Suikerstructuren op Phytophthora-glyco-eiwitten
Krol, A.R. van der - \ 2007
aardappelen - solanum tuberosum - phytophthora infestans - aardappeleiwit - fysicochemische eigenschappen - glycogeen - ziektepreventie - potatoes - solanum tuberosum - phytophthora infestans - potato protein - physicochemical properties - glycogen - disease prevention
Onderzoek naar mogelijk nieuwe aanknopingspunten ter bestrijding van Phytophthora te vinden, welke eiwitten de plant uitscheidt en welke eiwitten tijdens infectie door P.infestans worden uitgescheiden
Occurrence and physico-chemical properties of protease inhibitors from potato tuber (Solanum tuberosum)
Pouvreau, L.A.M. - \ 2004
Wageningen University. Promotor(en): Fons Voragen, co-promotor(en): Harry Gruppen; G.A. van Koningsveld. - [s.l.] : S.n. - ISBN 9789085040231 - 157
aardappelen - aardappeleiwit - proteïnaseremmers - fysicochemische eigenschappen - potatoes - potato protein - proteinase inhibitors - physicochemical properties
Potato proteins are present in a by-product of the potato starch industry, the so-called potato juice. They are recovered by an acidic heat-treatment of the potato juice. This results in a completely irreversible precipitation of the proteins, with a complete loss of functionality for food applications. This explains that so far potato proteins are only used in low-value applications such as feed.
The aim of this research was to investigate and understand the thermal unfolding behaviour of potato protease inhibitors. The first step was to make an inventory of the relative abundance and inhibitory activity of the protease inhibitors present in potato tuber. The second step was to investigate the stability of the most abundant and, therefore, representative protease inhibitors in potato juice, as a function of temperature and pH. This information should be of help to understand the mechanism of the irreversible precipitation occurring in industrial processes, thereby creating possibilities to obtain soluble and/or biologically active potato proteins that can be used in food and pharmaceutical applications.
In contrast to patatin, the most abundant protein in potato juice, the protease inhibitors are a more heterogeneous group of proteins. In chapter 2, protease inhibitors from potato juice (cv. Elkana) were purified and quantified. The protease inhibitors represent approximately 50 % of the total soluble proteins in potato juice. They were classified in seven different groups: Potato Inhibitor I (PI-1), Potato Serine Protease Inhibitor (PSPI formerly called 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' (OSPI). The most abundant groups were the PSPI and PCPI, representing 22 and 12 % of the total protein in potato juice, respectively. In chapter 3, the gene of the most abundant protease inhibitor in potato (cv. Elkana ) was isolated and sequenced . The amino acid sequence deduced from this gene showed 98 % identity with Potato Serine Protease Inhibitor (PSPI), a member of the Kunitz-type inhibitor, and not, as was assumed in literature, with PI-2. It can be concluded that, in cv. Elkana , not PI-2 but PSPI is the most abundant group of proteases inhibitors. Potato protease inhibitors inhibit an extraordinary broad spectrum of enzymes. All the groups (except PCI) inhibited also trypsin and/or chymotrypsin. PSPI isoforms exhibit 82 and 50 % of the total trypsin and chymotrypsin inhibiting activity, respectively. A strong variation within the activities was observed within one group as well as between the protease inhibitor groups. 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 showed that presumably approximately 70% of the protease inhibitors present in potato juice belongs to the Kunitz-type inhibitor.
In chapter 4, PSPI isoforms were shown to have a highly similar structure at both the secondary and tertiary level. From the results described, PSPI is classified as aβ-II protein based on: (1) the presence of sharp peaks in the near UV spectra, indicating a rigid and compact protein, (2) the sharp transition from the native to the unfolded state upon heating (only 6°C) and (3) the similarity in secondary structure to soybean trypsin inhibitor, a knownβ-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 to 7.5. Upon lowering the pH to 3.0, only 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.
In chapter 5, the unfolding behaviour of PSPI was studied in detail 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 two parts of the protein unfold more or less independently. 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.
In chapter 6, the conformational stability of Potato Cysteine Protease Inhibitor (PCPI), the second most abundant protease inhibitor group in potato tuber, was investigated, at ambient temperature and upon heating, using far and near UV CD spectroscopy, fluorescence spectroscopy and DSC. The PCPI isoforms investigated were shown to have a highly similar structure at both the secondary and tertiary level. PCPI isoforms show structural properties similar to those of Potato Serine Protease Inhibitor and the Kunitz-type soybean trypsin inhibitor. Therefore, PCPI isoforms are also classified as members of theβ-II protein subclass. Results show that the thermal unfolding of PCPI isoforms also does not follow a two-state mechanism, and that at least one intermediate is present. The occurrence of this intermediate is most apparent in the thermal unfolding of PCPI 8.3, as indicated by the presence of two peaks in the DSC thermogram. Additionally, the formation of large aggregates (>100 kDa), especially at low scan rates, increases the apparent cooperativity of the unfolding and makes the system again kinetically rather than thermodynamically controlled.
In chapter 7, the structural properties of potato protease inhibitor 1 (PI-1) were studied as a function of temperature, in order 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 literature. The recombinant protein seems to have either a predominantly unordered structure or also belongs to theβ-II proteins. DSC 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 o C. Similar results were obtained for a partially purified pool of native PI-1 from cv. Bintje .
In chapter 8, the common structural characteristics of potato protease inhibitors from different groups are discussed and compared to those of soybean trypsin inhibitor, a Kunitz-type inhibitor. This leads to the conclusion that all these proteins belong to theβ-II protein sub-class and have a more or less commonβ-trefoil fold. A scheme is introduced, defining the main characteristics, which should be of help to classify any unknown protease inhibitor in the correct family. Finally, the pH and the thermal stability of the protease inhibitors are discussed in relation with the aggregation and precipitation processes occurring in industrial potato juice.
Physico-chemical and functional properties of potato proteins
Koningsveld, G.A. van - \ 2001
Wageningen University. Promotor(en): P. Walstra; A.G.J. Voragen; M.A.J.S. van Boekel; H. Gruppen. - S.l. : S.n. - ISBN 9789058084446 - 147
aardappelen - aardappeleiwit - chemische precipitatie - oplosbaarheid - chemische structuur - schuimen - emulgering - potatoes - potato protein - chemical precipitation - solubility - chemical structure - foaming - emulsification
Key words: potato proteins, patatin, protease inhibitors, solubility, structure, pH, temperature, ethanol, ionic strength, phenolic compounds, foams, emulsions
In potato starch manufacture an aqueous byproduct remains that is called potato fruit juice (PFJ). On a dry matter basis PFJ contains about 20-25 % protein and amino acids, 15 % sugars, 20 % minerals, 14 % organic acids and other components, such as phenolic compounds. Potato protein has a relatively high nutritional quality, comparable to that of whole egg, and it therefore has high potential for utilization in food applications. Protein recovery from industrial PFJ is presently achieved through heat coagulation by steam injection after pH adjustment. This method is very efficient in removing protein from solution. However, it leads to protein precipitates that exhibit a poor solubility, which hampers potential food applications.
An economic method to efficiently recover soluble potato protein would considerably increase its possibilities for use in food and add to its commercial value. Therefore, the important question resulting in this study was: can potato proteins be recovered from PFJ in such a way that they retain their functional properties, most importantly their solubility? This recovery method should be applicable at a large scale and result in a high yield. Potato protein recovery was expected to be complicated by the presence of and the interactions with non-protein components in PFJ. The objective in this study was to examine how extrinsic factors like pH, ionic strength and temperature would influence the structure of potato proteins, this in relation to the functionality of the proteins in making and stabilizing foams and emulsions.
Three groups of potato proteins can be distinguished in PFJ. Patatin, the major potato tuber protein, comprises 38 % of the protein in PFJ from cultivar Elkana . The protease inhibitors make up about 50 % and other proteins up to 12 % of total protein in PFJ from cultivar Elkana .
In Chapter 2 the effects of pH and various additives on the precipitation and (re)solubility at pH 7 of potato proteins from industrial PFJ are studied. Addition of various strong and weak acids caused the same extent of protein precipitation, which comprised at the most 60 % of total protein at pH 3. The use of weak acids, however, resulted in an increase in the resolubility of the precipitates at pH 7, as compared to strong acids. At pH 5 addition of FeCl 3 or ZnCl 2 increased both precipitation and resolubility. The largest increase in precipitation and resolubility was achieved by using organic solvents, resulting in a maximum precipitation (pH 5) of 91 % of total protein and a maximum resolubility of 91 % of precipitated protein. The results described in Chapter 2 lead to the hypothesis that precipitation and resolubilization of potato proteins from PFJ is not so much determined by their isoelectric pH but by their interactions with low molecular weight components.
In Chapter 3 it was shown, using DSC and both far-UV and near-UV CD spectroscopy, that potato proteins unfold between 55°C and 75°C. Increasing the ionic strength from 15 to 200 mM generally caused an increase in denaturation temperature. It was concluded that the dimeric protein patatin unfolds either in its monomeric state or that 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 best soluble at neutral and strongly acidic pH. At mildly acidic pH the overall potato protein solubility was dependent on ionic strength and the presence of unfolded patatin.
In Chapter 4 a protein isolate with a high solubility at neutral pH prepared from industrial PFJ by precipitation at pH 5 in the presence of ethanol is described. 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 preparation of this potato protein isolate should be done at low temperature to retain a high solubility. In the presence of ethanol the thermal unfolding of the tertiary and the secondary structure of patatin were shown to be almost completely decoupled. Even at 4°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 preparations.
In Chapter 5 foam forming and stabilizing properties of potato proteins are described; whipping or sparging was used to make foam. The performed whipping tests showed that less foam could be formed from untreated patatin than from the protease inhibitors, but also that patatin foam was much more stable against coalescence, Ostwald ripening and drainage. 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 concentrations, also indicated that foams made with an ethanol precipitated protein isolate (PPI) were more stable against Ostwald ripening and drainage than those made withβ-casein andβ-lactoglobulin. More generally it was concluded that when proteins are used as a foaming agent, a high concentration is required, because the available protein is inefficiently used. Also, the different methods used to make foam, result in changes in the mutual differences in foaming properties between the various protein preparations and may induce different instabilities to become apparent in foams made at the same conditions.
In Chapter 6 emulsions made with various potato protein preparations were characterized with respect to average droplet size, plateau surface excess and the occurrence of droplet aggregation. The average droplet size of the emulsions made with potato proteins appeared to be determined by the lipolytic release of surface active fatty acids and monoglycerides from the tricaprylin oil phase during the emulsification process. It was concluded that only trace amounts of patatin, the lipase activity of which has been strongly underestimated in literature, sufficed to liberate significant amounts of these surfactants. The plateau surface excess of emulsions made with patatin was found to be 2.6 mg/m 2 , while emulsion droplets made with protease inhibitors showed a significantly smaller surface excess. Of the various solvent conditions and treatments applied only heat treatment resulted in a significant increase in surface excess. Droplet aggregation in emulsions made with potato protein preparations other than patatin, could in contrast to at pH 5 and at pH 7 be prevented at pH 3.
In Chapter 7 the relations between potato protein structure, solubility and foam and emulsion forming and stabilizing properties are discussed. Also, the different mechanisms by which phenolic compounds may affect protein solubility are discussed in relation to the solubility and resolubility behavior of potato proteins in PFJ and when separated. A summary of the most important differences in the properties of patatin and protease inhibitors is also given.
Physico-chemical properties and thermal aggregation of patatin, the major potato tuber protein
Pots, A.M. - \ 1999
Agricultural University. Promotor(en): A.G.J. Voragen; P. Walstra; H. Gruppen. - S.l. : S.n. - ISBN 9789058080455 - 123
aardappeleiwit - potato protein
In potato tubers patatin is the most abundant protein, it is a 43 kDa glycoprotein with a lipid acyl hydrolase activity. Next, different classes of potato protease inhibitors are present. The content and biological activity of patatin and a fraction of potato protease inhibitors of molecular size 20-22 kDa were monitored as a function of storage time of whole potato tubers. It was observed that the amount of buffer-extractable protein decreased gradually during storage of whole potatoes of the cultivars Bintje and Desiree whereas, for Elkana, after an initial decrease it increased after approximately 25 weeks.
Patatin can be divided into two mass isomers, that each can be divided into various isoforms with a slightly differing primary sequence (genetic variants). The isoforms appeared to be of highly homogenous character, therefore, patatin can be studied as a single protein species.
Isolated patatin at room temperature is a highly structured molecule at both secondary and tertiary level as indicated by fluorescence, circular dichroism and Fourier transform infrared spectroscopy. Patatin unfolds partly upon heating or lowering the pH. At low pH, when the starting conformation is already irreversibly unfolded to a certain extent, only minor changes occur upon heating. The unfolding of patatin coincides with its precipitation in the potato fruit juice. The acid or heat precipitation of patatin when present in this juice may be enhanced by so far unknown components.
The thermal aggregation of patatin was studied by dynamic lightscattering and chromatographic analysis of the proportions of non-aggregated and aggregated patatin as a function of incubation time and temperature. The aggregation of patatin requires the unfolding of the protein and can accurately be described quantitatively with a two-step model. The course of aggregation suggested a mechanism of slow coagulation, limited by both reaction and diffusion.