from purple sweet potato increases initially and decreases afterwards,with the highest activity of PPO on the third day.And BD increases very quickly as time goes by,especially in the first three days.The relativity between PPO activity and browning degree during storage is negative.Relativity coefficient is-0.8747.This result shows that the browning of purple sweet potato is mainly due to polyphenol oxidase in it.The effects of various inhibitors on the activity of PPO are also assayed.The activity of PPO from purple sweet potato can be restrained effectively by phytic acid(PA),L-cysteine (L-Cys),citric acid(CA)and ascorbic acid(Vc)with different concentrations to different extent except EDTA-2Na.With catechol as the substrate,V_C and PA showed strong inhibitory effects,with V_C of the highest effect and EDTA-2Na the lowest.The effect of decreasing PPO activity is not ideal by using these four different inhibitors solely.The most effective combination of browning inhibitors,based on orthogonal experiment,selected for fresh-cut purple sweet potato is 0.05%L-Cys+0.03%PA+0.3%V_C+0.3%CA.The effect of four factors on inhibiting PPO activity is in the following order:V_C＞PA＞L-Cys＞CA.Variance analysis of orthogonal experiment results shows that V_C is the most effective inhibitor(F value is above F_0.05(2.2)).It is significance to reduce the processing cost by using this multiple-inhibitor,which had been proved in this paper to be safe,effective and low-cost.9 kinds of phenolic compounds are used as the substrates of PPO extracted from fresh purple sweet potato for specificity study.The results shows that the substrates of PPO included phenol,guaiacol,catechol,resorcin,hydroquinone,L-tyrosine,chlorogenic acid, pyrogallic acid and 1,3,5-hydroxybenzene,because there are absorption apexes within the range of 300～400 nm.The maximum absorption wavelength of the products catalyzed by PPO are respectively 372,368,370,360,358,374,384,370and362nm.The Michaelis constants(K_m)for these 9 kinds of phenolic substrates are determined by Hanes-Woolf diagram.They are 15.68,15.01,12.06,14.71,21.31,17.12,10.23,13.22 and 13.46mmol/L.The combining capability of the PPO with various substrates is in the order of chlorogenic acid＞catechol＞pyrogallic acid＞l,3,5-hydroxybenzene＞resorcin＞guaiacol＞phenol＞L-tyrosine＞hydroquinone.The optimum substrate identified for PPO from purple sweet potato is chlorogenic acid,then catechol.According to the molecular structures of 9 kinds of phenolic substrates,it can be inferred that it is easy for adjoining-hydroxide to combine with PPO from purple sweet potato and it is difficult for opposite-hydroxide.And the substitutional groups and their positions influence directly the ability of phenolic substrates combining with PPO from purple sweet potato.With catechol as a substrate,the Michaelis constants(K_m)which determined by Hanes-Woolf diagram is 12.06mmol/L,maximum velocity of the reaction catalyzed by PPO is 4.366×10^-2mol/L·min,which is near to 4.366×10^-2mol/L-min depending on Bi-reciprocal diagram of Lineweaver-Burk.According to Arrhenius formula,the activation energy of the PPO-catalyzed reaction is 22.93kJ/mol.The effects of four various inhibitors on PPO activity are different.With catechol as the substrate,ascorbic acid(V_C)and phytic acid(PA)show strong inhibitory effects,with V_C of the highest effect and citric acid the lowest.This conclusion is the same as the one showed in the best multiple inhibitor orthogonal experiment.Among these inhibitors,citric acid is a reversible competitive inhibitor with K_I=1.34×10^-2mol/L,which is significant and instructive to the preservation of fruit and vegetables.At the same time,the experimental result is consistent with the conclusion by reasoning in theory.The same purple sweet potato is used as the material for the p-amylase study.P-amylase is extracted from purple sweet potato by using Phosphate Buffer Solution as a solvent which contains reducing agent and sodium chloride,with DNS colorimetry as determination method. And the effects of extracting temperature,extracting time,acidity,dosage of extraction solution onβ-amylase activity are studied.Choose mentioned four factors above,the optimization for the extraction ofβ-amylase from purple sweet potato is chosen by orthogonal experiment,the result shows that the best technology is that extracting temperature 40℃,extracting time 1 hour,PBS and raw material ratio 5:1,pH 6.5.Two kinds of educing agents are added separately to Phosphate Buffer Solution and optimum dosage of sodium bicarbonate(0.5g/L)is affirmed by studying its effect on p-amylase activity.In addition,sodium chloride(9g/L)is also added.It is known that P-amylase existing in purple sweet potato peel is more than that in purple sweet potato inner-tissue.Soβ-amylase can be extracted from purple sweet potato peel so that comprehensive utilization ratio of purple sweet potato can be increased and environment can also be protected at the same time.The result of the storage stability ofβ-amylase from purple sweet potato shows that amongβ-amylase which added separately 0.12 percent of sodium benzoate,0.12 percent of CMC-Na and 0.2 percent of maltose,the one added 0.12 percent of sodium benzoate preserves the best compared with the one added no preservative.Theβ-amylase from purple sweet potato is extracted by phosphate buffer and purified first by means of ion-exchange chromatography using DEAE Cellulose-52 column,and then by gel filtration on Sephadex G-200 column.SDS-PAGE shows only one band at the end of separation which shows the enzyme system is pure.Molecular weight of p-amylase is estimated 170kD by SDS polyacrylamide gel electrophoresis with the separating glue concentration of 10%.The specific activity of the purifiedβ-amylase increases from 78.9u/mg to 1478u/mg,which is 18.73 folds over the crude extraction with 0.92%recovery.The method of P-amylase purification in this experiment is feasible.A systematic research on the characteristics of purified P-amylase from purple sweet potato is also made by spectrophotometry.As shown by the results,its optimum temperature is 40℃,the temperature range of stability is from 40℃to 50℃,its optimum pH is 6.8,and the pH range of stability is from 5.0 to 7.0.The purified P-Amylase deactivates quickly at extreme acidic or alkali condition.The Michaelis constant(K_m)for P-amylase which determined by Bi-reciprocal diagram of Lineweaver-Burk is 2.25×10^-5mol/L.It shows that its combining capability with starch is powerful.It also shows that hydrolysis rate of starch reaches its highest level within 4 hours and it is stable after the reaction starts 6 hours.Effect of various metal ions on P-amylase activity shows that the activity is promoted by Mg²⁺,Cd²⁺and Ca²⁺,while the activity slightly reduced by Cu²⁺and Fe³⁺and strongly inhibited by Al³⁺and Zn²⁺by 70%or more even at low concentration.The contents of starch and reducing sugar in purple sweet potato are determined after 6 kinds of different processing treatments including sun-drying,oven-drying,cooking,baking, microwave heating and frying.The results shows that starch content is significantly decreased by 1.02%～9.98%and reducing sugar content significantly increased by 2.60%～31.07% after processing.Oven-drying results in starch content decrease and reducing sugar content increase more effectively than sun-drying,but cooking and baking do not effectively reduce starch content in comparison with drying.The purple sweet potato p-amylase activity leads to different change in starch and reducing sugar contents during processing.