Conformational stability of the potato serine protease inhibitor group

The thermal unfolding of potato serine protease inhibitor (PSPI), the most abundant protease inhibitor group in potato tuber, was measured using far UV CD spectroscopy, fluorescence spectroscopy, and DSC. The results indicate that the thermal as well as the guanidinium-induced unfolding of PSPI occurs via a non-two-state mechanism in which at least one stable intermediate is present. Additionally, the occurrence of aggregation, especially at low scan rates, increases the apparent cooperativity of the unfolding and makes the system kinetically rather than thermodynamically controlled. Aggregate formation seems to occur via a specific mechanism of which PSPI in a tetrameric form is the end product and which may involve disulfide interchanges.     

Tentative assignment of the potato serine protease inhibitor group as beta-II proteins based on their spectroscopic characteristics

Potato serine protease inhibitor (PSPI) is the most abundant protease inhibitor group in potato tuber. The investigated PSPI isoforms have a highly similar structure at both the secondary and the tertiary level. From the results described, PSPI is classified as a beta-II protein based on (1) the presence in the near-UV spectra of sharp peaks, indicating a rigid and compact protein; (2) the sharp transition from the native to the unfolded state upon heating (only 6 degrees C) monitored by a circular dichroism signal at 222 nm; and (3) the similarity in secondary structure to soybean trypsin inhibitor, a known beta-II protein, as indicated by a similar far-UV CD spectrum and a similar amide I band in the IR spectrum. The conformation of PSPI was shown also to be stable at ambient temperature in the pH range 4-7.5. Upon lowering the pH to 3.0, some minor changes in the protein core occur, as observed from the increase of the intensity of the phenylalanine peak in the near-UV CD spectrum.     

Relative abundance and inhibitory distribution of protease inhibitors in potato juice from cv. Elkana.

Protease inhibitors from potato juice of cv. Elkana were purified and quantified. The protease inhibitors represent ca. 50% of the total soluble proteins in potato juice. The protease inhibitors were classified into seven different families: potato inhibitor I (PI-1), potato inhibitor II (PI-2), potato cysteine protease inhibitor (PCPI), potato aspartate protease inhibitor (PAPI), potato Kunitz-type protease inhibitor (PKPI), potato carboxypeptidase inhibitor (PCI), and "other serine protease inhibitors". The most abundant families were the PI-2 and PCPI families, representing 22 and 12% of all proteins in potato juice, respectively. Potato protease inhibitors show a broad spectrum of enzyme inhibition. All the families (except PCI) inhibited trypsin and/or chymotrypsin. PI-2 isoforms exhibit 82 and 50% of the total trypsin and chymotrypsin inhibiting activity, respectively. A strong variation within the latter activities was shown within one family and between protease inhibitor families.     

Effects of pH and heat treatments on the structure and solubility of potato proteins in different preparations

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.     

Structural characterization of potato protease inhibitor I (Cv. Bintje) after expression in Pichia pastoris

In the present study the structural properties of potato protease inhibitor 1 (PI-1) were studied as a function of temperature to elucidate its precipitation mechanism upon heating. A cDNA coding for PI-1 from cv. Bintje was cloned and expressed in Pichia pastoris. Using the recombinant PI-1 it was suggested that PI-1 behaves as a hexameric protein rather than as a pentamer, as previously proposed in the literature. The recombinant protein seems either to have a predominantly unordered structure or to belong to the beta-II proteins. Differential scanning calorimetry analysis of PI-1 revealed that its thermal unfolding occurs via one endothermic transition in which the hexameric PI-1 probably unfolds, having a dimer instead of a monomer as cooperative unit. The transition temperature for the recombinant PI-1 was 88 degrees C. Similar results were obtained for a partially purified pool of native PI-1 from cv. Bintje.     

The most abundant protease inhibitor in potato tuber (cv. Elkana) is a serine protease inhibitor from the Kunitz family

The gene of the most abundant protease inhibitor in potato cv. Elkana was isolated and sequenced. The deduced amino acid sequence of this gene showed 98% identity with potato serine protease inhibitor (PSPI), a member of the Kunitz family. Therefore, the most abundant protease inhibitor was considered to be one of the isoforms of PSPI. The PSPI group represents approximately 22% of the total amount of proteins in potato cv. Elkana and is composed of seven different isoforms that slightly differ in isoelectric point. Antibodies were raised against the two most abundant isoforms of PSPI. The binding of these antibodies to PSPI isoforms and protease inhibitors from different groups of protease inhibitor in potato showed that approximately 70% of the protease inhibitors present in potato juice belong to the Kunitz family.     

Thermodynamics and structural features of the yeast Saccharomyces cerevisiae external invertase isoforms in guanidinium-chloride solutions

Recently, four external invertase isoforms (EINV1, EINV2, EINV3, and EINV4) have been isolated from S. cerevisiae. However, there is nothing known about their structural features and thermodynamics of unfolding. Since this information is essential for understanding their functioning at the molecular level as well as applicable in the food industry, we investigated guanidinium-chloride induced structural changes of the isoforms by CD and fluorescence spectroscopy. The resulting unfolding curves measured for each isoform at different temperatures were described simultaneously by a reversible two-state model to obtain the corresponding thermodynamic parameters. Here, we show that they are different for different isoforms and demonstrate that they correlate with the surface charge density of the native isoforms which follows the order EINV1 < EINV2 < EINV3 < EINV4. It appears that at physiological temperatures the thermodynamic stability of the isoforms follows the same order, while above 55 °C, the order is the opposite EINV1 > EINV2 > EINV3 ≈ EINV4. This suggests that increasing the efficiency of the food industry processes involving invertase would require the application of EINV3 and/or EINV4 at physiological temperatures and EINV1 at elevated temperatures.     

Conformational study of globulin from common buckwheat (Fagopyrum esculentum Moench) by Fourier transform infrared spectroscopy and differential scanning calorimetry

Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) were used to study changes in the conformation of globulin from common buckwheat (Fagopyrum esculentum Moench) (BWG) under various environmental conditions. The IR spectrum of the native BWG showed several major bands from 1691 to 1636 cm(-1) in the amide I' region, and the secondary structure composition was estimated as 34.5% beta-sheets, 20.0% beta-turns, 16.0% alpha-helices, and 14.4% random coils. Highly acidic and alkaline pH conditions induced decreases in beta-sheet and alpha-helical contents, as well as in denaturation temperature (Td) and enthalpy of denaturation (DeltaH), as shown in the DSC thermograms. Addition of chaotropic salts (1.0 M) caused progressive decreases in ordered structures and thermal stability following the lyotropic series of anions. The presence of several protein structure perturbants also led to changes in IR band intensities and DSC thermal stabilities, suggesting protein unfolding. Intermolecular antiparallel beta-sheet (1620 and 1681 cm(-1)) band intensities started to increase when BWG was heated to 90 degrees C, suggesting the initiation of protein aggregation. Increasing the time of the preheat treatment (at 100 degrees C) caused progressive increases in Td and pronounced decreases in DeltaH, suggesting partial denaturation and reassociation of protein molecules.     

Effects of ethanol on structure and solubility of potato proteins and the effects of its presence during the preparation of a protein isolate

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.     

Effects of protein composition and enzymatic activity on formation and properties of potato protein stabilized emulsions

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.     

Formation and stability of foam made with various potato protein preparations

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.     

Molecular structure, physicochemical characterization, and in vitro degradation of barley protein films

Barley protein films were prepared by thermopressing using glycerol as a plasticizer. The combined effects of heating temperature and amount of plasticizer interacted to determine protein conformation and, subsequently, the properties of the film matrix. The film barrier and mechanical properties were systematically investigated using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), SDS-PAGE, and protein solubility tests. These experiments demonstrated that heat treatment induced barley protein unfolding and then protein aggregation and the formation of covalent disulfide bonds to enhance film strength. Increasing the amount of plasticizer reduced protein denaturation and limited protein interactions, resulting in significantly improved film flexibility at the cost of reduced film moisture barrier property and tensile strength. In vitro degradation experiments demonstrated that barley films were resistant in gastric conditions, yet can still be completely degraded by intestinal enzymes, and they possess low cytotoxicity to Caco-2 cells. The prepared barley films have potential for development as delivery systems for gastric-sensitive bioactive compounds to the intestine for release.     

Effect of heat-induced aggregation on the IgE binding of patatin (Sol t 1) is dominated by other potato proteins

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.     

Kunitz-type serine protease inhibitor from potato (Solanum tuberosum L. cv. Jopung)

An antifungal protein, AFP-J, was purified from tubers of the potato (Solanum tuberosum cv. L Jopung) by various chromatographic columns. AFP-J strongly inhibited yeast fungal strains, including Candida albicans, Trichosporon beigelii, and Saccharomyces cerevisiae, whereas it exhibited no activity against crop fungal pathogens. Automated Edman degradation determined the partial N-terminal sequence of AFP-J to be NH2-Leu-Pro-Ser-Asp-Ala-Thr-Leu-Val-Leu-Asp-Gln-Thr-Gly-Lys-G lu-Leu-Asp-Ala-Arg-Leu-. The partially sequence had 83% homology with a serine protease inhibitor belonging to the Kunitz family, and the protein inhibited chymotrypsin, pepsin, and trypsin. Mass spectrometry showed that its molecular mass was 13 500.5 Da. This protease inhibitor suppressed over 50% the proteolytic activity at 400 microg/mL. These results suggest that AFP-J is an excellent candidate as a lead compound for the development of novel antiinfective agents.     

Comparison of Amylose Determination Methods and the Development of a Dual Wavelength Iodine Binding Technique

It has long been recognized that limitations exist in the analytical methodology for amylose determination. This study was conducted to evaluate various amylose determination methods. Purified amylose and amylopectin fractions were obtained from corn, rice, wheat, and potato and then mixed in proportion to make 10, 20, 30, 50, and 80% amylose content starch samples for each source. These samples, considered amylose standards, were analyzed using differential scanning calorimetry (DSC), high-performance size-exclusion chromatography (HPSEC), and iodine binding procedures to generate standard curves for each of the methods. A single DSC standard equation for cereal starches was developed. The standard curve of potato starch was significantly different. Amylose standard curves prepared using the iodine binding method were also similar for the cereal starches, but different for potato starch. An iodine binding procedure using wavelengths at 620 nm and 510 nm increased the precision of the method. When HPSEC was used to determine % amylose, calculations based on dividing the injected starch mass by amylose peak mass, rather than calculations based on the apparent amylose/amylopectin ratio, decreased the inaccuracies associated with sample dispersion and made the generation of a cereal amylose standard curve possible. Amylose contents of pure starch, starch mixtures from different sources with different amylose ranges, and tortillas were measured using DSC, HPSEC, iodine binding, and the Megazyme amylose/amylopectin kit. All the methods were reproducible (±3.0%). Amylose contents measured by these methods were significantly different (P < 0.05). Amylose measurements using iodine binding, DSC, and Megazyme procedures were highly correlated (correlation coefficient >0.95). DSC and traditional iodine binding procedures likely overestimated true amylose contents as residual butanol in the amylose standards caused interference. The modified two-wavelength iodine binding procedure seemed to be the most precise and generally applicable method. Each amylose determination method has its benefits and limitations.     

超声波和环氧丙烷改性马铃薯全粉的制备与性能研究

为开发优良马铃薯全粉资源,本试验利用超声波和环氧丙烷分别处理马铃薯全粉,采用扫描电镜、差示量热扫描、傅里叶变换红外光谱和X-射线衍射等方法,探究改性全粉在加工性能、热力学和结构性质等方面的差异。结果表明,与未改性马铃薯全粉(PF)相比,超声波改性马铃薯全粉(UF)和羟丙基马铃薯全粉(HF)的溶解度分别显著提高至18.33%和17.33%,析水力均显著降低至0.28%,透明度显著提高至76.90%和90.14%,碘蓝值(8.10和6.61)也显著下降(P <0.05)。3种马铃薯全粉衍射峰峰形相似,反应主要发生在无定型区,电镜扫描表明,较PF而言,UF和HF碎片和絮状形态消失,均出现了明显的黏结现象。综上所述,2种改性处理均提高了马铃薯全粉的结晶度、溶解度、冻融稳定性、透明度和热稳定性,且降低了全粉的碘蓝值,在一定程度上提高了马铃薯全粉的加工品质。本研究为马铃薯全粉的开发利用提供了理论依据。     

Inhibition of Angiotensin converting enzyme I caused by autolysis of potato proteins by enzymatic activities confined to different parts of the potato tuber

Autolysis of protein isolates from vascular bundle and inner tuber tissues of potato (Solanum tuberosum) enhanced the inhibition of the angiotensin converting enzyme I (ACE), a biochemical factor affecting blood pressure (hypertension). The physiological age of the tuber affected the strength of ACE inhibition, the rate of its increase during autolysis, and the tuber tissue where ACE inhibition was most pronounced. The highest inhibitory activities (50% reduction in ACE activity achieved following autolysis at a protein concentration of 0.36 mg mL (-1)) were measured in tubers after 5-6 months of storage prior to sprouting. The rate of ACE inhibition was positively correlated with protease activity in tuber tissues. Amendment of the autolysis reaction with protein substrates from which bioactive ACE-inhibitory peptides may be released, for example, a purified recombinant protein or a concentrate of total tuber proteins, also enhanced ACE inhibition. Many tuber proteins including aspartic protease inhibitors were degraded during autolysis. The data provide indications of differences in the enzymatic activities confined to different parts of the potato tuber at different physiological stages. Results suggest that native enzymes and substrate proteins of potato tubers can be utilized in search of dietary tools to manage elevated blood pressure.     

Physicochemical and structural properties of maize and potato starches as a function of granule size

Chemical composition, molecular structure and organization, and thermal and pasting properties of maize and potato starches fractionated on the basis of granule size were investigated to understand heterogeneity within granule populations. For both starches, lipid, protein, and mineral contents decreased and apparent amylose contents increased with granule size. Fully branched (whole) and debranched molecular size distributions in maize starch fractions were invariant with granule size. Higher amylose contents and amylopectin hydrodynamic sizes were found for larger potato starch granules, although debranched molecular size distributions did not vary. Larger granules had higher degrees of crystallinity and greater amounts of double and single helical structures. Systematic differences in pasting and thermal properties were observed with granule size. Results suggest that branch length distributions in both amylose and amylopectin fractions are under tighter biosynthetic control in potato starch than either molecular size or amylose/amylopectin ratio, whereas all three parameters are controlled during the biosynthesis of maize starch.     

Gelatinizing,Pasting, and Gelling Properties of Potato and Amaranth Starch Mixtures

Physicochemical properties of mixtures of native potato and native amaranth (Amaranthus cruentus), heat‐moisture treated (HMT) potato and heat‐moisture treated amaranth, cross‐linked potato and cross‐linked amaranth, native potato and heat‐moisture treated amaranth, and heat‐moisture treated potato, and native amaranth were tested at different ratios. Two peaks were noticed in the pasting curves when large differences of swelling factor and amylose leaching existed between individual components in the mixture. It seems that amylose leaching from one starch in a mixture may affect the swelling and much of the granular break down of the other. The mixtures showed stabilities in hot pastes that were higher than the less stable components in a mixture. Some mixtures such as HMT potato and native amaranth showed very specific nonadditive pasting behavior. Mixing 10% of native amaranth to HMT potato starch caused a large reduction of peak viscosity and cold paste viscosity, resulting in a very soft gel. In the differential scanning calorimeter, each component of a mixture gelatinized independently, showing two peaks corresponding to the individual components. When transition temperatures of both components were similar in DSC, the result was a single endotherm. Dramatic changes of pasting and subsequent gel properties resulted when thermal transition of the two components occurred in the same temperature range. Retrogradation enthalpies as measured by DSC were between the two individual components in all tested mixtures.     

Molecular properties and activities of tuber proteins from starch potato cv. Kuras

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.