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1.
Lipid acyl hydrolase (LAH; patatin) was purified from potato tubers by ammonium sulfate fractionation followed by anion-exchange and affinity chromatography. The major protein band of 40-43 kDa on SDS-PAGE appeared to be patatin, and it stained positive for lipase activity on native PAGE. Selectivity of a Celite-immobilized potato LAH in esterification reactions with n-acyl fatty acids (FA; C4, C6, C8, C10, C12, C14, C16, and C18) and alcohol acceptors (n-propanol, 2-propanol, 1,3-propanediol, and glycerol; 1,2-propanediol was not sufficiently reactive) was studied in isooctane. Immobilized LAH was highly selective for medium chain FAs (C8/C10) with a secondary optimum for chain lengths of C14/16. Water activity (a(w)) influenced activity and FA selectivity of the enzyme. Initial rates of ester synthesis were greatest at a(w) of 0.90 for all alcohol acceptors except for glycerol, where greatest initial rates were observed at a(w) of 0.19. Immobilized LAH preparations exhibited a bell-shape pH profile with optimum activity at pH 6-7 for ester synthesis, and no effect of pH on FA selectivity was observed.  相似文献   

2.
摘要：将马铃薯(Solanum tuberosum )块茎特异蛋白patatin classⅠ基因cDNA与CaMV 35S启动子融合，构建了植物表达载体pBSSP。用电激法将表达载体导入根癌农杆菌（Agrobacterium tumefaciens）LBA4404，然后转化烟草(Nicotiana tabacum )叶片获得再生植株。经PCR、PCR-Southern、Northern杂交和蛋白质检测，证明patatin classⅠ基因cDNA已整合到烟草基因组中并在转基因植株中转录和表达。酯酰水解酶（lipid acylhydrolase, LAH）活性分析显示，转基因烟草植株的叶片中LAH活性明显提高。  相似文献   

3.
The potato (Solanum tuberosum L.) tuber storage protein, patatin, was purified to homogeneity with a molecular mass of 45 kDa. The purified patatin showed antioxidant or antiradical activity by a series of in vitro tests, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (half-inhibition concentration, IC(50), was 0.582 mg/mL) scavenging activity assays, anti-human low-density lipoprotein peroxidation tests, and protections against hydroxyl radical-mediated DNA damages and peroxynitrite-mediated dihydrorhodamine 123 oxidations. Using electron paramagnetic resonance (EPR) spectrometry for hydroxyl radical detections, it was found that the intensities of the EPR signal were decreased by the increased amounts of patatin added (IC(50) was 0.775 mg/mL). Through modifications of patatin by iodoacetamide or N-bromosuccinimide, it was found that the antiradical activities of modified patatin against DPPH or hydroxyl radicals were decreased. It was suggested that cysteine and tryptophan residues in patatin might contribute to its antioxidant activities against radicals.  相似文献   

4.
Potato starch production leaves behind a huge amount of juice. This juice is rich in protein, which might be exploited for food, biotechnological, and pharmaceutical applications. In northern Europe cv. Kuras is dominant for industrial starch production, and juice protein of freshly harvested mature tubers was fractionated by Superdex 200 gel filtration. The fractions were subjected to selected activity assays (patatin, peroxidase, glyoxalases I and II, alpha-mannosidase, inhibition of trypsin, Fusarium protease, and alcalase) and protein subunit size determination by SDS-PAGE and mass spectrometry. Proteins present in SDS-PAGE bands were identified by tryptic peptide mass fingerprinting. Protein complexes such as ribosomes and proteasomes eluted with the void volume of the gel filtration. Large proteins were enzymes of starch synthesis dominated by starch phosphorylase L-1 (ca. 4% of total protein). Five identified dimeric patatin variants (25%) coeluted with four monomeric lipoxygenase variants (10%) at 97 kDa. Protease inhibitor I variants (4%) at 46 kDa (hexamer) inhibited alcalase. Fourteen Kunitz protease inhibitor variants (30%) at 19 kDa inhibited trypsin and Fusarium protease. Carboxypeptidase inhibitor variants (5%) and defensins (5%) coeluted with phenolics. The native sizes and molecular properties were determined for 43 different potato tuber proteins, several for the first time.  相似文献   

5.
The interaction of the major potato allergen patatin, Sol t 1, with IgE was investigated on a quantitative level as a function of heat treatment at different temperatures. On the basis of a number of publications, potato is considered to be a heat-labile allergen, but the molecular explanation for this behavior was not given. In this work, heat treatment of patatin in the absence and presence of other potato proteins mimicking the proteinaceous environment of the potato was studied. Using far-UV circular dichrosim spectroscopy, tryptophan fluorescence spectroscopy, and differential scanning calorimetry, the molecular transitions during heating of patatin were investigated. It was found that as long as patatin is not aggregated, denaturation of patatin on a secondary or tertiairy folding level is reversible with only a minor effect on the IgE affinity. Aggregation of patatin results in a nonreversible unfolding and a concomitant important decrease in affinity for IgE (25-fold). Aggregation of patatin in the presence of other potato proteins results in a less condensed aggregate compared to the situation of isolated patatin, resulting in a more pronounced decrease of affinity for IgE (110-fold). It is concluded that the heat lability of patatin-IgE interaction is explained by aggregation of patatin with other potato proteins rather than by denaturation of patatin itself.  相似文献   

6.
In the present study, foam-forming and -stabilizing properties of potato proteins were studied using whipping and sparging tests. The soluble potato proteins are mainly composed of patatin and protease inhibitors. The whipping tests showed that less foam was formed from untreated patatin than from the protease inhibitors, but patatin foam was much more stable. The foam-forming properties of patatin could be strongly improved by partial unfolding of the protein. Whipping tests, at both low (0.5 mg/mL) and high (10 mg/mL) protein concentration, also indicated that foams made with an ethanol-precipitated protein isolate were more stable than those made with beta-casein and beta-lactoglobulin. More generally, it can be concluded that when proteins are used as a foaming agent, a high concentration is required, because the protein available is inefficiently used. Also, there are several variables that may all, in different ways, affect both foam formation (amount of foam, bubbles size distribution) and foam stability. These variables include the type and concentration of protein, solvent conditions (pH, I), and the method used to make the foam.  相似文献   

7.
Trypsin inhibitors (TIs), root storage proteins, were purified from sweet potato (Ipomoea batatas[L.] Lam cv. Tainong 57) roots by trypsin affinity column according to the methods of Hou and Lin (Plant Sci. 1997, 126, 11-19 and Plant Sci. 1997, 128, 151-158). A single band of 33 kDa TI was obtained by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. This purified 33 kDa TI had scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. There was positive correlation between scavenging effects against DPPH (2 to 22%) and amounts of 33 kDa TI (1.92 to 46 pmol). The scavenging activities of 33 kDa TI against DPPH were calculated from linear regression to be about one-third of those of glutathione between 5 and 80 pmol. Using electron paramagnetic resonance (EPR) spectrometry for hydroxyl radical detection, it was found that 33 kDa TI could capture hydroxyl radical, and the intensities of EPR signal were significantly decreased from 1.5 to 6 pmol of 33 kDa TI compared to those of the controls. It is suggested that 33 kDa TI, one of the sweet potato root storage proteins, may play a role as an antioxidant in roots and may be beneficial to health when it is consumed.  相似文献   

8.
The soluble potato proteins are mainly composed of patatin and protease inhibitors. Using DSC and both far-UV and near-UV CD spectroscopy, it was shown that potato proteins unfold between 55 and 75 degrees C. Increasing the ionic strength from 15 to 200 mM generally caused an increase in denaturation temperature. It was concluded that either the dimeric protein patatin unfolds in its monomeric state or its monomers are loosely associated and unfold independently. Thermal unfolding of the protease inhibitors was correlated with a decrease in protease inhibitor activities and resulted in an ionic strength dependent loss of protein solubility. Potato proteins were soluble at neutral and strongly acidic pH values. The tertiary structure of patatin was irreversibly altered by precipitation at pH 5. At mildly acidic pH the overall potato protein solubility was dependent on ionic strength and the presence of unfolded patatin.  相似文献   

9.
The composition of seed storage proteins (SSPs) in olive endosperm and cotyledon has been analyzed. Precursor forms of these proteins are made up of individual proteins, which have been purified to homogeneity and further named p1-p5 (20.5, 21.5, 25.5, 27.5, and 30 kDa, respectively). N-terminal sequences of p1 and p2 proteins displayed relevant homology to the basic subunit of the 11S family of plant SSPs (legumins). Two-dimensional polyacrylamide gel electrophoresis experiments allowed us to verify the basic character of p1 and p2 and the acidic character of p3, p4, and p5 proteins. In addition, the putative presence of highly similar isoforms or posttranslational modifications of these polypeptides was detected. As a result, a model describing the putative association of p1-p5 proteins into subunits of alpha(acidic)/beta(basic) type has been proposed. Solubility experiments have shown that the majority of these olive seed proteins from the 11S storage protein family are extracted with aqueous alcohol and only partially with water and diluted saline solutions, therefore suggesting their similarity to prolamines. Moreover, no visible differences were found in either subunit composition or 11S proteins mass among six olive cultivars examined. This result suggests that the synthesis of storage proteins is highly conserved in this plant species. By using a rabbit antiserum raised to p1 protein, the proteins have also been immunolocalized in olive seed tissues, showing that they accumulate in conspicuous protein bodies present in both the endosperm and the cotyledon.  相似文献   

10.
不同马铃薯品种用于加工油炸薯片的适宜性   总被引：4，自引：2，他引：4  
中国马铃薯品种数量繁多,品质指标差异性大,这些指标很大程度上决定了油炸薯片制品的品质,为研究不同马铃薯品种加工油炸薯片的适宜性,试验以国内外广泛种植的74个品种的马铃薯为原材料,分别测定原料的8个加工指标（水分、淀粉、还原糖、总糖、灰分、可溶性固形物、维生素C、蛋白质）及油炸薯片的4个品质指标（蛋白质、感官得分、脆性、白度）,随机选取56个样品为校正集,其余18个样品为验证集,应用相关性分析、主成分分析、逐步回归分析方法建立薯片综合评价指标与马铃薯原料加工指标X1～X8之间的回归模型,模型决定系数R2=0.607,调整后R2=0.585,F=26.815,sig.=0.000,拟合度较高,回归模型显著。同时利用验证集样品对回归模型进行验证,结论表明薯片综合评价指标预测值与真值相关系数为0.502,显著性为0.034,相关关系显著,该模型可用于实际油炸薯片综合品质评价。通过K-means聚类法对74个马铃薯品种加工适宜性进行初步划分,筛选出适宜加工薯片的品种15个,评价结果与实际应用现状相符。所建模型可应用于实际马铃薯油炸薯片加工适宜性评价,为中国马铃薯产业选择特定薯片加工品种具有一定的指导意义。  相似文献   

11.
Patatin has, so far, been considered a homogeneous group of proteins. A comparison of the isoforms in terms of structural properties or stability has not been reported. A method to obtain various isoform fractions as well as a comparison of the physicochemical properties of these pools is presented. Patatin could be separated in four isoform pools, denoted A, B, C, and D, representing 62%, 26%, 5%, and 7% of the total amount of patatin, respectively. These isoforms differed in surface charge, resulting in a different behavior on anion exchange chromatography, isoelectric focusing, native polyacrylamide gel, and capillary electrophoresis. All isoforms of the patatin family contained proteins with two molecular masses of approximately 40.3 and 41.6 kDa, respectively. The size of this difference in the molar mass (1300 Da) is on the order of one carbohydrate moiety. Despite the biochemical differences given above, no variations in the structural properties nor in the thermal conformational stability could be observed using far-ultraviolet circular dichroism, infrared, and fluorescence spectroscopy.  相似文献   

12.
The influence of genotype and environment on a soluble wheat dough liquor proteome was studied for four cultivars grown under field conditions and under hot/dry and cool/wet regimes by two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole time-of-flight mass spectrometry. Although the four cultivars had similar patterns, differences in the relative abundances of some components were observed. Similarly, some differences were observed between the control samples and the samples grown under cool/wet and hot/dry conditions. These included differences in the abundances of storage proteins belonging to the 7S globulin (vicilin-like) and alpha-globulin families and of protective proteins including members of the serpin, described as allergens, and chitinase families. A number of novel annotations were made as compared to previous work on the dough liquor of cv. Hereward, including two 19 kDa alpha-globulins, precursors of endochitinases A and C, and several polypeptides belonging to the 7S globulin (vicilin-like) family.  相似文献   

13.
In the present study emulsions were made with various potato protein preparations, which varied in protease inhibitor and patatin content. These emulsions were characterized with respect to average droplet size, plateau surface excess, and the occurrence of droplet aggregation. Droplet aggregation occurred only with potato protein preparations that contained a substantial amount of protease inhibitors and could be prevented only at pH 3. The average droplet size of the emulsions made with potato proteins appeared to be related to the patatin content of the preparation used. Average droplet size was found to be dominated by the patatin-catalyzed lipolytic release of surface active fatty acids and monoglycerides from the tricaprylin oil phase during the emulsification process. Addition of monoglycerides and especially fatty acids, at concentrations representative of those during emulsification, was shown to cause a stronger and much faster decrease of the interfacial tension than that with protein alone and to result in a drastic decrease in droplet size. The patatin used was shown to have a lipolytic activity of 820 units/g with emulsified tricaprylin as the substrate. Because of the droplet aggregating properties of the protease inhibitors, the patatin-rich potato preparations seem to be the most promising for food emulsion applications over a broad pH range, provided the lipolytic activity can be diminished or circumvented.  相似文献   

14.
The applicability of amino acid analysis for accurate quantitation of reference standard preparations of proteins has been evaluated. This approach is very useful since, in addition to absolute quantitative information, it also provides a measure of composition, partial identity, and purity in a single experiment. Comparisons with Kjeldahl nitrogen assay and/or UV measurements shows that amino acid analysis is reliable for the quantitation of small-to-medium size proteins in the molecular weight range of 6-22 kDa. For larger proteins such as immunoglobulins (150 kDa), amino acid analysis may "underestimate" the total protein concentration. These results also show the effect of recovery of individual residues on protein quantitation. As expected, the recovery of more than one stable residue could be used to calculate total protein content of samples, which is in good agreement with the results obtained by Kjeldahl nitrogen assay. However, the protein concentrations calculated from the total mass of the recovered residues appear to give relatively low estimates in almost all cases. Thus, it is concluded that amino acid analysis is an appropriate reference method only when stable residues are employed for quantitation, particularly for highly purified proteins of rDNA origin.  相似文献   

15.
In this study, antioxidant activities in free-radical-mediated oxidative systems and the genotoxic/antigenotoxic effects of two proteins with molecular mass around 17 kDa, purified from Solanum betaceum fruits (cyphomine) and Solanum tuberosum tubers (solamarine), were investigated. Both proteins inhibited uric acid formation with IC(50) values between 55 and 60 μg/mL, and both proteins were able to reduce oxidative damage by scavenging hydroxyl radicals and superoxide anion in a dose-dependent manner. Furthermore, the DPPH? reduction assay showed SC(50) values of 55-73 μg/mL. Cyphomine and solamarine were able to retain their antioxidant activity after heat treatment at 80 °C for 15 min. Allium cepa and Salmonella /microsome assays showed no genotoxic and mutagenic effects. Solamarine showed an antimutagenic effect against a direct mutagen (4-nitro-o-phenylenediamine). Consequently, the present study showed that the investigated proteins are promising ingredients for the development of functional foods with a beneficial impact on human health and an important source for the production of bioactive peptides.  相似文献   

16.
Dioscorin, the tuber storage protein of yam (Dioscorea batatas Decne), was purified successively by ammonium sulfate fractionation, DE-52 ion exchange chromatography, and Sephadex G-75 column. Two protein bands (82 and 28 kDa) were found under nonreducing conditions after SDS-PAGE; but only one band (32 kDa) was detected under reducing conditions. The first 21 amino acids in the N-terminal region of the 28 kDa form were VEDEFSYIEGNPNGPENWGNL, which was highly homologous to deductive sequence of dioscorin from cDNA of another yam species (Dioscoreacayenensis Lam) reported by Conlan et al. (Plant Mol. Biol. 1995, 28, 369-380). Hewett-Emmett and Tashian (Mol. Phylogenet. Evol. 1996, 5, 50 -77) mentioned that, according to DNA alignments, dioscorin from yam (D. cayenensis) was alpha-carbonic anhydrase (alpha-CA) related. In this report, we found that the purified dioscorin showed both CA dehydration activity using sodium bicarbonate as a substrate and CA activity staining after SDS-PAGE. A polyclonal antibody, which was raised against trypsin inhibitor (TI), a storage protein of sweet potato (Ipomoea batatas [L.] Lam var. Tainong 57), cross-reacted with dioscorin, which also showed TI activity determined by both activity staining after SDS-PAGE and trypsin inhibition determination.  相似文献   

17.
The primary structures of high molecular weight glutenin subunits (HMW-GS) of 5 Triticum durum Desf. cultivars (Simeto, Svevo, Duilio, Bronte, and Sant'Agata), largely cultivated in the south of Italy, and of 13 populations of the old spring Sicilian durum wheat landrace Timilia (Triticum durum Desf.) (accession nos. 1, 2, 3, 4, 7, 8, 9, 13, 14, 15, SG1, SG2, and SG3) were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high performance liquid chromatography/nanoelectrospray ionization mass spectrometry (RP-HPLC/nESI-MS/MS). M(r) of the intact proteins determined by MALDI mass spectrometry showed that all the 13 populations of Timilia contained the same two HMW-GS with 75.2 kDa and 86.4 kDa, whereas the other durum wheat cultivars showed the presence of the expected HMW-GS 1By8 and 1Bx7 at 75.1 kDa and 83.1 kDa, respectively. By MALDI mass spectrometry of the tryptic digestion peptides of the isolated HMW-GS of Timilia, the 1Bx and 1By subunits were identified as the NCBInr Acc. No AAQ93629, and AAQ93633, respectively. Sequence verification for HMW-GS 1Bx and 1By both in Simeto and Timilia was obtained by MALDI mass mapping and HPLC/nESI-MSMS of the tryptic peptides. The Bx subunit of Timila presents a sequence similarity of 96% with respect to Simeto, with differences in the insertion of 3 peptides of 5, 9, and 15 amino acids, for a total insertion of 29 amino acids and 25 amino acid substitutions. These differences in the amino acidic sequence account for the determined Δm of 3294 Da between the M(r) of the 1Bx subunits in Timilia and Simeto. Sequence alignment between the two By subunits shows 10 amino acid substitutions and is consistent with the Δm of 148 Da found in the MALDI mass spectra of the intact subunits.  相似文献   

18.
In this study, a protein isolate with a high solubility at neutral pH was prepared from industrial potato juice by precipitation at pH 5 in the presence of ethanol. The effects of ethanol itself and the effects of its presence during precipitation on the properties of various potato protein fractions were examined. The presence of ethanol significantly reduced the denaturation temperature of potato proteins, indicating that the preparation of this potato protein isolate should be performed at low temperature in order to retain a high solubility. In the presence of ethanol, the thermal unfolding of the tertiary and the secondary structure of patatin was shown to be almost completely independent. Even at 4 degrees C, precipitation of potato proteins in the presence of ethanol induced significant conformational changes. These changes did, however, only result in minor changes in the solubility of the potato protein fractions as a function of pH and heat treatment temperature.  相似文献   

19.
This study reports quali-quantitative analyses on isoprenoids, phospholipids, neutral lipids, phytosterols, and proteins in purified plastids isolated from fresh fruits of traditional (Donald and Incas) and high-pigment (Kalvert and HLY-18) tomato cultivars at four ripening stages. In all of the investigated cultivars, lycopene, β-catotene, lutein, and total carotenoids varied significantly during ripening. Chromoplasts of red-ripe tomato fruits of high-pigment cultivars accumulated twice as much as lycopene (307.6 and 319.2 μg/mg of plastid proteins in Kalvert and HLY-18, respectively) than ordinary cultivars (178.6 and 151.7 μg/mg of plastid proteins in Donald and Incas, respectively); differences in chlorophyll and α-tocopherol contents were also evidenced. Phospholipids and phytosterols increased during ripening, whereas triglycerides showed a general decrease. Regardless of the stage of ripening, palmitic acid was the major fatty acid in all cultivars (ranging from 35 to 52% of the total fatty acids), followed by stearic, oleic, linoleic, linolenic, and myristic acids, but their relative percentage was affected by ripening. Most of the bands detected on the SDS-PAGEs of plastid proteins were constantly present during chloroplast-to-chromoplast conversion, some others disappeared, and only one, with a molecular weight of ~41.6 kDa, was found to increase in intensity.  相似文献   

20.
Peroxidases (POD; EC 1.11.1.7) can cross-link cell wall polymers and may have an impact on the final textural quality of potato tubers. Because heat treatments are important during processing, the thermal properties of isoPODs from soluble and ionically and covalently bound fractions were studied from both potato tubers and sprouts. For both tissues, the ionically bound fraction was the most thermostable; approximately 20% of POD activity remained after a heat treatment of 10 min at 90 degrees C (for sprouts). The temperature profile of the ionically bound sprout fraction appeared to be nonlinear and suggested the presence of a very thermostable POD, which still showed activity after a heat treatment at 100 degrees C. Visualization by using isoelectric focusing confirmed the occurrence of a thermostable isoPOD with an IEP of 9.5, which displayed regeneration of activity after heat inactivation. This cationic POD was further purified by chromatography techniques, and by SDS-PAGE its molecular mass was estimated at 38 kDa.  相似文献   

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