Occurrence and physico-chemical properties of protease inhibitors from potato tuber (Solanum tuberosum)

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.