PROCESS FOR THE MODIFICATION OF TOXIC AND/OR OFF-FLAVOURED COMPOUNDS

Abstract: The present invention describes a process wherein toxic and/or off-flavoured compounds are modified upon hydrolysis with a single enzyme. The modification results in a reduction of the toxicity and/or off-flavour of said compounds. The toxic and/or off-flavoured compounds preferably originate from Solanaceae and are glycosides. The enzyme cleaves said compounds at the covalent bond between the aglycone and the glycosidic part. More preferably the enzyme has an alpha-chaconinase activity, degrades alpha-chaconine to liberate the solanidine aglycone and a trisaccharide and originates from Aspergillus niger. The enzymatic hydrolysis of the invention can be applied in, for instance, the recovery of protein from potato fruitwater after separation of the starch granules.

Description: Process for the modification of toxic and/or off-flavoured compounds Field of the invention The present invention relates to enzymatic processes for the modification of undesirable compounds, especially in plant- derived material. Background of the invention A wide variety of plants contain so called Anti-Nutritional Factors (ANF's) which are claimed to provide a natural protection against attacks of moulds, bacteria, insects and birds. Well known ANF's include trypsin and chymotrypsin inhibitors, lectins, tannins, saponins and (glyco) alkaloids. In commercially important crops like soy, alfalfa and potato, the presence of non-proteinaceous ANF's like for example saponins and glycoalkaloids represent a considerable problem, because these compounds are toxic and bitter tasting. Unfortunately, these compounds are also very heat stable and, due to their hydrophobicity, difficult to extract. For instance, the family of Solanaceae, which comprise potato and tomato, are known to contain several types of glycoalkaloids . These glycoalkaloids are normally present in potatoes at low levels, but can accumulate to high levels in greened and damaged potatoes. The highest levels occurs in the peel of tubers. The potato juice released in starch manufacture contains a considerable amount of water-soluble organic compounds, of which 25-30% is protein. The protein of the potato has a remarkably good amino acid composition in the sense that it is relatively rich in lysine. As such, potato protein could compensate for relatively low lysine levels found in cereals. Furthermore, the functional properties of pure and undenatured potato protein are of interest because of their thermo-gelling and emulsifying properties. Unfortunately, the economic importance of potato protein is severely limited due to the presence of substantial quantities of glycoalkaloids which can cause gastro-intestinal disturbances and even death. At lower levels, the astringent, bitter taste evoked by the glycoalkaloids represents a serious drawback. As humans appear to be much more susceptible than animals to poisoning by glycoalkaloids, it would be of utmost importance to have a method to selectively reduce the glycoalkaloid levels in potato protein preparations. Solanine is a general term used to describe the glycoalkaloids present in potato. The major compounds which form part of solanine are α-solanine and α-chaconine. Both substances have a common steroidal alkaloid part, the so-called aglycone or, more specifically, solanidine, but they differ in their sugar moieties . Reportedly, the aglycone solanidine is considerably less toxic than the glycoside (Nishie et al. (1971) , Toxicology and Applied Pharmacology 1981-1992) . To remove glycoalkaloid compounds from plant material such as potato, several extraction procedures are conceivable. A serious drawback in this approach is that the glycoalkaloids chaconine and solanine are insoluble in many organic solvents and also practically insoluble in water. The acid extraction methods which are described have the disadvantage of protein denaturation so that the functional properties of the protein are lost. Moreover, an acid extraction method tends to raise the level of metal ions in the protein preparation and the residual amount of glycoalkaloid after extraction still can be high enough to create off-flavours. Thus, it is desirable to develop a method for the removal or inactivation of toxic and/or off-flavoured glycoalkaloid compounds from plant material, which produces a sufficiently low level of said glycoalkaloid compounds . In citrus fruit processing, an enzymatic process for the inactivation of a bitter-tasting glycoside has been described. Narengin, a bitter-tasting glycoflavonoid compound, can be converted to the tasteless naringenin by the subsequent action of two glycosidases, α-rhamnosidase and β-glucosidase (Askar and Bielig (1976) , Alimenta 15, 155-159) . Furthermore, in European Patent EP 0 332 281 is described that the generation of aroma compounds (in wine) from their glycosidic precursors (glycosylated terpenols) also requires the subsequent action of several enzymes. Thus, in both enzymatic processes described above, several enzymes are required for the sequential removal of monomeric sugar residues. Summary of the invention The present invention discloses a process of for degrading a compound comprising an aglycone part covalently joined to a. glycosidic part, by contacting said compound with an enzyme capable of cleaving the covalent bond between said aglycone and glycosidic parts. Preferably, the process is used for degrading a toxic compound to a less toxic product, or for degrading an off- flavoured compound to a less off-flavoured product. Preferably, the compound is part of plant-derived material. The plant-derived material preferably originates from the family Solanaceae, more preferably it originates from potato. The compound is preferably a glycoalkaloid, more preferably α-chaconine. The process according to the invention is applied in, for instance, the recovery of protein from potato after starch removal. The present invention also relates to nucleic acids which can be used to produce the enzyme used for degrading the compound. Detailed description of the invention Thus the invention provides for the first time a substantially purified enzyme capable of cleaving a covalent bond between an aglycone and a glycoside, i.e. without the express need for additional enzymes. Hitherto only mixtures of two or more enzymes were found to be capable of cleaving glycosidic compounds. It will be clear that this provides a great advantage in terms of production and application. For example the enzyme may be produced by a microorganism capable of producing it, under conditions wherein no or substantially no other enzymes are induced. This is beneficial in terms of productivity of the production process, as well as in terms of the specific activity of the enzyme that is being produced. According to a particular embodiment of the invention, the enzyme according to the invention is produced by being expressed in a host cell, such as an animal cell, a plant cell, a fungal cell a yeast cell or a bacterium, transformed with DNA encoding the said enzyme, or a precursor thereof. The enzyme according to the invention may then be recovered from the cells, or the culture medium if secreted by the host cell, in large quantities, and substantially devoid of any other activities. The present invention describes a process to modify toxic and off-flavoured compounds which are present in plant-derived material. The process is characterized in that it comprises the use of enzymes. Plant-derived material is treated with an enzyme, wherein the enzyme modifies said toxic and/or off- flavoured compounds in such a way that the toxicity and/or the off-flavour of the said compounds is reduced. Additionally, the properties of the enzyme-modified product are changed in such a way that its removal from the plant-derived material is facilitated. A sufficiently low level of toxic and/or off-flavoured compounds is reached when the plant-derived material or a product containing the plant-derived material, subjected to the process of the present invention, does not longer have a typical off-flavour and further complies with legislative rules on the Maximum Tolerable Levels of said compounds in the relevant products. The plant-derived material comprises any substance or product derived from a plant or a plant part, or the plant or plant part itself, whereby said plant or plant part contains a toxic and/or an off-flavoured compound. The plant-derived material containing a toxic and/or off- flavoured compound can originate from any plant, provided that said compound is present in the original plant or plant part or that said compound is formed during industrial processing of the plant or plant part. Preferably, the plant-derived material originates from plants belonging to the family of Solanaceae . More preferably, it originates from potato or tomato. The toxic and/or off-flavoured compounds which are subject to the process of the present invention are characterized in that said compounds are modified upon enzymatical treatment in such a way that their toxic properties and/or their off-flavour are reduced. Preferably, said toxic and/or off-flavoured compounds are modified upon enzymatical deglycosylation. At the same time, the solubility of the resulting deglycosylated compound is reduced as compared to the glycoside, thereby facilitating complete removal of said deglycosylated compounds . The glycosylated toxic and/or off-flavoured compounds preferably are selected from the group of glycoalkaloids, glycoterpenoids and glycosteroids. More preferably, they are selected from the group of solanine, most preferably they are α-solanine and/or α-chaconine. Preferably, the process of the present invention is applied on plant material derived from potato, containing the glycoalkaloids α-solanine and α-chaconine. The enzymes which are applicable in the process of the present invention are glycosidases which are capable of hydrolysing the glycosidic bond present in the glycoalkaloids, glycoterpenoids and/or glycosteroids. In the process of the present invention, the hydrolysis of glycoalkaloids, glycoterpenoids and/or glycosteroids preferably proceeds through the action of a single glycosidase . The glycosidase acts upon a glycosidic precursor by removal of the complete sugar moiety. The term "enzyme" or "glycosidase" defines here any means capable of obtaining the corresponding enzymatic activity. The enzymes employed in the process according to the present invention can be of any origin: bacterial, fungal, yeast, plant, animal or synthetic. Preferably, the enzymes employed originate from fungi, more preferably they originate from Aspergillus, most preferably from Aspergillus niger. The enzyme employed in the process of the present invention can be part of a complex enzyme preparation, said complex enzyme preparation comprising additional enzymes which are not critical for the process of the present invention. The enzyme employed in the process of the present invention can be purified from said complex enzyme preparation by fractionation techniques well-known to the person skilled in the art. The thus-purified enzyme can subsequently be characterized in more detail. In addition, the availability of the purified enzyme enables the person skilled in the art to isolate the corresponding gene or cDNA. Enzymes produced by genetic manipulations using host microorganisms are preferably encompassed according to the invention. Microorganisms can thus be modified by techniques known to those versed in the art for producing the enzyme which can be usable in the process of the invention. Since preferably a single enzyme is involved in separating the sugar moiety from the aromatic aglycone, production strains (over) expressing only one desired enzyme need to be generated. The use of such strains will result in lower costs and less side reactions. The process of the present invention can be used in a process to obtain a product from a plant, said plant containing toxic and/or off-flavoured compounds, such that the level of said toxic and/or off-flavoured compounds in the desired product is reduced. Since the aglycone exhibits a lower toxicity and/or a reduced off-flavour and/or can be more easily extracted, the economic advantages of the application of the process of the present invention in the treatment of, for example, potato protein are obvious. Preferably, the process of the present invention can be applied in the recovery of a protein-rich product from potato fruitwater obtained after separation of the starch granules . Example 1 Hydrolysis of the aglycone-sugar bond of α-chaconine α-chaconine (obtained from Sigma Chemicals) was dissolved at a concentration of 1 mg/ml in D 2 0, buffered with 100 mmol/1 sodium acetate at pH 5. To this solution several commercially available enzyme preparations were added. 15 μl of the liquid enzyme preparations and 2 mg of the powdered enzyme preparations were added per ml chaconine solution. Incubation took place at 37°C for 8 hours. The effect of the various enzyme preparations on the sugar moiety of α-chaconine was established by means of 600 MHz 1 H NMR. Of the many enzyme preparations tested in this way, only two preparations (i.e. the Aspergillus preparations AR 2000™ and Cytolase ® PCL5, both available through Gist-brocades, Seclin - France) exhibited a very efficient cleavage of the sugar- aglycone bond. After incubation with these two enzyme preparations, the only reducing sugar resonances that could be detected originated from a terminal glucose moiety. Because no β-resonances of rhamnose could be detected, it is inferred that the complete and intact tri-saccharide has been split-off by the two enzyme preparations . To confirm the outcome of this experiment, the pH value of the reaction mixture was adjusted to pH 10 followed by extraction with deuterated chloroform. The NMR spectrum of the extract showed to be identical to the spectrum of the pure aglycone solanidine (obtained from Sigma Chemicals) , proving that upon enzymatic hydrolysis α-chaconin has been split into solanidine and a tri-saccharide. These data implicate that the enzyme preparations AR 2000™ and Cytolase ® PCL5 contain an enzymatic activity which is able to cleave the β-1,3 bound between glucose and solanidine. This finding further implicates that the major toxic glycoalkaloid fraction present in potato can be modified into the considerably less toxic aglycone compound by incubation with selected enzymes. Example 2 Action of Emulsin on α-chaconine Further enzyme incubations were carried out incorporating the enzyme preparations AR 2000™ as well as Emulsin, a known source of β-glucosidase from almond and available through Sigma Chemicals. Experimental conditions were identical to those described in Example 1. The β-glucosidase activity present in the almond extract Emulsin was not able to split-off the tri-saccharide moiety of α-chaconin. This finding demonstrates that a special class of enzymes is present in AR 2000™ and Cytolase ® PCL5 which is involved in α-chaconine hydrolysis. Example 3 Purification of the chaconinase from the Aspergillus preparation AR2000 ® AR2000 ® (available through Gist-brocades, Seclin, France) was dissolved in buffer A (20% ethanol, 25 mM sodium citrate pH 3.4) (40 grammes in a 800 ml volume) . After centrifugation to remove dissolved material, the supernatant was applied to a 220 x 50 mm cation exchange column (S Sepharose ® Fast Flow, Pharmacia) ; flow rate 5 ml/min. The column was washed with buffer A until the absorption at 280 nm was about zero. A 2400 ml gradient from 0-0.3 M NaCl in buffer A was applied to the column. Fractions of different size were collected and the absorbance was measured at 280 nm. Chaconinase activity in the different fractions was assayed as an adaptation of the method described by Kudou et al . (Agric. Biol. Chem. (1990) 54: 1341-1342) in 80 mM sodiumacetate pH 5.0 with 80 μg α-chaconine (Sigma) as substrate in a volume of 100 μg at 37°C. After 30-90 min. the reaction product was extracted with 30 μl chloroform. TLC analysis was performed on a Silica 60 F 254 plate (Merck) with a solvent system of chloroform-methanol-water (65:35:10, v/v low layer) . 20 μl of the chloroform layer was applied to the Silica plate. Pure solanidine (Sigma) was used as a reference. The active fractions were pooled (700 ml) . One half of the pooled fractions was diluted five times with buffer A and applied to a second S Sepharose ® Fast Flow column (110 c 26 mm, flow rate 2 ml/min) . A gradient of 300 ml 0-0.3M NaCl in buffer A was applied to the column and fractions were collected. Activity was measured as described above. All active fractions were pooled and concentrated to about 8.5 ml (Amicon YM10 filter) and applied to a gel filtration column (Sephacryl ® S300, Pharmacia) ; 550 x 50 mm, flow rate 5 ml/min. in 50 mM sodiumacetate pH 5.0. Fractions were collected from the time the 280 nm signal increased. Chaconinase activity was measured and the fractions showing the highest activity were further concentrated to about 1.5 ml. 500 μl of this solution was applied to an anion exchange column (mono Q ® HR Pharmacia; 50 x 5 mm, flow rate 1 ml/min) in 50 mM sodiumacetate pH 5.0. A gradient of 0-0.3M NaCl in acetate buffer was applied to the column and 1 ml fractions were collected. Chaconinase activity was measured as described above. The fraction displaying this activity eluted before the bulk of the protein. Example 4 Properties of the chaconinase present in the Aspergillus preparation AR2000 ® The protein fraction containing chaconinase activity showed a single band in the SDS-PAGE. Using a protein mixture containing proteins of known molecular weight, the molecular weight of the purified chaconinase was estimated to be 120 kDa. The internal sequence of the chaconinase was determined by EUROSEQUENCE (Groningen, the Netherlands) . Edman degradation was performed on an automated sequenator (Model 477A, Applied Biosystems) coupled to an HPLC (Model 120A ABI) for analysis of the phenylthiodantoin (PTH) amino acids. The chaconinase has the following internal amino acid sequence (SEQ ID N0:1) : Gln-Leu-Gly-Ser-Leu-Tyr-Trp-Gln-Leu-Glu-Asp-Ile- (Trp) -Gln- Ala-Pro-Ser-Trp-Ala-Gly- (lie) The chaconinase was incubated under conditions as described in Example 1 in the presence of solanine and in the presence of a substance that contained soja saponin. The none of the incubation mixtures solanidine could be detected. This demonstrates the substrate specificity of the purified enzyme. /3-glucosidase and 3-galactosidase activity were measured throughout the purification procedure, using p-nitrophenyl- / S-D- glucopyranosidase and p-nitrophenyl-3-D-galactopyranoside respectively, as substrates. The purified chaconinase showed no 3-glucosidase activity or 3-galactosidase activity. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Gist-Brocades B.V. (B) STREET: Wateringseweg 1 (C) CITY: Delft (D) STATE: Zuid-Holland (E) COUNTRY: The Netherlands (F) POSTAL CODE (ZIP) : NL-2611 XT (G) TELEPHONE: 31.15.2799111 (H) TELEFAX: 31.15.2793957 (ii) TITLE OF INVENTION: Process for the modification of toxic and/or off- flavoured compounds (iii) NUMBER OF SEQUENCES: 1 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: EP 95201980.0 (B) FILING DATE: 18-ϋ * UL-1995 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Aspergillus niger (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 13 (D) OTHER INFORMATION:/note= "uncertain" (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION:21 (D) OTHER INFORMATION: /note= "uncertain" (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1: Gin Leu Gly Ser Leu Tyr Trp Gin Leu Glu Asp lie Trp Gin 1 5 10 Ala Pro Ser Trp Ala Gly lie 15 20

Claims: Claims 1. A process for degrading a compound comprising an aglycone part covalently joined to a glycosidic part, by contacting said compound with an enzyme capable of cleaving the covalent bond between said aglycone and glycosidic part . 2. A process according to claim 1, wherein said compound is a toxic compound and said aglycone part is less toxic. 3. A process according to claim 1, wherein said compound is an off-flavoured compound and said aglycone part is less off-flavoured. 4. A process according to claims 1-3, wherein said compound is part of plant-derived material. 5. A process according to claim 4, wherein said plant-derived material originates from the family Solanaceae . 6. A process according to claim 5, wherein said plant-derived material originates from potato. 7. A process according to claim 1-7, wherein said compound is a glycoalkaloid. 8. A process according to claim 7, wherein said compound is α-chaconine. 9. A process according to claim 1, wherein said degradation is part of a process for preparing a protein-rich product. 10. A preparation enriched in α-chaconinase activity. 11. A substantially pure polypeptide having α-chaconinase activity. 12. A substantially pure polypeptide according to claim 11 comprising an internal amino acid sequence corresponding to SEQ ID N0:1. 13. An isolated nucleic acid encoding a polypeptide according to claims 11 or 12. 14. A recombinant nucleic acid comprising a nucleic acid according to claim 13. 15. A host cell comprising recombinant nucleic acid according to claim 14. 16. The use of an enzyme capable of cleaving a covalent bond between an aglycon and a glycosidic part of a compound, for degrading glycoalkaloids, glycoterpenoids orglycosteroids. 17. The use of an enzyme according to claim 17, wherein said enzyme is capable of cleaving α-chaconine. 18. The use of an enzyme for degrading glycoalkaloids.

Inventors: EDENS LUPPO, NAN HELENA MARIA, SCHIPPER DIRK, SOUPPE JEROME, EDENS, LUPPO, NAN, HELENA, MARIA, SCHIPPER, DIRK, SOUPPE, JEROME
Applicants: EDENS LUPPO, GIST BROCADES BV, NAN HELENA MARIA, SCHIPPER DIRK, SOUPPE JEROME, EDENS, LUPPO, GIST-BROCADES B.V., NAN, HELENA, MARIA, SCHIPPER, DIRK, SOUPPE, JEROME
Application year: 1996
Main classification: C12N15
IPC codes: C12N9, C12N15, C12P17, C12P19