澳洲青苹多酚氧化酶的分离纯化研究

采用丙酮粉沉淀抽滤法从澳洲青苹中提取多酚氧化酶.提取的粗酶液经30%～90%饱和度硫酸铵分级沉淀、透析除盐、交联葡聚糖G-75凝胶色谱层析、后经PEG 6000浓缩,得到纯化的多酚氧化酶,纯化倍数为7.49.纯化后的酶液经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳,采用考马斯亮蓝R-250染色显示一条电泳带.通对过PPO与邻苯二酚反应产物的波谱扫描显示,其产物在λ=410nm下有最高吸收.     

PURIFICATION OF PEACH POLYPHENOL OXIDASE IN THE PRESENCE OF ADDED PROTEASE INHIBITORS1

Crude preparations of peach fruit (Prunus persica Batsch cv. Redskin) polyphenol oxidase (PPO) showed many apparent isoenzyme forms. Some of these forms were probably the result of proteolytic action of peach proteases while other forms were the result of association of PPO with carbohydrate materials. In the presence of protease inhibitors, Trasylol and phenylmethylsul-fonyl fluoride, three apparent isoenzyme forms of PPO were purified to homogeneity. The purification scheme included hydrophobic chromatography on phenyl sepharose CL-4B, hydroxylapatite chromatography, DEAE cellulose chromatography, and gel filtration on Ultrogel AcA 34. Minor contaminants remaining after these steps were separated from PPO by gel electrophoresis. The major PPO isoenzyme form (A) was purified 44 fold with an overall yield of 5.6% and contained no detectable carbohydrates. Isoenzyme forms A' and A' were purified 104 and 67 fold respectively, but still were associated with carbohydrate material. Cesium chloride centrifugation partially removed the carbohydrates associated with PPO A' and A'. Purified peach PPO A showed greater activity toward D-catechin (539%) and pyrogallol(l82%) than to catechol (100%). An apparent K₃ of 4, 0.3, and 2 mM was obtained with D-catechin, pyrogallol and catechol, respectively. The enzyme was severely inhibited by 10 μM 2,3-naphthalenediol (91%) and by 10 pM diethyl dithiocarbamate (100%).     

PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM POTATO: I. PURIFICATION AND PROPERTIES

Polyphenol oxidase (Isozyme I) from potato was extracted and purified with ammonium sulfate, cation-exchange (Bio-Rad Bio-Scale S2) and Sephadex G-100 column chromatography. The enzyme was purified 11.8 fold resulting in a specific activity of 250.3 units/mg. Optimum pH of the enzyme was 6.6. Optimum temperature of the enzyme was 40C and its half-life was 0.8 min at 70C. The K_mfor catechol, pyrogallol, 4-methyl catechol, caffeic acid and L-DOPA were 4.11 mM, 0.61 mM, 0.78 mM, 0.50 mM and 32 mM, respectively. However, monophenols such as tyrosine, p-cresol and 1-naphtol did not show any activity. Data for V_max/K_m which represents catalytic efficiency show that 4-methyl catechol has the highest value. The molecular weight of the active enzyme was 86,000 Da, composed of two identical subunits. The number of Cu²⁺ ions bound was found to be 2 per enzyme molecule.     

AB-8大孔树脂分离提纯无花果总黄酮的研究

研究了AB-8大孔树脂对无花果总黄酮的吸附和解吸的特性，并考察了相关的工艺条件。结果表明：采用0．5mg／mL流速上样，80％乙醇1．0mg／mL流速洗脱的工艺条件下，AB-8大孔树脂可较好的分离纯化无花果黄酮。     

INHIBITION OF o-DIPHENOL OXIDASE BY DICHLORODIFLUOROMETHANE

Dichlorodifluoromethane (f-12) can effectively and irreversibly inhibit the catalytic activity of o-diphenol oxidase (tyrosinase, polyphenol oxidase) in a simple buffered system. The degree of inhibition was influenced by concentration of and duration of exposure to f-12 and by time and vigor of agitation. Under the conditions tested, maximum inhibition of o-diphenol oxidase was obtained using 2.9 mole % f-12 and agitating samples at room temperature and 180 cpm for 20–40 min.     

酶法提取大米蛋白的研究

采用4种蛋白酶水解大米蛋白,比较提取率后得出碱性蛋白酶为最优酶.采用单因素实验分别考察温度、加酶量、料液比、水解时间和pH对该酶提取大米蛋白的影响.通过正交实验确定了最佳工艺条件为:温度60℃、加酶量(E/S)1.5%、pH9.5、料液比1:6、水解时间4h.在此条件下,蛋白质的提取率可达76.42%.     

THE β-AMYLASE ENZYMES OF BARLEY AND MALT: I. PURIFICATION OF THE MALT ENZYMES*

Two β-amylase enzymes from malted barley were purified by successively fractionating an extract on CM-cellulose, Sephadex G-75, and DEAE-cellulose. The procedure resulted in a net recovery of 10% of the initial activity and a 10-fold and 16-fold purification of the individual enzymes. Each enzyme migrated as a single protein band during disc electrophoresis at pH 4·5, but each purified protein extract produced two active protein bands at pH 8·9. This heterogeneity within components appeared to be due to oxidation of essential SH groups at high pH that caused loss of enzymic activity.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM FRESH-CUT CHINESE WATER CHESTNUT

The characteristics of polyphenol oxidase (PPO) from Chinese water chestnut (CWC) and its potential inhibitors for browning reactions were investigated. PPO was isolated from fresh‐cut CWC and was purified on a Sephadex G‐100 column, with a yield of total activity close to 10%. The molecular weight, Michaelis constant (K_m), substrate specificity, optimal pH and temperature of CWC PPO were examined. Kinetic studies indicated that the K_m and V_max values of CWC PPO for catechol were 10.32 mmol/L and 6.452 × 10⁴ U/min, respectively. The optimal pH and temperature for CWC PPO was 6.5 and 40C, respectively. Among the browning inhibitors tested, 4‐hexylresorcinol, at a concentration of 0.3 mmol/L, showed the strongest inhibition (70%) against the PPO activity of CWC, followed by 3.0 mmol/L N‐acetyl‐L‐cysteine with an inhibition of 53%.     

PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM POTATO: II. INHIBITION AND CATALYTIC MECHANISM

KCN and ascorbic acid showed competitive inhibition patterns with K_is values of 0.032 and 0.27 mM, respectively. Uncompetitive inhibition patterns were obtained with sodium azide, L-cysteine and NaCl with K_ii values of 3.3 mM, 0.12 mM and 0.3 M, respectively. A noncompetitive inhibition pattern was obtained for thiourea with 0.067 mM for K_is and 0.59 mM for K_ii. Cu₂⁺ increased the activity about 2.5 fold at or above 40 μM and K⁺ decreased the enzyme activity about 33% at 0.4 M. Other metal ions did not have any effects on the activity. Two pK values of 5.8 and 8.0 were obtained from V_max profile and two pK values of 5.9 and 8.1 from V_max/K_m profile. The data suggest that cysteine is likely to be involved in catalysis and histidine in binding. Data from chemical modification show that cysteine was completely inactivated at 1.74 mM o-methylisourea, and histidine and tryptophan were modified at much higher concentrations of diethylpyrocarbonate and N-bromosuccinimide, respectively. It is suggested that the protonated cysteine works as a general base, tryptophan as a substrate binding residue and histidine as a oxygen binding residue.     

鲫鱼水解条件的研究

对鲫鱼水解条件进行研究,考察了各因素对水解效果的影响,通过正交实验确定木瓜蛋白酶水解鲫鱼的最佳条件.结果表明,鲫鱼的最佳水解条件为:pH值7.0,酶用量为1600U/g,底物浓度为4%,在温度为60℃,酶解4h.在此条件下鲫鱼的水解度可达到24.54%.     

柱层析法纯化番茄红素的研究

用氧化铝作柱层析固定相，三步洗脱分离提纯番茄红素油树脂，得番茄红素晶体，经高压液相色谱检测知番茄红素的纯度为90％，同时还得到β-胡萝卜素、叶黄素等产品。     

树脂法提取纯化草莓色素的研究

研究以草莓为原料、用大孔树脂提取和纯化草莓色素的工艺条件.试验结果表明,最佳的工艺条件是:提取液为0.1 mol/L的盐酸溶液;最佳树脂是AB-8树脂;最佳吸附条件是:50℃,pH=1.5的条件下吸附,吸附流速为1 BV/h;最佳解吸条件是:用pH=1.5时,50%的乙醇在室温条件下洗脱,洗脱流速为0.5 BV/h.     

大孔吸附树脂分离纯化白英果红色素的研究

比较AB-8、S-8、X-5、NKA-9、D-3520、NKA6种吸附剂对白英果红色素类化合物的吸附及脱附性能。在静态吸附试验研究的基础上,筛选出效果较好的X-5树脂进行动态试验研究。结果表明：X-5树脂在室温下对白英果红色素动态吸附-脱附较优的工艺参数为：上柱液自然pH值（5．12）,上柱速度3BV／h,溶液处理量6BV,次;脱附剂为95％乙醇,脱附剂的流速3BV／h,脱附剂用量6BV／次。此工艺条件能够满意地分离纯化白英果红色素,树脂可重复使用30次以上。     

PURIFICATION AND CHARACTERIZATION OF JERUSALEM ARTICHOKE (HELIANTHUS TUBEROSUS L) POLYPHENOL OXIDASE

Polyphenol oxidase (EC 1.14.18.1) has been purified from Jerusalem artichoke tubers by immobilized copper affinity chromatography. The enzyme is primarily an o-dihydroxyphenol oxidase with apparent K_m values of 1.9, 3.5 and 3.9 mM for chlorogenic acid, 4-methylcatechol, and catechol, respectively. Several compounds exhibited inhibitory action for the enzyme in the order of: sodium metabisulfite > sodium diethyldithiocarbamate > 2,3-naphthalenediol > thioglycollate. Multiple forms were identified by gel filtration and SDS-gradient polyacrylamide gel electrophoresis: two aggregates with apparent MW of 120 and 86 K and two monomeric subnits of 40–42 and 32–34 K, respectively. Concentration dependent association-dissociation phenomena most likely determine the multimeric state of this enzyme. While the aggregated forms exhibited specificity towards mono-, di- and polyhydroxyphenols, the low MW subunits were found active only with o-dihydroxyphenols. The isoelectric points of the various enzyme species were within the range of 4.0 to 10.0. The enzyme was found to contain appreciable amounts of associated carbohydrate material.     

EFFECTS OF PROCESSING ON ADSORPTION OF OFF-FLAVORS ONTO SOY PROTEIN1

Heats of adsorption and adsorption coefficients for the reversible adsorption of several aliphatic alcohols, aldehydes, and ketones on soy protein were measured using gas chromatography. The physical state of protein samples was changed by heating at 100°C and 121°C at moisture contents of 29%and 40%. A comparison was made with values for untreated soy isolate and sheared soy isolate, and the effects of moisture, temperature, and their interaction were examined by analysis of variance. Heats of adsorption were unchanged, but changes in adsorption coefficients of the treated samples demonstrated a significant decrease in binding that can be attributed to protein denaturation. The response depended on the interaction of moisture and temperature effects.     

大孔吸附树脂纯化刺嫩芽皂甙的工艺研究

以辽宁产刺嫩芽为试验材料,通过筛选树脂的种类、研究pH值、流速和洗脱剂浓度对吸附过程的影响,确定出大孔树脂纯化刺嫩芽皂甙的新方法.结果表明:大孔树脂AB-8对刺嫩芽皂甙的吸附量大,解析率高,纯化效果较好;最佳柱纯化条件:洗脱液pH值为8.0、洗脱流速为O.6mL/min、洗脱剂浓度为70%.将洗脱液浓缩,真空干燥即得高纯度刺嫩芽皂甙,纯度达90%以上,为纯化皂甙研究出一种好方法.