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1.
The inhibition of mushroom tyrosinase by cucumber extracts was evaluated. The inhibitory effect was measured by both polarographic and spectrophotometric methods. The commercial aldehyde, trans,cis-2,6-nonadienal, described as a major volatile compound of cucumber, was characterized as a noncompetitive inhibitor against 4-tert-butylcatechol oxidation by mushroom tyrosinase. The K(I) obtained was 3.4 mM. Polyphenol oxidase (PPO) activity was not detected in cucumber skin extracts. However, the presence of PPO was revealed by Western blot; a single band was found with a M(r) of 53 kDa. These results support the assumption that the enzyme PPO is present in the cucumber skin, but its activity is inhibited. Peroxidase (PO) was also found in cucumber skin extracts. This enzyme was detected in the soluble fraction but not in the membrane fraction. The kinetic characterization of PO was carried out. Native isoelectric focusing revealed several acidic PO isoenzymes with a pI in the range between 5 and 6, a basic isoenzyme, and one principal neutral isoenzyme of pI = 7.2.  相似文献   

2.
Polyphenol oxidase (PPO) has been extracted from beet root, in both soluble and membrane fractions. In both cases, the enzyme was in its latent state, and it was activated by sodium dodecyl sulfate. PPO was purified to apparent homogeneity. The soluble PPO purification was achieved by hydrophobic interaction chromatography and gel filtration chromatography, with apparent molecular mass of 55 kDa. The membrane PPO purification was achieved by anion exchange chromatography and gel filtration with apparent molecular mass of 54 kDa. A totally denaturing SDS-PAGE indicated the presence of a single polypeptide with an apparent molecular mass of 60 kDa for both fractions, with the band also revealed by Western blot. A partially denaturing SDS-PAGE stained a single active 36 kDa band for both fractions. Under native isoelectric focusing, a major acidic band of pH 5.2 was detected in both fractions. Kinetic characterization of PPO on the natural substrate l-dopa was carried out.  相似文献   

3.
Color is a key quality trait of wheat products, and polyphenol oxidase (PPO) is implicated as playing a significant role in darkening and discoloration. In this study, total and soluble PPO activities were characterized in whole kernel assays and bran extracts. In whole kernel assays similar to AACC Approved Method 22–85, four wheat cultivars were ranked the same for both total and soluble (leached) PPO activity with L‐DOPA (diphenol) as the substrate. Total kernel PPO activity was much greater than soluble PPO activity in three hexaploid wheat cultivars, indicating that insoluble PPO was the major contributor to kernel PPO measurements. Tyrosine (monophenol) was an excellent PPO substrate in kernel assays as expected but had no activity as a substrate for soluble PPO. However, soluble PPO activity with tyrosine was activated by the addition of the diphenols chlorogenic acid and caffeic acid. When PPO was assayed in homogenized bran, 89–95% of total PPO activity remained insoluble, associated with the bran particles. The kernel assay detected <2% of PPO measured in an equivalent amount of homogenized bran. However, total PPO activity was 2‐fold higher in Klasic than in ID377s, both when measured in the kernel assay and in homogenized bran, indicating that the kernel assay was an accurate predictor of relative total extracted PPO activity in these two cultivars. Adding detergents (0.1% SDS plus 0.2% NP‐40) to the bran extraction buffer increased both soluble and insoluble PPO activity. Results indicate that relative PPO activities among wheat cultivars are similar in whole kernel and kernel leachate assays, and that the predominant insoluble fraction of PPO, which is relatively uncharacterized, may be largely responsible for wheat product discoloration.  相似文献   

4.
There is a growing interest to develop environmentally friendly surfactants for utilization with supercritical carbon dioxide (scCO2), which is a "green" solvent with many industrial applications. The goal of the present work was to separate the commonly used soybean lecithin into a phospholipid-rich fraction, acetylate this fraction, and then test its solubility in scCO2 to gauge its suitability as a surfactant for potential scCO2-based applications. Soybean lecithin was first purified by fractionation using acetone and ethanol and then acetylated with acetic anhydride. The acetylated lecithin was further purified by fractionation with acetone to separate the acetylated fraction from the nonacetylated fraction. High-performance liquid chromatography and electrospray ionization mass spectrometry were utilized to characterize these fractions. The various acetylated phospholipid fractions were then tested for solubility in scCO2 under various pressures and temperatures using both a cloud-point and a Fourier transform infrared apparatus. Acetylation was found to increase the solubility of the phospholipids in scCO2, and N-acetylated phosphatidylethanolamine (NAc-PE) was found to be the most soluble component of the acetylated phospholipids.  相似文献   

5.
The effect of aluminum (Al) on phosphate homeostasis and induction of acid phosphatase activity and isoenzymes was analyzed in root tips of Lotus japonicus. The rapid uptake of phosphate as well as the decrease of soluble inorganic phosphate in tissues suggested, that the presence of Al in root apoplast causes precipitation of the most of absorbed phosphate. Acid phosphatase (ACP) isoenzymes are affected differentially during low pH and Al stress and both Al induced increases and decreases of ACP isoenzyme activities were observed.  相似文献   

6.
Studies were conducted to compare polyphenol oxidase (PPO) specific activities in various milling fractions of a variety of wheat cultivars and determine the levels of activities in a number of cultivars from different localities and harvesting seasons. Substrate specificities were also investigated. Bran was singled out as the richest source of PPO activity, which may also influence the activity in the other milling fractions that are known to have some proportion of bran content. We showed by gel electrophoresis and spectrophotometrically that the protein responsible for PPO activity apparently exists as a single isoform in bran and that the observed enzyme activity is likely to be a tyrosinase type, not a laccase or peroxidase. The specific activity was not significantly different between the reduction shorts and break shorts from the same cultivar, indicating a similar level of bran contamination in these fractions. Very low levels of PPO activity were recorded in the flour of all cultivars studied. Bran was used, therefore, to determine the varietal differences in the PPO activities in a number of cultivars from different localities and seasons of harvest. Results showed that the most significant determinant of PPO activity was the genotype, and this may be influenced by seasonality. We also determined that, apart from substrate preferences by the PPO enzyme, some phenolic acids actually inhibit PPO. Furthermore, we found that bran of some cultivars extracted with acidified methanol inhibited PPO activity substantially, whereas other extracts had less inhibitory properties. Thus, these unknown compounds in wheat may inhibit endogenous PPO activity.  相似文献   

7.
This paper analyzes the kinetic and structural characteristics of polyphenol oxidase (PPO) from peach cv. Catherina. The PPO was obtained in a latent state in both the soluble and membrane-bound forms, and both forms were activated by acid shock and the detergent SDS. Plant defense is the main function assigned to PPO, which would be activated by the acid environment resulting from tissue damage. On the other hand, it has been suggested that, physiologically, the role played by SDS may be fulfilled by lipids. Native isoelectric focusing identified two acid isoforms of pI 5.7 and 5.8 for the soluble form and one isoform with pI 5.7 for the membrane-bound form. A partially denaturing SDS-PAGE revealed two very close bands of activity in both cases, but the Western blot performed on a totally denaturing SDS-PAGE, using polyclonal antibodies against bean PPO, revealed a single band in the membrane-bound fraction with a molecular mass of 60 kDa.  相似文献   

8.
The impact mechanism of endoxylanases in straight dough bread-making was investigated in fractionation-reconstitution experiments. To this end, two European flours with different bread-making characteristics were separated in gluten, prime starch, a squeegee fraction (SQF), and a water-extractable fraction. Whereas the former fractions contained negligible levels of arabinoxylan (AX), the latter contained, respectively, most of the water-unextractable AX (WU-AX) and all of the water-extractable AX (WE-AX). In vitro modification with a Bacillus subtilis endoxylanase allowed controlled solubilization of WU-AX from SQF and controlled degradation of solubilized AX and WE-AX from the water-extractables. It followed from bread-making tests with the reconstituted flours that endoxylanases exert positive loaf volume effects in bread-making by lowering the concentration of WU-AX and increasing that of total soluble AX. Limited degradation of WE-AX and significant breakdown of solubilized AX by endoxylanases, on the other hand, resulted in volume losses when compared to their nondegraded counterparts. The volume increasing effects of endoxylanases are therefore related to their ratio of solubilizing to degrading activity and thus to their substrate specificity.  相似文献   

9.
The specific antiradical activity against the hydroxyl radical of the water soluble components in green and dark roasted Coffea arabica and Coffea robusta coffee samples, both in vitro by the chemical deoxiribose assay and ex vivo in a biological cellular system (IMR32 cells), were determined. All the tested coffee solutions showed remarkable antiradical activity. In the deoxiribose assay, all the tested solutions showed similar inhibitory activity (IA%) against the sugar degradation (IA values ranged from 45.2 to 46.9%). In the cell cultures, the survival increase (SI%) ranged from 197.0 to 394.0% with C. robusta roasted coffee being significantly more active than the other samples. The coffee solutions underwent dialysis (3500 Da cutoff membrane) to fraction their components. In both systems, the dialysates (MW < 3500 Da) either from green or roasted coffee, showed antiradical activity, while the only retentates (MW > 3500 Da) from the roasted coffee samples were active. The preparative gel-filtration chromatography of roasted coffee C. robusta dialysate gave three fractions active in the biological system, all containing chlorogenic acid derivatives. The most active fraction was found to be that containing the 5-O-caffeoilquinic acid, which shows a linear relation dose-response ranging from 0.02 to 0.10 mM. The results show that both green and roasted coffee possess antiradical activity, that their more active component is 5-O-caffeoyl-quinic acid, and moreover that roasting process induces high MW components (later Maillard reaction products, i.e., melanoidins), also possessing antiradical activity in coffee. These results could explain the neuroprotective effects found for coffee consumption in recent epidemiological studies.  相似文献   

10.
Polyphenol oxidase (PPO) was extracted from five Sicilian varieties of tomato fruit [Pizzutello, Naomi (Hazera), F1 PS212 (Peto seed), Rosa Maletto, and PO228] and assayed with a method using 3-methylbenzothyazolinone hydrazone (MBTH) as chromophore coupling agent. 3,4-Dihydroxyphenylacetic acid was chosen for tomato PPO activity determination. The tomato PPO had maximum activity at pH 4.8. The pH of juice in ripe fruits is between 4.1 and 4.4, a range in which PPO relative activity is between 74 and 87%. The optimum temperature of activity for tomato PPO was 40 degrees C; the enzyme showed a good relative activity (55% of the maximum) at cold-storage temperature (4 degrees C). PPO retained 82% relative activity at an NaCl concentration of 0.1 M; at higher concentrations the PPO became gradually inactivated. The commercial variety Naomi is more susceptible to enzymatic browning than the local varieties Pizzutello, Rosa Maletto and PO228, due to higher PPO activity levels. This result confirms the suitability of these local tomato varieties to national markets. Results from storage tests seem to relate PPO activity with color changes associated with browning and lycopene degradation, because lycopene is an antioxidant agent that reconstitutes the polyphenols oxidized by the action of PPO.  相似文献   

11.
Polyphenol oxidases (PPOs) from several plant species, including wheat, have been implicated in undesirable brown discolorations of food products. It has been demonstrated that these enzymes are often present in a latent form or are membrane‐associated, necessitating detergent or other treatments to obtain fully active preparations. Here, the influence of different detergents on wheat meal and flour PPOs was investigated. Extraction in presence of 50 mM SDS led to a 5‐ to 15‐fold increase in PPO activity, making quantitative assays in flour from low‐PPO lines more robust. Among a series of additional nonionic, anionic, and cationic detergents tested, only n ‐lauroylsarcosine increased extractable PPO activity to a degree comparable to that of SDS. Additional experiments suggested that a large fraction of wheat meal PPOs may be membrane‐associated and that SDS is able to activate PPOs extracted from high‐activity but not from low‐activity wheat lines. PPO activities assayed after SDS extraction of meal and flour were highly correlated with each other and with activity determined in whole (intact) kernels in absence of SDS. Correlation coefficients between PPO activities measured with all these methods and noodle brightness were about equal, indicating that activities assayed after SDS extraction are useful for germplasm screening and quality prediction.  相似文献   

12.
Six Manitoba fruits were analyzed for their phytochemical content and antioxidant activity in order to increase their production and marketability. The major proanthocyanidins (flavanols) present in whole fruit, juice, and pulp of strawberry, Saskatoon berry, raspberry, wild blueberry, chokecherry, and seabuckthorn were measured. The extraction and purification were facilitated using flash column chromatography, while separation and identification were accomplished by using HPLC and LC-MS techniques. The total proanthocyanidin contents varied from 275.55 to 504.77 mg/100 g in the whole fruit samples. Raspberry contained the highest content, and seabuckthorn showed the lowest content of total flavanols. The highest concentration of proanthocyanidin in juice was found in Saskatoon berry (1363.34 mg/100 mL) and the lowest value in strawberry (622.60 mg/100 mL). HPLC and LC-MS results indicated that epicatechin was the most abundant flavanol followed by B2 in the berry samples, while no catechin or B1 was detected in these fruits. A series of oligomers and polymers were detected in all samples. The recovery percentage was obtained from the ratio of the unspiked samples to the area of spiked samples. Monomers, dimers, oligomers, and polymers gave recovery ranges of 83-99%. The lipophilic and hydrophilic antioxidant capacities of whole fruit, juice, and pulp extracts were measured by the oxygen radical absorbance capacity (ORAC) procedure. In whole fruits, the ORAC values varied from 135 to 479 mg/100 g TE in the MeOH fraction. The corresponding ORAC values varied from 115.30 to 733.15 mg/100 g for the acetone fraction. In juice, all berries showed the same antioxidant capacity (P > 0.05) (133.0-312.0 mg/100 g) in the MeOH fraction, with the exception of raspberry (603.0 mg/100 g). Overall, MeOH fractions mainly contained monomers and dimers with smaller amounts of oligomers and polymers when compared to the acetone fractions. Acetone fractions mainly contained polymers and some oligomers. Although acetone fractions contained a higher quantity of total proanthocyanidins, their antioxidant capacities were lower than MeOH fractions.  相似文献   

13.
Summary Extracellular benzoyl-l,-argininamide (BAA)-hydrolysing protease was extracted with neutral pyrophosphate from an arable soil and fractionated by membrane ultrafiltration. There were three fractions: A1 (molecular weight > 105), AII (molecular weight 104–105), and R (molecular weight < 104). Analytical isoelectric focusing (IEF) of the fractions was carried out on polyacrylamide gels with a restricted pH gradient of 4.0 to 5.0. Two extracellular proteases characterized soil extract E, with one peak (Ip 4.44) bound to a large amount of humic matter and the other (Ip 4.06) bound to a small amount of humus. Following ultrafiltration, the humus-enzyme complex of extract E (Ip 4.44) split into the fractions AI, AII, and R, and was displaced at Ip values that depended on the electrophysical properties of bound organic matter, whereas that at Ip 4.06 was completely removed from the extract E and accumulated only into the low-molecular-weight fraction R. High recoveries of absolute activity were obtained after IEF of the whole extract E, and fractions AII and R, but only about 50% was recovered from fraction AI.It appears that humic substances have reversible inhibitory effects on extracellular proteases, since the maximum recoveries of activity were obtained from fractions where high amounts of protease non-active organic matter had been removed by IER IEF was able to fractionate humic molecules and purify humic-protease complexes on the basis of smaller differences in Ip, and even smaller differences of 0.05 pH units. The present results show that BAA hydrolysing proteases were preferentially linked with a specific class of humic molecules with an Ip of close to 4.44.Joint program CNR (Italy) — C.S.I.C. (Spain), no. 7, 1985–1986. This paper is part of the doctoral thesis of Prof. M. Bonmati  相似文献   

14.
不同处理对水稻病程相关蛋白和过氧化物酶的影响   总被引：3，自引：0，他引：3  
以稻白叶枯菌 (Xanthomonasoryzaepv.oryzae.XOO)弱毒株 75 1和Ni(NO3) 2 作为诱导因子处理水稻幼苗。表明不同处理均在第 2d表现出最大诱导效果 ,Ni(NO3) 2处理后的相对诱导效果达 50 8% ;可溶性蛋白质IEF和SDS PAGE显示 ,诱导蛋白一般在诱导后 2 4h及 48h出现。Ni(NO3) 2 处理后 48h ,SDS PAGE发现上位叶有新增谱带 (MW分别为 54 95和 3 2 3 6kD) ;IEF显示上位叶出现pI5 1谱带 ,诱导叶出现pI5 2 6谱带 ;POD同工酶诱导叶新增加 4条酶带 (Rf分别为 0 62、0 63、0 64及0 73 ) ,上位叶新增加两条酶带 (Rf为 0 63和 0 64)。不同诱导因子处理后 ,可溶性蛋白质含量增加 ,诱导后 2 4～ 48h增幅最大 ;还显示不同诱导因子对同一植物诱导PRP的效力不同  相似文献   

15.
Different nitrogen (N) fractions from 14 sediments from the shallow lakes in the middle and lower reaches of Yangtze River area before and after N release experiments were investigated, and the content of different N fractions, and contribution of different N fractions to the N released from sediments were also studied. Ion-exchangeable form (IEF–N), carbonate form (CF–N), iron-manganese oxide form (IMOF–N) and organic matter-sulfide form (OSF–N) accounted for 2.72～17.67%, 0.47～4.43%, 1.18～3.49% and 31.05 to 71.61% to total N, respectively. The N released was higher than 50% from IEF–N, approximately 35% from OSF–N, 6 and 8% from CF–N and IMOF–N on the average. Approximately 27.32～70.02% of IEF–N, 10.37～32.11% of CF–N, 11.37～33.43% IMOF–N and 2.02～8.19% OSF–N were released. For the sediments that were slightly polluted, IEF–N was the main N fraction that may be released and its contribution to total N released was more than 63.07%, for the sediments that TN was higher than 3,540.27 mg·kg?1, OSF–N would become the main N fraction that can be released and its contribution to total N released was more than 45%.  相似文献   

16.
Influence of the amount of sulfur supplied on the soluble leaf protein, the synthesis and the specific activity of some enzymes of young sunflower plants The influence of a sulfur supplement on the pattern of leaf proteins, soluble in 0.1 M phosphate buffer, pH 7.5, was studied in experiments with young sunflower plants. The leaves were harvested after growing periods of 14 and 21 days, respectively. In addition, the electrophoretic distribution of isoenzymes of some enzyme systems and their specific activities have been analyzed. 1. Plant fresh weight was not affected by the level of sulfur supply, although symptoms of S-deficiency were observed in the low S-treatment. 2. In contrast to this finding, a clear effect of S-deficiency on the electrophoretic patterns of the leaf proteins was detected. This was specially the case in plants. grown for three weeks at a low sulfur level. Increased contents of low molecular protein fractions were found, whereas the relative amounts of high molecular proteins were reduced. 3. In plants, which were kept S-deficient, the number of isoenzyme components was lower in comparison to plants, which were adequately supplied with sulfur. The isoenzymes of malate dehydrogenase and acid phosphatase, however, were synthesized to about the same degree in both S-treatments. 4. The specific activities of most of the enzymes studied in these investigations were lower in the plants, grown at S-deficiency in comparison with the control. These findings were particularly evident after a growth period of 3 weeks. The specific activities of glutaminate dehydrogenase, succinate dehydrogenase and a-ketoglutarate dehydrogenase were not significantly influenced by the level of S-supply. The specific activities of acid phosphatase and malate dehydrogenase were relatively high even in those plants, which were treated with low amounts of sulfur.  相似文献   

17.
重金属对水稻过氧化物酶同功酶的影响   总被引：15，自引：2，他引：13  
对重金属胁迫条件下扬稻 6号体内POD活性测定和POD同功酶的聚丙烯酰胺浓度梯度凝胶电泳分析表明 ,0 0 5mmol L的Cu2 +、Cd2 +和Hg2 +诱导扬稻 6号叶片和根系POD同功酶活性较对照提高 ,但≥ 0 5mmol L的Cu2 +、Cd2 +和Hg2 +抑制叶片分子量较小的POD同功酶的表达。 0 5～ 1 0mmol L的Cu2 +、Cd2 +和Hg2 +强烈抑制根系POD同功酶的表达。 0 0 5～1 0mmol L的Cu2 +、Cd2 +和Hg2 +显著诱导穗中POS同功酶的表达。  相似文献   

18.
In this paper lipoxygenase (LOX) presence was investigated in coffee berries to determine its involvement in lipid degradative metabolism of plants grown in organic and conventional cultivations. An immunochemical analysis has evidenced a ca. 80 kDa protein, cross-reacting with an anti-LOX antibody, only in the pulp fraction of berries obtained from plants of both cultivations. LOX activity in this fraction could be monitored either as conjugated diene formation or reaction products (determined by HPLC) and was mainly associated with a heavy membrane fraction (HMF, enriched in tonoplast, endoplasmic reticulum, plasma membrane, and mitochondria) and a light membrane fraction (LMF, enriched in plasma membrane and endoplasmic reticulum, with low levels of tonoplast and mitochondria). The LOX activity of LMF from berries of both cultivations showed an optimum at pH 8.0. The HMF exhibited a different activity peak in samples from conventional (pH 8.0) and organic (pH 5.5) cultures, suggesting the presence of different isoenzymes. These findings were also confirmed by variation of the ratio of 9- and 13-hydroperoxides in organic (1:1) and conventional cultivations (1:10), indicating that the organic one was subjected to an oxidative stress in the coffee pulp fraction leading to the expression of an acidic LOX. Such de novo synthesized LOX activity could be responsible for the production of secondary metabolites, which may interfere with the organoleptic profile of coffee.  相似文献   

19.
In the present study, we successively extracted the Pu-erh tea with acetone, water, chloroform, ethyl acetate, and n-butanol, and the extracts were then isolated by column chromatography. Our study demonstrates that the Pu-erh tea ethyl acetate extract, n-butanol extract, and their fractions had superoxide anion and hydroxyl radical scavenging activity: fractions 2 and 8 from the ethyl acetate extract and fractions 2, 4, and 5 from the n-butanol extract showed protective effects against hydrogen peroxide-induced damage in human fibroblast HPF-1 cells and increased the cells' viability under normal cell culture conditions. In addition, it is found that these fractions, except fraction 5 from the n-butanol extract, decreased the accumulation of intracellular reactive oxygen species in hydrogen peroxide-induced HPF-1 cells. Interestingly, the antioxidant effect of fraction 8 from the ethyl acetate extract on the above four systems was much stronger than that of the typical green tea catechin (-)-epigallocatechin-3-gallate, but there were almost no monomeric polyphenols, theaflavins, and gallic acid in fraction 8.  相似文献   

20.
土壤多酚氧化酶性质研究及意义   总被引：27，自引：2，他引：27  
土壤多酚氧化酶是一类以铜、锰为活性中心的氧化还原酶。本文以滨江带生态湿地土壤为材料,系统研究了土壤多酚氧化酶活性随着干湿状态、溶解氧、pH值、温度而变化的情况。结果表明,湿土壤比风干土壤和烘干土壤活性高;氧气充足时活性高于缺氧时活性;多酚氧化酶活性在强酸强碱条件下失活,pH值(9~11)或温度45℃时活性最高。通过对土壤多酚氧化酶性质研究,对一些现象进行了初步解释,如荷花出淤泥而不染成因;为加快土壤多酚氧化酶在环境修复中应用和开发它的广阔应用前景奠定基础。  相似文献   

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