WO1998038316A1 - Beta-glucosidases, methods for obtaining same, preparations containing said beta-glucosidases and uses thereof - Google Patents

Abstract

The invention provides substantially pure peptides having the following amino acid sequence: (1) Glu-Ala-Tyr-Gln-Xaa1-Tyr-Leu-Val-Thr-Glu-Pro-Asn-Xaa2-Gly, wherein Xaa1 and Xaa2 may be any DNA encodable amino acid; (2) Glu-Ala-Tyr-Gln-Xaa1-Tyr-Leu-Val-Thr-Glu-Pro-Asn-Xaa2-Gly or (3) NH2 - Leu Ser Val Ser Phe Pro His Tyr Val Gly Asp Leu Pro Ile Tyr Tyr Asp Tyr Leu - COOH, and β-glucosidases comprising parts having the said amino acid sequences. Also provided are the uses of said peptides for design probes to detect, isolate and/or amplify nucleic acid sequences coding for the said β-D-glucosidases, expression constructs, and transformed host cells capable of producing the said β-D-glucosidases in amounts higher than usual. Also uses of the said β-D-glucosidases are claimed in processes of modifying aroma precursor compounds and/or anthocyanidin-glucosides, in processes of making food, feed or a beverage. The β-D-glucosidases according to the invention are especially suitable for use in making wine and champagne.

Description

BETA-GLUCOSIDASES , METHODS FOR OBTAINING SAME , PREPARATIONS CONTAINING SAID- BETA-GLUCOSIDASES AND USES THEREOF

Technical Field The present invention relates to an enzyme having β-glucosidase activity, methods for obtaining same and preparations containing said enzyme. The invention is further concerned with the use of said enzyme in the food and feed industry, in wine making and the like.

Background of the invention

Commercial enzyme preparations are complex mixtures of pectinases, cellulases, hemicellulases and glycosidases (Rombouts, F.M. & Pilnik, W. (1978) Enzymes in fruit and vegetable juice technology, Process Biochem. 13, 9-13.; Voragen, F.G.J., Heutink, R. & Pilnik, W. (1980) Solubilization of apple cell walls with polysaccharide-degrading enzyme, J. Appl. Biochem. 2, 452-468; Haight, K.G. & Gump, B.H. (1994) The use of macerating enzymes in grape juice processing, Am. J. Enol. Vitic. 45, 113-116). They are used in the processing of fruits to degrade pectins, reduce the viscosity (Sreenath, H.K. & Santhanam, K. (1992) Comparison of cellulolytic and pectinolytic treatment of various fruit pulps, Chem. Mikrobiol. Technol. Lebensm. I 46-50.; Wrolstad, R.E., Wightman, J.D. & Durst, R.W. (1994) Glycosidase activity of enzyme preparations used in fruit juice processing, Food Technol. 1L, p. 90-98.), and improve the filterability and clarification of juices (Rombouts and Pilnik, 1978, supra; Haight and Grump, 1994, supra). They also degrade fruit cell walls to increase juice yields, as well as colour and flavour extraction (Chang, T.S., Siggiq, M., Sinha, N.K. & Cash, J.N. (1994) Plum juice quality affected by enzyme treatment and fining, J. Food Sci. 59„ 1065-1069.; Spagna, G. & Pifferi, P.G. (1994) The purification of a commercial pectinlyase from Aspergillus niger. Food Chem. 50, 343-349; Wightman, J.D. & Wrolstad, R.E. (1995) Anthocyanin analysis as a measure of glycosidase activity in enzymes for juice processing, J. Food Sci. 60_^ 862-867), β-D-glucosidase (EC 3.2.1.21) present in commercial enzyme preparations participate in cellulolysis through the hydrolysis of cellobiose and in the enhancement of flavour (particularly in wine) by hydrolysing terpenyl-β-D-glucosides into terpenols (Shosheyov, O., Bravdo, B.A., Siegel, D., Goldman, A., Cohen, S., Shosheyov, L. & Ikan, R. (1990) Immobilized endo-β-glucosidase enriches flavor of wine and passion fruit juice, J. Agric. Food Chem. 38j 1387-1390; Gunata, Z. (1994) Etude et exploitation par vote enzymatique des precurseurs d'arόmes du raisin de nature glycosidique, Revue des Oenologues 74, 22-27; Vasserot, Y., Arnaud, A. & Galzy, P. (1995) Monoterpenyl glycosides in plants and their biotechnological transformation, Acta Biotechnol. Λ5, 77-95). In red fruit juices, they can induce a loss of colour (Wrolstad et al., 1994, supra; Wightman and Wrolstad, 1995, supra) since the aglycons (anthocyanidins), released by enzymic hydrolysis of anthocyanidin-β-D-glucosides (anthocyanins) degrade spontaneously to colourless products (Huang, H.T. (1955) Fruit color destruction: Decolorization of anthocyanins by fungal enzymes, J. Agric. Food Chem. 3_, 141-146). The undesired discolorating effect of fungal preparations from Aspergillus niger on raspberry (Jiang, J., Paterson, A. & Piggott, J.R. (1990) Short communication: Effects of pectolytic enzyme treatments on anthocyanins in raspberry juice, International Journal of Food Science & Technology 25„ 596-600, 1990), strawberry (Blom, H. (1983) Partial characterization of a thermostable anthocyanin-β-glycosidase from Aspergillus niger, Food Chem. 12, 197-204), blackberry (Yang, H.Y. & Steele, W.B. (1958) Removal of excessive anthocyanin pigment by enzyme, Food Technol. 12, 517-519), cranberry (Wrolstad et al., 1994, supra) and grape (Yang and Steele, 1958, supra; Fu-Mian, C, Pifferi, P.G., Setti, L., Spagna, G. & Martino, A. (1994) Immobilization of an anthocyanase (β-glucosidase) from Aspergillus niger. Ita. J. Food Sci. 6, 31-42) anthocyanins has been reported. In grape juices and wines, the decolorization is due to β-D-glucosidases, as the main anthocyanins are anthocyanidin-β-D-glucosides (Timberlake, C.F. & Bridle, P. (1975) Anthocyanins, in: The Flavonoids (Harborne J.B., Marby T.J. & Marby H., Eds.), 115-149, Chapman & Hall London; Ribereau-Gayon, P. (1982) The anthocyanins of grapes and wines, in: Anthocyanins as food colors (Markakis, P., Ed), 209-242, Academic Press.Inc).

Fungal β-D-glucosidases have been isolated from Fusarium oxysporum (Christakopoulos, P., Goodenough, P.W., Kekos, D., Macris, B.J., Claeyssens, M. & Bhat, M.K. (1994) Purification and characterization of an extracellular β-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum, Eur. J. Biochem. 224. 379-385), Gliocladium virens (Todorovic, R., Grujic, S., Kandrac, J. & Matavulj, M., 1989, Some properties of β-glucosidase from Gliocladium virens C2R1, Biotechnol. Appl. Biochem. II, 459-463), Neocallimatix frontalis (Li, X. & Calza, R.E. (1991) Purification and characterization of an extracellular β-glucosidase from the rumen fungus Neocallimastix frontalis EB188, Enzyme Micro. Technol. ii, 622-28), Botrytis cinerea (Sasaki, I. & Nagayama, H. (1995), Purification and characterization of β-glucosidase from Botrytis cinerea, Biotech. Biochem. 59_, 100-101; Guegen, Y., Chemardin, P., Arnaud, A. & Galzy, P. (1995) Purification and characterization of an intracellular β-glucosidase from Botrytis cinerea, Enzyme Micro. Technol. I_7, 900-906); Talaromyces emersonii (McHale, A. & Coughan, M.P. (1982) Properties of the β-glucosidases of Talaromyces emersonii, Journal of General Microbiology 128. 2327-31), Trichoderma reesei (Fadda, M.B., Curreli, N., Pompei, R., Rescigno, A., Rinaldi, A. & Sanjust, E. (1994) A highly active fungal β-glucosidase: Purification and properties, Appl. Biochem. Biotechnol. 44, 263-270), and Aspergillus aculeatus (Sakamoto, R., Arai, M. & Murao, S. (1985) Enzymic proprietes of three β-glucosidases from Aspergillus aculeatus No. F-50, Agric. Biol. Chem. 49, 1283-1290), foetidus (Hang, Y.D. & Woodams, E.E. (1994) Apple pomace: A potential substrate for production of β-glucosidase by Aspergillus foetidus, Lebens. Wiss. u. Technol. 27_, 587-589), roseus (Vodjani, G., Le Dizet, P. & Petek, F. (1992) Purification et propri t s de deux (1 → 4) β-D-glucosidases d 'Aspergillus roseus, Carbohydr. Res. 236. 267-279), terreus (Araujo, A. & D'Souza, J. (1986) Characterization of cellulolytic enzyme components from Aspergillus terreus and its mutants, J. Ferment. Technol. 64_, 463-467), and niger (Watanabe, T., Sato, T., Yoshioka, S., Koshijima, T. & Kuwahara, M. (1992) Purification and properties of Aspergillus niger β-glucosidase, Eur. J. Biochem., 209, 651-659; Hoh, Y.K., Yeoh, H.H. & Tan, T.K.

(1992) Properties of β-glucosidase purified from Aspergillus niger mutants USDB 0827 and USDB 0828, Appl. Microbiol. Biotechnol. 37, 590-593; Witte, K. & Wartenberg, A. (1989) Purification and properties of two β-glucosidases isolated from Aspergillus niger, Acta Biotechnol. 9, 179-190; Himmel, M.E., Adney, W.S., Fox, J.W., Mitchell, D.J. & Baker, J.O. (1993) Isolation and characterization of two forms of β-glucosidase from Aspergillus niger, Appl. Biochem. Biotechnol. 39-40, 213-225; Unno. T., Ide, K., Yasaki, T., Tanaka, Y., Nakakuki, T. & Okada, G.

(1993) High recovery purification and some properties of β-glucosidase from Aspergillus niger, Biosci. Biotechnol. Biochem. 57, 2172-2173. All β-D-glucosidases from Aspergillus niger reported so far have a molecular weight in the range 90-130 kDa, an optimum pH about 4, an optimum temperature between 50 and 70°C and an acidic isoelectric point in the pH range 3.5-4.5. Most A. niger glucosidases have been studied for their activity on -NP-β-D-glucoside and cellobiose but very few studies (Fu-Mian, C, Pifferi, P.G. & Spagna, G. (1992) Partial purification and characterization of anthocyanase (β-glucosidase) from Aspergillus niger, Cerevisiae & Biotechnology 17, 20-27; Blom, 1983, supra) concerned the degradation of anthocyanidin-β-D-glucosides. As far as we are aware, no study reported the effect of a purified β-D-glucosidase on several substrates representing the different technological targets of β-D-glucosidases: cellobiose (cellolysis), terpenyl-β-D-glucosides (flavour enhancing), and anthocyanidin-β-D-glucosides (decolorization) in comparison with the "reference" activity on -nitrophenyl-- β-D-glucoside. It is an object of the present invention to provide a β-glucosidase in substantially pure form, at least free from anthocyanase activity, which can be suitably used in the food industry without the associated disadvantages of the known β-glucosidases such as decolorization of fruit juices and red wine, or must.

Summary of the invention

The present invention provides a substantially pure peptide having the following amino acid sequence

Glu-Ala-Tyr-Gln-Xaal-Tyr-Leu-Val-Thr-Glu-Pro-Asn-Xaa2-Gly, wherein Xaal and Xaa2 may be any DNA encodable amino acid. Preferably, in said sequence Xaal is Asp, and/or Xaa2 is Asn.

According to another embodiment a substantially pure polypeptide having β-glucosidase activity but substantially no anthocyanase activity is provided, preferably one which comprises a part having an amino acid sequence Glu-A- la-Tyr-Gln-Xaal-Tyr-Leu-Val-Thr-Glu-Pro-Asn-Xaa2-Gly, wherein Xaal and Xaa2 may be any DNA encodable amino acid.

The invention also provides a composition comprising a polypeptide having β-glucosidase activity, characterised in that said composition is low in β-glucosidase activity on an anthocyanidin-glucoside substrate selected from the group consisting of malvidin-3-glucoside, delphinidin-3-glucoside, cyanidin-3-glucoside, petunidin-3-glucoside and peonidin-3-glucoside. Preferably, the said composition is characterised in that the said β-glucosidase activity on an anthocyanidin-glucoside substrate selected from the group consisting of malvidin-3-glucoside, delphinidin-3-glucoside, cyanidin-3-glucoside, petunidin-3-glucoside and peonidin-3-glucoside amounts to 20% or less, preferably 10% or less, of the total β-glucosidase activity in said composition. More preferably the said composition is one, wherein said polypeptide having β-glucosidase activity comprises an amino acid sequence Glu-Ala-Tyr-Gln-Xaal-Tyr-Leu-Val-Thr-Glu-Pro-Asn-Xaa2-Gly, wherein Xaal and Xaa2 may be any DNA encodable amino acid.

The invention also provides for the use of a substantially pure polypeptide according to the invention or a composition, in a process for releasing aromatic compounds from their glycosidic precursors, such as a process for making a beverage or a food product for human consumption or a feed product for animal consumption. Furthermore, said process may be one for making a starting material for a beverage a food or feed, or a process for making an ingredient for use in a beverage, food or feed product. Preferred according to the invention is a use wherein said beverage product is red grape juice, or red wine.

According to yet another aspect of the invention the use is provided of a substantially pure peptide according to the invention in a process of making a nucleic acid primer or primers and/or a nucleic acid probe which can be used to detect and/or amplify and/or isolate a nucleic acid sequence coding for at least part of a polypeptide having β-glucosidase activity.

According to a specially preferred embodiment, a composition is provided comprising a polypeptide having β-glucosidase activity, characterised in that the k_at of the said β-glucosidase for malvidin-3-glucoside represents less than 1% of the k_cat for cellobiose or -NP-β-D-glucoside.

A further embodiment of the invention is a substantially pure peptide having anthocyanase activity comprising an amino acid sequence: NH₂ - Leu Ser Val Ser Phe Pro His Tyr Val Gly Asp Leu Pro He Tyr Tyr Asp

Tyr Leu - COOH. and use of a said pure peptide in a process of making a nucleic acid primer or primers and/or a nucleic acid probe which can be used to detect and/or amplify and/or isolate a nucleic acid sequence coding for at least part of a polypeptide having anthocyanase activity.

Further provided is an isolated nucleic acid sequence encoding a said peptide. According to another aspect of the invention a substantially pure peptide having anthocyanase activity comprising an amino acid sequence:

NH₂ - Leu Ser Val Ser Phe Pro His Tyr Val Gly Asp Leu Pro He Tyr Tyr Asp Tyr Leu - COOH (SEQIDNO: 3), is provided. The invention further provides for the use of a said substantially pure peptide in a process of making a nucleic acid primer or primers and/or a nucleic acid probe which can be used to detect and/or amplify and/or isolate a nucleic acid sequence coding for at least part of a polypeptide having anthocyanase activity.

According to yet a further aspect of the invention an isolated nucleic acid sequence encoding a peptide as above is provided. Also provided are methods of use of a substantially pure polypeptide having anthocyanase activity and which comprises an amino acid sequence as depicted in SEQIDNO: 3. to formulate an enzyme preparation. Further uses of said polypeptide are contemplated in a process for making a food, feed or beverage, such wine, fruit juice or champagne.

Also provided is a use of a substantially pure polypeptide according to the invention having anthocyanase activity and which comprises an amino acid sequence according to SEQIDNO: 3, or an enzyme preparation according comprising it in a process of hydrolysing an anthocyanidin-precursor.

The invention is illustrated by the following Figures.

Description of the Figures

Fig.l. Chemical structure of malvidin-3-glucoside and geranyl-β-D-glucoside.

Fig.2. Chemical structure of D-glucose and two β-D-glucosidase inhibitors.

Fig.3. Lineweaver-Burk (A), Hanes (B) and Scatchard (C) plots for determination of the kinetic constants on cellobiose. The enzyme was incubated 20 min. at 40°C in 50 mM sodium acetate pH 4.2, containing cellobiose at various concentrations from 0.2 to 0.6 mM.

Fig.4. Protein elution and enzyme recovery on various columns during the purification of the β-D-glucosidase from Aspergillus niger. Anion-exchange chromatography on Q-Sepharose (A) and D-Zephyr pH 7.2 (B). ( — ♦ — ) and

( — o — ) represent A280 and β-D-glucosidase activity. Insert, ( ) NaCl elution gradient.

Fig.5. Diagram of purification of the β-D-glucosidase from Aspergillus niger. Dotted arrow indicated the purification of a second β-D-glucosidase from the same pectinase preparation.

Fig.6. Effect of temperature and pH on β-D-glucosidase activity. (A) Temperature optimum; the enzyme was incubated 20 min. in 50 mM sodium acetate pH 4.2, containing 2mM jc-NP-β-D-glucoside at a range temperature from 20 to 80°C. (B) pH optimum; the enzyme was incubated 20 min. at 40°C in universal buffer from pH 2.0 to 12.0, containing 2 mM pNPG. (C) Stability of the β-D-glucosidase towards temperature. The enzyme was incubated 60 min. in 50 mM sodium acetate pH 4.2 at the various temperature and then the β-D-glucosidase activity was measured. (D) Stability of the β-D-glucosidase towards pH. The enzyme was incubated 60 min. at 40°C in universal buffer from pH 2.0 to 12.0 and then the β-D-glucosidase activity was measured.

Fig. 7 Protocol of extraction of the free aroma fraction

Fig. 8 (A-C) Gas Chromatography of wine A (control), wine B (treated with α-L-arabinofuranosidase (B) and wine C (incubated with a mixture of -L-arabinofuranosidase and the purified β-D-glucosidase); peak 1: 4-nonanol; peak 2, linalool; peak 3: geraniol; peak 4: 3,7-dimethylocta-l,5-diene-3,7-diol; peak 5: 4-vinyl-gaiacol; peak 6: 3,7-dimethylocta-l,5-diene-3,8-diol; peak 7: geranic acid.

Fig.9 Diagram of purification of the β-D-glucosidase from Aspergillus niger. Dotted arrow indicated the purification of a second β-D-glucosidase from the same pectinase preparation.

Fig. 10 Protein elution and enzyme recovery at anion exchange chromatography step s on D-Zephyr column (pH 6.5) during the purification of the β-D-glucosidase and the anthocyanase fraction from A. niger — ♦ — , --□-- , and — o— represent A280, the purified β-D-glucosidase activity and the anthocyanase activity, respectively. represents the NaCl-gradient.

ιo Fig.11 Effect of temperature and pH on β-glucosidase activity of the anthocyanase. (A) Temperature optimum; the enzyme was incubated for 20 min in 50 mM sodium acetate pH 4.2, containing 2 mM p-NP-β-D-glucoside at a temperature range from 20 to 80°C. (B) pH optimum; the enzyme was incubated for 20 min at 40°C in universal buffer from pH 2.0 to 12.0, containing 2 mM p-NP-β-D-glucoside. (C) is Stability of the β-D-glucosidase towards temperature. The enzyme was incubated for 60 min in 50mM sodium acetate pH 4.2 at the various temperatures and then the β-D-glucosidase activity was measured. (D) Stability of the β-D-glucosidase towards pH. The enzyme was incubated 60 min at 40°C in universal buffer from pH 2.0 to 12.0 and then the β-D-glucosidase activity was measured.

20

Fig. 12 Chemical structures of malvidin-, peonidin- and cyanidin-3-glucoside.

Fig. 13. Lineweaver-Burk (A), Hanes (B) and Scatchard (C) plots for determination of kinetic constants on malvidin-3-glucoside. The enzyme was incubated 30 min at 25 40°C in 50 mM sodium acetate pH 3.6, containing malvidin-3-glucoside at various concentrations from 0.075 to 0.3 mM.

Fig. 14 Anthocyanin composition of the cabernet franc extract; peak 1: delphinidin-3-glucoside (10% of total content); peak 2: cyanidin-3-glucoside (5%); 30 peak 3: petunidin-3-glucoside (15%); peak 4: peonidin-3-glucoside (15%); peak 5: malvidin-3-glucoside (42%).

Fig. 15 Change in Absorption at 520 nm (A₅₂₀) of red wine treated with anthocyanase

( — o — ), purified β-glucosidase ( — ■ — ) and non-treated wine ( — Δ— ) during vinification.

Fig. 16 Decolorisation of red wine during vinification due to the presence of the purified β-D-glucosidase (A) and the purified anthocyanase (B) expressed in percentage relative to the control wine.

Fig. 17 Table summarising the purification profiles of the β-D-glucosidase and the anthocyanase purified from Rapidase^® PCL5, an enzyme preparation from Aspergillus niger.

Fig. 18 Changes in absorption at 520 nm (a) and hue (b) during the ageing of red wines treated with the purified anthocyanase B ( — o — ) and non-treated wine (Δ).

Detailed description

A number of important aroma precursor compounds and anthocyanins in natural substrates share the property that they are a target for β-D-glucosidases which are frequently present in commercially available enzyme preparations. It is the merit of the present invention to identify the existence of β-D-glucosidases with basically different catalytic preferences; those that have high catalytic activity on aroma precursors of a glucosidic nature, but no or very little activity on anthocyanins, and those that show high catalytic activity on anthocyanins and less activity on other substrates. Thus, for the first time, it is possible to separate these enzymatic functions, thereby increasing the applicability and the versatility of β-glucosidase in the food, feed and beverages industry.

According to one aspect, the present invention provides a method for the enzymatic hydrolysis of non-volatile precursors of aroma and flavour (enhancing) compounds, whereby volatile aroma (enhancing) compounds are released and add to the flavour and/or aroma of a food, feed or beverage. Examples of such precursors are terpenols and alcohols of a glucosidic nature, such as those present in fruit, including but not limited to all sorts of berries, grapes and the like. The method is especially advantageous when practised on food or food ingredients containing anthocyanidin-glucosides, such as red grapes, red currants, black currants, cranberries, cherries, raspberry, strawberries, and the like. An overview of the occurrence of anthocyanins (anthocyanidin-glycosides) is given in Timberlake C.F. & Bridle, 1975, Chapter 5 "Anthocyanins, In: The flavonoids S.B. Harborne. T.J. Marby, S.H. Marby Eds., Chapman & Hall, London, pp. 115-149. The β-glucosidase according to the invention finds particular use in aroma enhancing enzyme compositions, such as those described in EP 332 381 and EP 0 416 713, without the concomitant loss of color observed when these preparations are being used indiscriminately. For example, it is now possible to use a much higher dosage of β-glucosidase activity, or allow the β-D-glucosidase to act longer, or in a later stage of the food or beverage making process than before.

Examples of the use of β-glucosidases to increase aroma release from glycosidic precursors can be found in European patent application EP 0 416 713 Al, published March 13, 1993 and EP 0 332 381, published on September 13, 1989. It will be clear from the instant application, that it is advantageous to work with well characterised enzymes, rather than crude enzymes. It is known from these patent applications, that the subsequent action of α-arabinofuranosidases and/or α-rhamnosidases and/or α-l,6-apiofuranosidases, and other β-glycosidases assist the β-glucosidase according to the invention in liberating the volatile aroma compounds from their sugar moieties. Hence, the β-D-glucosidase according to the invention is advantageously used in conjunction with the mentioned β-glycosidases. Relatively pure enzymes may be mixed prior to use, or added to their substrates sequentially. Large amounts of relatively pure enzyme may be obtained by the cloning and over-expression of DNA sequences coding for α-arabinofuranosidase, as disclosed in WO 92/17592, published on October 15, 1992; the relevant parts whereof are incorporated herein by reference.

In a manner substantially the same as disclosed in WO 92/17592, the DNA sequence coding for the purified β-glucosidase may be isolated and over-expressed in any suitable host, such as a plant, a fungus, such as a Aspergillus, Trichoderma, Fusarium, species, a yeast, such as a Saccharomyces, Kluyveromyces, Pichia or a Yarrowia species, a bacterium species, such as Bacillus or any other microorganism suitable for the purpose. -- On the basis of these amino acid sequences oligonucleotide sequences have been designed which can be used in a suitable cloning strategy to obtain a cDNA or genomic DNA sequence encoding the β-D-glucosidases according to the invention, or a precursor form thereof. Any of the following methods may suitably be used (for a general reference to nucleic acid hybridisation see for example: Hames & Higgins, 1985, in: Nucleic Acid Hybridisation - a practical approach, IRL press, Washington DC):

1. probing a cDNA library, optionally after having determined suitable hybridisation and washing conditions using a Southern blot approach on Aspergillus niger DNA, in order to clone a cDNA coding for part of the purified β-glucosidase according to the invention; or,

2. probing a genomic library, substantially as described above; or

3. expression cloning of cDNA in a microbial host, such as E. coli or Kluyveromyces lactis, followed by detection of putative positive clones, using a suitable substrate for detecting the presence of the β-glucosidase, or using an antibody capable of detecting the purified β-glucosidase; or

4. amplification of DNA coding for a fragment of the β-glucosidase gene using a set of primers as disclosed herein; alternatively, the strategy of inverse-PCR may be used, well known to those of skill in the art. Any of the above techniques, or combinations, will lead to the gene coding for the Aspergillus niger β-glucosidase according to the invention. Once the gene, or a cDNA, has been cloned, it may be overexpressed under the control of its own regulatory elements, or regulatory elements, such as promoters, enhancers, terminators, introns, deemed more suitable for overexpression in the host of choice. Dependent of the choice of the host and the purpose of recombinant production, the β-glucosidases according to the invention may be redirected to specific compartments within the cell, or outside the cell. For production purposes in, e.g. a microbial host, it will be useful to secrete the β-glucosidase into the culture medium. Those of skill in the art are well aware of the need for a signal peptide functional in the host of choice, in order to achieve secretion.

The choice of regulatory elements and other expression tools is well within reach of those of ordinary skill in the art of molecular biology. Reference may be made to the following references and patent documents for further guidance: Concerning genetic engineering of Yeasts: EP 0 190 119 published as WO 85 05632 on 19-12-1985 for "CHIMERIC PLASMIDS THAT REPLICATE IN BACTERIA AND YEAST AND MICROORGANISMS TRANSFORMED THEREWITH"; EP 0 068 740 (5-01-1983) "RECOMBINANT DNA CLONING VECTORS AND THE EUKARYOTIC AND PROKARYOTIC TRANSFORMANTS THEREOF", illustrating the use of yeast selectable markers, such as hygromycin-B, G418- resistance and the like;

EP 0 077 689 (27-04-1983) "METHOD OF GENE MANIPULATION USING AN EUKARYOTIC CELL AS THE HOST", illustrating the use of a 3'-leader for expression of DNA in higher eukaryotic cell, such as yeasts and fungi;

EP 0 088 632 (14-09-1983) "EXPRESSION, PROCESSING AND SECRETION OF HETEROLOGOUS PROTEIN BY YEAST", illustrating the use of homologous signal peptides for secretion in Saccharomyces species; EP 0 096 910 (28-12-1983) "YEAST OF THE GENUS KLUYVEROMYCES MODIFIED FOR THE EXPRESSION OF PREPROTHAUMATIN OR ITS VARIOUS ALLELIC AND MODIFIED FORMS OR THEIR MATURATION FORMS" illustrating the cloning and expression of foreign genes in the yeast Kluyveromyces; EP 0 095 986 (7-12-1983) "NOUVEAU VECTEUR DE CLONAGE ET D'EXPRESSION, LEVURE TRANSFORME PAR CE VECTEUR ET LEUR APPLICATION", illustrating the use of the Kluyveromyces lactis pKdl plasmid; EP 0 123 811 (12-06-1991) "THE USE OF THE GAL 1 YEAST PROMOTER"; EP 0 127 304 (5-12-1984) "PROCESS FOR PRODUCING HETEROLOGOUS PROTEIN IN YEAST, EXPRESSION VEHICLE THEREFOR, AND YEAST TRANSFORMED THEREWITH", illustrating secretion in Saccharomyces using the invertase signal peptide;

EP 0 123 544 (31-10-1984) "PROCESS FOR EXPRESSING HETEROLOGOUS PROTEIN IN YEAST, EXPRESSION VEHICLES AND YEAST ORGANISMS THEREFOR", illustrating the use of the alfa-factor leader/signal peptide in Saccharomyces cerevisiae;

"EP 0 139 383 (2-05-1985) "METHOD FOR EXPRESSING FOREIGN GENES IN SCHIZOSACCHAROMYCES POMBE AND THE USE IN THERAPEUTIC FORMULATIONS OF THE PRODUCTS, DNA CONSTRUCTS AND TRANSFORMANT STRAINS OF SCHIZOSACCHAROMYCES POMBE USABLE

IN SUCH METHOD AND THEIR PREPARATION";

US 4,745,062 (PLASMID VECTORS FOR CLONING AND EXPRESSION OF A

PROTEIN IN A MICROORGANISM, COMPRISING AT LEAST ONE PROMOTER FOR EXPRESSION OF B-GLUCOSIDASE IN YEASTS;

MICROORGANISMS CONTAINING THESE PLASMIDS; A FERMENTATION

PROCESS AND THE ENZYMES OBTAINED", illustrating over-expression of a β-D-glucosidase in Saccharomyces cerevisiae;

EP 0 164 556 (18-12-1985) "ENHANCED YEAST TRANSCRIPTION EMPLOYING HYBRID PROMOTER REGION CONSTRUCTS", illustrating the use of hybrid promoters in yeast gene expression;

EP 0 707 068 (17-04-1996) "Yeast vector", illustrating a yeast selection marker, the

G418 resistance marker of Tn903;

EP 0 163 491 (4-12-1985) "YEAST VECTOR", illustrating the transformation of non-haploid industrial strains of the yeasts Saccharomyces cerevisiae and carls- bergensis;

EP 0 183 070 (9-10-1991) "TRANSFORMATION OF YEASTS OF THE GENUS

PICHIA", illustrating cloning and expression of genes in the yeast Pichia, using histidine auxotrophic mutants; EP 0 213 593 (10-04-1991) "REPRESSIBLE YEAST PROMOTERS", illustrating the use of some repressible yeast promoters in heterologous protein production, such as the hybrid acid phosphatase (PHO5):: GAPDH promoter;

US 4,997,767" "YEAST SHUTTLE VECTOR", illustrating yeast shuttle vectors operable in Saccharomyces cerevisiae and E.coli; EP 0 241 435 (2-12-1992) "VECTORS FOR THE CLONING AND EXPRESSION

OF HETEROLOGOUS GENES IN YEAST AND THE YEAST STRAINS

TRANSFORMED BY SAID VECTORS", illustrating the use of the vector pkDl and the URA3 marker in Kluyveromyces transformation;

EP 0 301 669 (1-02-1989) "DNA CONSTRUCTS CONTAINING A KLUYVER- OMYCES ALPHA-FACTOR LEADER SEQUENCE FOR DIRECTING

SECRETION OF HETEROLOGOUS DERIVATIVES" and EP 0 301 670

(1-02-1989) "KLUYVEROMYCES AS A HOST STRAIN", illustrating secretion of foreign proteins in Kluyveromyces using the various leaders and signal peptides, such as the alpha-factor leader from Kluyveromyces;

WO 90 05787 (31-05-1990) "POSITION-SPECIFIC INSERTION VECTORS AND METHOD OF USING SAME", illustrating yeast integration vectors based on Ty3 in Saccharomyces cerevisiae; EP 0 394 538 (31-10-1990) "A YEAST CELL OF THE GENUS SCHWANNI- OMYCES", illustrating cloning and expression of foreign genes in the yeast Schwanniomyces;

WO 90/14423 (29-11-1990) "MICROORGANISM TRANSFORMATION", illustrating yeast transformation by the use of integration by linear vector having regions of homology at the extremities of the linearised vector;

EP 0 481 008 (24-01-1991) "PROCESS FOR PREPARING A PROTEIN BY A FUNGUS TRANSFORMED BY MULTICOPY INTEGRATION OF AN EXPRESSION VECTOR, illustrating multiple integration of foreign DNA in 4 to 40 using a deficient selection marker such as LEU2d, URA3d and TRPld; NL 9001159 (16-12-1991) "Methode om de efficientie van de secretie van eiwitten door gistcellen te vergroten", illustrates Kluyveromyces and Saccharomyces mutants with improved secretion due to permeable cell walls;

EP 0 531 181 (18-02-1993) "HIGHLY STABLE RECOMBINANT YEASTS FOR THE PRODUCTION OF RECOMBINANT PROTEINS", illustrates another high copy system in yeast, using a essential gene glycolytic gene deficient strain;

EP 0 531 187 (10-03-1993) "PROMOTEUR DE LEVURE ET SON UTILISATION", discloses the yeast ADH4 (alcohol dehydrogenase) promoter for foreign gene expression in yeast, such as Kluyveromyces lactis; WO 94 01570 (20-01-1994) "K. lactis rp28 RIBOSOMAL PROTEIN GENE PROMOTER AND USE THEREOF", WO 94 01569 (20-01-1994) "K.lactis PYRUVATE DECARBOXYLASE PROMOTER GENE AND USE THEREOF", WO 94 03618" (17-02-1994) "K. lactis TRANSALDOLASE GENE PROMOTER AND USE THEREOF" and WO 94 13821 (23-06-1994) "THE USE OF THE KLUYVEROMYCES MARXIANUS LNULINASE GENE PROMOTER FOR PROTEIN PRODUCTION", illustrate the use of other promoters for foreign gene expression in the yeast Kluyveromyces lactis;

EP 0 748 872 (18-12-1996) "STRAINS OF BAKERY YEAST CAPABLE OF EXPRESSING AND EXPORTING TO THE CELLULAR EXTERIOR THE ENZYME A-(l-4)-AMYLASE OF ASPERGILLUS ORYZAE: PRODUCTION PROCESS AND APPLICATION", illustrating improvement of Saccharomyces cerevisiae for bakery purposes using recDNA techniques, such as cloning and expression of alpha-amylase from Aspergillus oryzae in baker's yeast;

Concerning genetic engineering of Fungi: US 4,885,249 and EP 0 184 438 (l l-06-1986)"ASPERGILLUS NIGER TRANSFORMATION", illustrating the cloning and expression of genes in Aspergillus niger; EP 0 238 023 (23-09-1987) "PROCESS FOR THE PRODUCTION OF PROTEIN IN ASPERGILLUS ORYZAE AND A PROMOTER FOR USE IN ASPERGILLUS", illustrating the transformation and expression of foreign genes in Aspergillus oryzae using the TAKA-amylase promoter;

EP 0 730 655 and EP 0 730 656 (8-06-1995) "ASPERGILLUS FOETIDUS EXPRESSION SYSTEM" , illustrating the use of Aspergillus foetidus for hete- rologous enzyme production;

Concerning genetic engineering of Bacteria: EP 0 1929 and EP 0 1930, illustrating cloning and expression of foreign genes in E. coli;

EP 0 207 165 (7-01-1987) "POLYPEPTIDE SECRETION-CAUSING VECTOR, MICROORGANISMS TRANSFORMED BY SAID VECTOR, AND PROCESS FOR PREPARING POLYPEPTIDE USING SAID MICROORGANISMS", illustrating the use of E. coli for foreign gene expression using the vector pGH54, comprising the β-lactamase promoter, ribosome binding site and signal sequence;

Concerning the genetic engineering of plants: EP 0 120 516 on the use of binary vectors for the transformation of dicotyledonous plants; EP 0 131 620, EP 0 131 623 and EP 0 131 624 on the use of kanamycin resistance markers for the transformation of plants; WO 96/40951 and US 5,530,185 on the modification of color phenotype in recombinant plants; WO 95/04152 on the use of fruit specific promoters in e.g. tomato plants; WO 94/21803 and WO 94/21794 on the genetic modification of fruit properties. Concerning recombinant production of enzymes useful for flavour and/or aroma enhancement:

WO 96 29416 (26-09-1996) "ASPERGILLUS ARABINOFURANOSIDASE", illustrating the cloning of fungal enzymes having α-arabinofuranosidase activity and expression thereof in useful host organisms for production purposes and/or to improve their technological properties;

Wine yeast strain improvement using recombinant DNA techniques ES 2 059 280 (1-11-1994) "WINE YEAST CECT1973 RECOMBINANT FOR T. LONGIBRACHIATUM ENDOGLUCANASE", illustrates wine yeast strain improvement by cloning and expression of the endoglucanase from Trichoderma longibrachiatum in Saccharomyces cerevisiae under the control of the actin promoter;

WO 96/41888 (27-12-1996) "YEAST STRAINS HAVING A MODIFIED ALCOHOLIC SUGAR FERMENTATION BALANCE, USES THEREOF, AND VECTORS FOR PRODUCING SAID STRAINS", illustrating wine yeast strain improvement with a view to low alcohol wine production;

EP 0 751 997 (28-09-1995) "DNA ENCODING ENZYMES OF THE GLYCOL YTIC PATHWAY FOR USE IN ALCOHOL PRODUCING YEAST", illustrating yeast strain improvement by genetic engineering of the Entner-Doudoroff pathway;

WO 97/02341 (23-01-1997) "β-GLUCOSIDASE FROM FILAMENTOUS FUNGI, AND USES THEREOF", illustrating the use of glucose-insensitive β-D-glucosidase for the improvement of flavour and aroma in citrus fruit juices.

Other suitable microorganisms which are contemplated as host for the genes coding for the enzymes according ot the invention are microorganisms commonly used for malolactic fermentation during in wine making processes, such Lactococcus, Lactobacillus species and the like; the microorganisms so transformed preferably produce the enzymes and secrete it into the must/wine during malolactic or maloethanolic fermentation. Also wine yeast transformed by the genes coding for the enzymes according to the invention may advantageously be used to change the character (aroma and flavour) or color of the wine when used in the vinification process.

The above references merely serve to indicate the advanced state of the prior art with regard to gene cloning and expression in genetically modified organisms; they are in no way intended to indicate limitations to the way the enzymes according to the invention can be produced or used. Those of skill in the art will be able to readily recognise numerous other applications of the enzymes according to the invention.

The following examples serve to illustrate the invention.

Experimental Materials Enzvme preparation

A commercial enzyme preparation from Aspergillus niger Cytolase PCL5 (Gist-brocades-Seclin, France) was used as source for β-D-glucosidase.

Substrates for enzvme activity assays Cellobiose and -NP-β-D-glucopyranoside (p-NP-β-D-glucoside), pNP~ αL-glucopyranoside, pNP-β-D-galactopyranoside, pNP-αL-arabinofuranoside, pNP-αL-rhamnopyranoside, and pNP-β-D-cellobioside were purchased from Sigma (USA).

Geranyl-β-D-glucoside was a generous gift from Dr Z. Gϋnata (Laboratoria des Arόmes et des Substances Naturelles-IPV-INRA-Montpellier, France) (Fig. 1). The method to prepare Geranyl-β-D-glucoside and other synthetic substrates has been described in European patent application 89200592.7, published as EP 0 332 281 on September 13, 1989, pages 4 et seq..

Malvidin-3-β-D-glucoside was purified from an anthocyanin extract of Vitis vinifera var. grenache noir.

Anthocyanin extraction. Grape berries skins from Vitis vinifera var. Grenache noir were macerated 72 hours at 4°C in CH₃OH before crushing and final maceration 2 hours in CH₃OH. After centrifugation, the supernatant was recovered, acidified with HCOOH up to 0.5% and filtered before evaporation.

Extraction from grape berries skins from Vitis vinifera var. cabernet franc were isolated as described for var. Grenache noir, except that no evaporation was carried out. Anthocyanin separation. The anthocyanin extract was applied to a Polyclar AT (4x 34 cm) column equilibrated with water. Sugars, tannins and phenolic acids were first eluted with water (Glories, Y (1976) Conn. Vigne Vin 10, 51-57). Then anthocyanins were eluted with CH₃OH/H₂O/HCL (70:30:1; v/v/v) on the column. Malvidin-3-glucoside was eluted first and could be recovered separately, evaporated and freeze-dried.

Malvidin-3-glucoside analysis. Fractions were analyzed on a Lichrospher 100 RP 18 column (0.4 x 20 cm; Merck, Germany) with a gradient of two solvents: A, H₂O/HCOOH (98:2; v/v) and B, H₂O/CH₃CN/HCOOH (80:18:2; v/v/v): 3% B in 91% A (0-7 min); linear gradient up to 20% B (7-22 min); linear gradient up to 32 % B (22-30 min); linear gradient up to 35% (30-35 min); linear gradient up to 40% B (35-40 min); linear gradient up to 50% B (40-45 min); linear gradient up to 80% B (45-50 min); linear gradient up to 3% B (50-55 min). Products were detected with a Diode Array Detector 440 (Kontron) at 280, 313, and 515 nm. The malvidin-3-glucoside was estimated as 98% pure by multiwavelength analysis.

Inhibitors

Glucose, gluconolactone and deoxynojirimycin were purchased from Sigma (Fig. 2).

Enzyme assays

Enzvme activity towards p-NP-glvcosides

The enzymes were incubated 20 min. at 40°C in 50 mM sodium acetate pH

4.2 containing 2mM of the respective / NP-glucoside. Cellobiohydrolase activity was measured on 2mM -NP-β-D-cellobioside in the presence of 10 mM gluconolactone.

The reaction was stopped by addition of 1 M Na₂CO₃ and the activity was expressed in nkat*mg-' .

Enzvme activity towards cellobiose The enzymes were incubated 20 min. at 40°C in 50 mM sodium acetate pH

4.2, containing cellobiose at a concentration range 0.2-0.6 mM. The reaction was stopped by addition of NaOH up to 1 mM and reaction products were analyzed by high performance anion exchange chromatography (HPAEC) with a Dionex DX-300 chromatography system equipped with a PAD detector, using a CarboPac PA-1 column (0.4 x 25 cm; Dionex, USA) with a CarboPac guard column (0.4 x 5 cm) eluted at 1 ml min^"1 with 40 mM sodium acetate in 100 mM sodium hydroxide. The activity was expressed in nkat*mg^"1 as half of the glucose released per second.

Enzvme activity towards malvidin-3-glucoside

The enzymes were incubated 30 min. at 40°C in 50 mM sodium acetate pH 3.6, containing malvidin-3-glucoside at a concentration range 0.05-0.3 mM. The reaction was stopped by addition of HCl up to 0.1 N and the activity was measured by following the decrease of malvidin-3-glucoside peak measured by HPLC on a Lichrospher 100 RP 18 column (0.4 x 20 cm; Merck, Germany) with a gradient of two solvents: A, H₂0/HCOOH (98:2; v/v) and B, H₂0/CH₃CN/HCOOH (80:18:2; v/v/v). The peak area was reported to a calibration curve and the activity expressed in nkat*mg^'1.

Enzvme activity towards geranyl-β-D-glucoside

The enzymes were incubated 20 min. at 40°C in 50 mM sodium acetate pH

4.2, containing geranyl-β-D-glucoside at a concentration range 0.1-0.4 mM. The reaction was stopped by addition of NaOH up to 1 mM. The activity was measured by following the glucose released by HPAEC on the CarboPac PA-1 column but eluted with 100 mM sodium hydroxide and expression in nkat*mg^"'.

Decolorisation activity. During the purification, the decolorisation of an anthocyanin extract from Vitis vinifera var. cabernet franc was assayed for each fraction. The enzyme fraction was incubated for 30 min. at 40°C in 50 mM sodium acetate pH 3.6, containing 1% anthocyanin extract. The reaction was stopped by addition of HCl up to 0.1 M and the decolorisation was expressed in ΔA*min^"' by following the decrease of Absorbance at 515 nm.

Enzyme activity towards cyanidin- and peonidin-3-glucoside The enzymes were incubated for 30 min at 40°C in 50 mM sodium acetate pH 3.6, containing cyanidin-3-glucoside and peonidin-3-glucoside. The reaction was stopped by addition of HCl up to 0.1 M. The cyanidin-3-glucoside and peonidin-3-glucoside containing incubation media were prepared to obtain by HPLC the same peak area at 515 nm as a 0.4 mM malvidin-3-glucoside solution. The activity was expressed as relative activity compared to the activity on malvidin-3-glucoside.

s β-D-glucosidase purification

Unless otherwise indicated, all purification steps were performed at room temperature. The purification was followed by measuring the activity of each fraction on 2 mM -NP-β-D-glucopyranoside and on the anthocyanin extract from cabernet franc (decolorising activity). Elutions were performed with a Waters 650-E protein o purification system (Waters, USA).

Starting from Cytolase^®-PCL5: Step 1: ammonium sulphate precipitation. Solid (NH₄)₂SO₄ was added to 100 ml of crude enzyme preparation give 90% saturation and the solution was kept s overnight at 4°C. The precipitate was collected by centrifugation and washed two times with a 90% saturated (NH₄)₂SO₄ solution.

Step 2: hydrophobic interaction chromatography. The pellet was dissolved in 50 mM sodium phosphate pH 7.2 containing 1 M (NH₄)₂SO₄ and 0 applied to an Octyl-Sepharose CL-4B column (8 x 5 cm; Pharmacia, Sweden) equilibrated in the same buffer. Proteins were eluted at 2 ml min^"1 with the following gradient of (NH4)2SO4: isocratic at 950 mM (0-100 min); linear gradient 950->0 mM (100-300 min.).

s Step 3: anion-exchange chromatography. The unbound fraction was dialysed against 50 mM sodium acetate pH 4.8 and applied to a Q-Sepharose Fast-Flow column (2.6 x 10 cm; Pharmacia) equilibrated in the same buffer. Proteins were eluted at 5 ml min^"1 with the following gradient of NaCl: isocratic at 0 mM (0-10 min); linear gradient 0 ->200 mM (10 to 50 min); isocratic at 200 mM (50-80 0 min); linear gradient 200->300 mM (80-100 min); linear gradient 300->950mM (100-120 min).

Step 4: size exclusion chromatography. The fraction eluted at 200 mM NaCl was dialysed against 20 mM sodium acetate pH 4.8 and applied to a Sephadex S-200 HR column (1.6 x 95 cm; Pharmacia) equilibrated in the same buffer at 1 ml min^'1.

s Step 5: anion-exchange chromatography (D-Zephyr pH 6.5)

The excluded fraction was dialysed against 50 mM sodium acetate pH 6.5 and applied to a D-Zephyr/06 column (0.6 x 9 cm; Biosepra, France) equilibrated in the same buffer. Proteins were eluted at 1 ml min^"1 with the following gradient of NaCl; isocratic at 0 mM (0-15 min); linear gradient 0->200 mM (75-85 min); linear o gradient 200->400 mM (85-145 min); isocratic at 400 mM (225-240 min); linear gradient 400 -> 950 mM (240-250 min).

Step 6: anion-exchange chromatography (D-Zephyr pH 7.2)

The unbound fraction was dialysed against 50 mM sodium acetate pH 7.2 and s applied to the D-Zephyr/06 column, equilibrated in the same buffer. Proteins were eluted at 1 ml min^'1 with the following gradient of NaCl: isocratic at 0 mM (0-15 min); linear gradient 0->100 mM (15-75 min); isocratic at 100 mM (75-90 min); linear gradient 100->200 mM (90-150 min); isocratic at 200 mM (150-165 min); linear gradient 200-400 mM (165-225 min); isocratic at 400 mM (225-240 min); 0 linear gradient 400->950 mM (240-250 min). The β-D-glucosidase was eluted at 75 mM of NaCl.

Starting from AR2000^®: The same gradients were applied in hydrophobic interaction chromatography and anion-exchange chromatography on Q-Sepharose as for the purification of 5 β-glucosidase starting from Cytolase^®-PCL5 (above). Two chromatography steps were omitted: size-exclusion on Sephadex S-200 HR, and anion-exchange on

D-Zephyr at pH 6.5. The chromatography on D-Zephyr at pH 7.2 was modified as follows.

o step 4: anion-exchange chromatography (D-Zephyr pH 7.2)

The fraction eluting at 200 mM NaCl from the Q-sepharose Fast Flow column was dialysed against 50 mM sodium acetate pH 7.2 and applied to the D-Zephyr/0.6 column equilibrated in the same buffer. Proteins were eluted at 1 ml min^"1 with the following gradient of NaCl; isocratic at 0 mM (0-15 min); isocratic at 100 mM (15-45 min); isocratic at 120 mM (45-75 min); isocratic at 140 mM (75-105 min); isocratic at 160 mM (105-135 min); isocratic at 180 mM (135-165 min); isocratic at 200 mM (165-195 min); linear gradient 200 -> 400 mM (295-210 min); isocratic at 400 mM (210-220 min); linear gradient 400 -> 950 mM.

Enzyme purity and characterization

Protein determination. Protein concentrations were determined with ovalbumin as a standard (Lowry, O.H., Rosebrough, J., Farr, A.L. & Randall, R.J. (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265-276).

Molecular weight determination. Molecular weight was determined on a 6.5% polyacrylamide gel (Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage, Nature 227. 680-685) stained with Coomassie Brilliant Blue R-250 (Merck, Germany) and calibrated with molecular weight markers from 14.4 to 212 kDa (Electrophoresis LMW Calibration Kit, Pharmacia).

Temperature and pH optimum. The optimum temperature was determined with 20 minute (min) incubations on p-NP-β-D-glucoside in sodium acetate 50 mM pH 4.2 at temperature range 20-80°C. The optimum pH was determined using 20 min incubations at 40°C on p-NP-β-D-glucoside in universal buffer solutions from pH 2.0 to 12.0.

Temperature and pH stability. Temperature stability was studied after 60 min. incubations in sodium acetate 50 mM pH 4.2 at 20 to 80°C, pH stability was studied after 60 min. incubations in universal buffer from 2.0 to 12.0 at 25°C. The remaining activity was measured on p-NP-β-D-glucoside as described above.

Kinetic studies

Kinetic constants. Kinetic constants were determined on p-NP-β-D-glucoside, malvidin-3-glucoside, cellobiose and geranyl-β-D-glucoside. Affinity (K_m, mM) and catalytic (k^, nkat*mg^"') constants were calculated with three linear representations of the Michaelis-Menten equation: Lineweaver-Burk, Hanes and Scatchard (Fig. 3).

Inhibition constants. The inhibition of β-D-glucosidase activity on p-NP-β-D-glucoside by three competitive inhibitors of β-glucosidases, glucose, gluconolactone, and deoxynojirimycin, was studied (Winchester, B, (1992) Glycosidases and glycosyltransferases, Biochemical Society Transactions 20, 699-705); Neverova, I., Seaman, C.H., Srivastava, O.P., Szweda, R., Vijay, I.K. & Palcic, M.M. (1994) A spectrophotometric assay for glucosidase I, Anal. Biochem. 222, 190-195). Inhibition constants (K_:, mM) were determined at two concentrations with the three representations of the Michaelis-Menten equation.

Example 1 Purification of β-D-glucosidase from Cytolase^®-PCL5, an enzyme preparation from Aspergillus niger.

The first purification steps (ammonium sulphate precipitation and hydrophobic interaction chromatography) were required to eliminate the brown pigments from the crude enzyme preparation. However, these preliminary steps induced a 50% loss of total and specific activities. The specific activity of the β-D-glucosidase containing preparation increased from step 3 (anion exchange chromatography on Q-Sepharose Fast Flow column) (Table 1).

Table 1. Purification of β-D-glucosidase from Cytolase ^®-PCL5, an enzyme preparation from Aspergillus niger

Fraction Totact Totprot Spec.act. Purification

μkat mg nkat*mg^"' fold

Crude enzyme 334 1300 260 1.0

(NH₄)₂SO₄ 226 1150 2000 0.8

Octyl-sepharose 160 1150 140 0.5

Q-sepharose 42 150 280 1.1

Sephadex S-200 HR 25 53 460 1.8

D-Zephyr (pH = 6.5) 18 13 1420 5.5

D-Zephyr (pH = 7.2) 13 6 2170 8.4 As the enzyme was adsorbed on the anion exchange chromatography at pH 4.8, its isoelectric point is probably acidic and inferior to 4.0 (Fig. 4). The β-D-glucosidase activity was excluded on a Sephadex S-200 HR column indicating that its apparent molecular weight was greater than 200 kDa. Two additional anion exchange 5 chromatography steps on D-Zephyr were required to achieve the purification. The finally purified β-D-glucosidase has a specific activity of 2170 nkat*mg"¹ on p-NP-β-D-glucoside.

β-D-glucosidase properties o Homogeneity and molecular weight. The β-D-glucosidase was purified to homogeneity and SDS/PAGE analysis revealed an homogeneous protein band with an apparent molecular weight of 115 kDa (Fig. 6). The fact that the β-D-glucosidase fraction was excluded on Sephadex S-200 HR is consistent with a possible oligomeric nature. s General properties. The activity of the purified β-D-glucosidase was maximum at pH 4.0 and 60°C. The enzyme was stable between pH 2.0 and 9.0, and 20 to 50°C. A rapid loss of activity was obtained after 60 min. incubations at pH 10 or at 60°C (Fig. 7).

Activity on p-NP-glycosides. The purified enzyme was highly specific for 0 p-NP-β-D-glucoside since the activities on other p-NP-glycosides represented less than 1% of the β-D-glucosidase activity (Table 2).

Table 2. Activity and relative activity of β-glucosidase on p-NP-β-D-glycosides. Activity was measured by incubation of the enzyme 20 min at 40 °C in 50 mM s sodium acetate pH 4.2, containing 2 mM p-NP-β-D-glycoside.

Substrate Total activity Relative activity

nkat % 0 p-NP-β-D-glucopyranoside 12740 100 p-NP-α-D-glucopyranoside 0 0 p-NP-β-D-galactopyranoside 25 0.2 p-NP-α-L-arabinofiiranoside 2 0.02 p-NP-α-D-rhamnopyranoside 4 0.03 5 p-NP-β-D-cellobioside 56 0.4 The -enzyme had no detectable activity on p-NP-α-L-glucoside. The highest side-activity was measured on p-NP-β-D-cellobioside but this 0.4% relative activity may be due to an incomplete inhibition of β-D-glucosidase by 10 mM gluconolactone.

Kinetic constants on 0-D-glucosylated substrates. The purified β-D-glucosidase was also active on three substrates representing the technological targets of β-D-glucosidases: cellobiose, malvidin-3-glucoside, and geranyl-β-D-glucoside. The affinity constants (K_m) were similar and comprised between 0.43 and 0.5 mM (Table 3). However, the activity levels were very different.

Table 3. Kinetic constants of the /5-D-glucosidase on various substrates. Activity was measured by incubation of the enzyme 20 min at 40°C in 50 mM sodium acetate pH 4.2, contaimng p-NP-β-D-glucoside from 0.05 to 0.5 mM, cellobiose from 0.2 to 0.6 mM, malvidin-3-glucoside from 0.2 to 0.6 mM, malvidin-3-glucoside from 0.075 to 0.3 mM, and geranyl-β-D-glucoside from 0.10 to 0.35 mM.

mM nkat*mg^'' p-NP-β-D-glucoside 0.43 3240 cellobiose 0.50 3335 malvidin-3-glucoside 0.48 18 gerany 1- β-D-glucoside 0.44 470

A similar high catalytic constant k_at was calculated for cellobiose and p-NP-β-D-glucoside (about 3000 nkat mg). At the same concentration, glucose was released from the aroma precursor, geranyl-β-D-glucoside, but with a 15% relative activity when compared to p-NP-β-D-glucoside. Malvidin-3-glucoside was degraded very slowly, and the corresponding /_cat represented less than 1% of the value obtained for cellobiose or p-NP-β-D-glucoside.

Inhibition constants. The three inhibitors tested gave each a competitive inhibition of the β-D-glucosidase. The inhibition constant K_{ reflected that deoxynojirimycin was a strong inhibitor when compared to gluconolactone and glucose (Table 4).

Table 4. Inhibition constants of the β-D-glucoside on p-NP-β-glucoside. Activity was measured at various concentrations of p-NP-β-glucoside from 0.5 to 3 mM in the presence of glucose at 10 and 40 mM, gluconolactone at 0.5 and 1 mM, deoxynojirimycin at 0.005 and 0.01 mM.

Inhibitor K

mM glucose 2.5 gluconolactone 0.15 deoxynoj irimycin 0.002

Example 2

The activity of the purified β-glucosidase was tested on several anthocyanidin-glucosides. The results are presented in Table 5.

Table 5. Activity and relative activity of the purified β-D-glucosidase on anthocyanidin-glucosides: mv-3-glc, malvidin-3-glucoside; cya-3-glc, cyanidin-3-glucoside; peo-3-glc, peonidin-3-glucoside; mv-3-glc-coum, malvidin-3-p-coumaroyl-glucoside; cya-3-glc-coum, cyanidin-3-p-coumaroyl-glucoside; peo-3-glc-coum, peonidin-3-p-coumaroyl-glucoside; cya-3-rut, cyanidin-3-rutinoside

Substrate Total activity Relative activity

nkat % mv-3-glc 2 100 cya-3-glc 1.2 60 peo-3-glc 1 50 mv-3-glc-coum 0 0 cya-3-glc-coum 0 0 peo-3-glc-coum 0 0 cya-3-rut 0 0 Example 3

Decolorization of a total anthocyanin extract from Vitis vinifera var. cabernet franc

Anthocyanin composition. A Cabernet franc extract was analyzed by HPLC on a Lichrospher 100 RP 18 column (0.4 x 20 cm; Merck Germany) with a gradient of two solvents; A, H₂0/HCOOH (98:2 v/v) and B, H₂O/CH₃CN/HCOOH (80:18:2; v/v/v): 3% B in 97% A (0-7 min); linear gradient up to 20% B (7-22 min); linear gradient up to 32% B (22-30 min); linear gradient up to 35% B (30 35 min); linear gradient up to 40% B (35-40 min); linear gradient up to 50% B (40-45 min); linear gradient up to 80% B (45-50 min); linear gradient up to 3% B (50-55 min).

Table 6: Anthocyanin composition of Cabernet franc

anthocyanidins Relative amount % peak 1 delphinidin-3-glucoside 10 peak 2 cyanidin-3-glucoside 5 peak 3 petunidin-3-glucoside 15 peak 4 peonidin-3-glucoside 15 peak 5 malvidin-3-glucoside 42

Decolorizing activity of the purified β-glucosidase

In the experiment 5 μl of a solution of the purified β-glucosidase, representing 1.85 nkat of β-glucosidase activity (0.85 μg), was added to 1 ml of 1% anthocyanin extract in 50 mM sodium acetate pH 3.6, and incubated for 30 min at 40°C. Similarly, 0.2 μl of crude enzyme preparation, representing 0.67 nkat of β-glucosidase activity (2.6 μg protein) was added to 1 ml of 1% anthocyanin extract in 50 mM sodium acetate pH 3.6, and incubated for 30 min at 40°C. The reaction was stopped by addition of HCl up to 0.1 M and the decolorization was expressed in ΔA.min^'Vnkat β-glucosidase activity by following the decrease of absorbance at 515 nm. The specific decolorization activity of the purified β-glucosidase is 2.2 ΔA.min^'Vmg, which is equivalent to approximately 1 ΔA.min^"Vnkat β-glucosidase activity, compared to 2.7 ΔA.min^'Vmg for the crude extract, which is equivalent to 10.8 ΔA.min^'Vnkat β-glucosidase activity. This example indicates, that the purified β-glucosidase can advantageously be used in a vinification process using red grapes, in order to improve aroma release, without decolorization. The affinity of the purified preparation for anthocyanins, expressed as ΔA.min^"Vnkat β-glucosidase activity is decreased roughly by a factor 10, in this experiment.

Example 4 The performance of the purified β-glucosidase was followed in a microvinification experiment using the red grape variety Pinot noir, which is known for its high content in anthocyanidin-glucosides and its low content in the corresponding acylated and p-coumaroylated derivatives. The rationale behind this choice was, that β-glucosidases are generally less effective on acylated p-coumaroylated substrates (see Table 5).

The grapes have been harvested from the collection of the ENSA-M (Ecole Nationale Superieure d'Agronomie de Montpellier).

Microvinification: The vinification has been established at the station experimentale INRA de Pech Rouge Narbonne. Mature grapes were picked and destemmed. Three glass tanks were filled each with 1,5 kg of whole berries to perform the following small scale vinifications: wine T without enzyme addition (control wine), Wine A with addition of the purified β-glucosidase, and Wine B with addition of the crude enzyme preparation Cytolase^®-PCL5. The berries were crushed and sulfited (final concentration 16 mg/1 of S0₂).

After addition of 200 mg of wine yeast Fermivin^® (Gist-Brocades), the glass tanks were saturated with CO₂ and placed in a room at 23 °C for the alcoholic fermentation. The glass tanks were frequently stirred by manual shaking. Fermentation was complete after 15 days and the resulting wines (10.7 % alcohol) were obtained by direct pressing of the whole content of each tank.

As the malolactic fermentation had not started after 20 days, 10 ml of a Syrah wine in the process of malolactic fermentation was added to the glass tanks. After 7 days, as the malolactic fermentation had still not begun, the wine was centrifugated, bottled and lagered at 20°C.

Addition of enzyme: Enzymes were added after pressing of the grapes. The purified β-glucosidase was added in an amount of 0.06 mg, which is equivalent to 125 nkat, per litre must. The crude enzyme preparation (Cytolase^®-PCL5) was added in an amount of 40 mg/1 of must, giving a similar amount of 135 nkat β-glucosidase activity per litre must.

Color monitoring: color development was monitored spectrophotometrically at 520 nm. The samples were centrifugated and diluted in 50 mM sodium acetate buffer pH 3,6 prior to measurement.

Color development during vinification: The evolution of color development was followed in parallel in the three wines A, B and T: - a strong increase of the A₅₂₀ during the first days, associated with the release of colored matter due to maceration;

- a second increase of the intensity of the red color arising from the 15th day onward, corresponding to the end of the alcoholic fermentation and the pressing of the content of each glass tank. The addition of 10 ml of Syrah wine participated in the A₅₂₀-increase between day 33 and 39;

- a drop in the intensity of the color at day 39, in conjunction with the loss of part of the colored matter upon centrifugation prior to lagering;

- the effect on color of the anthocyanase present in the crude preparation (wine B) was evident right from the very first days: wine B showed a lower A₅₂₀ than the control and wine A. The percentage of discoloration of wine B tends to increase at the end: after 68 days it amounts to 42%.

Wine A, to which the purified β-glucosidase has been added, shows a little less color up to day 42, but after day 42 the red color of wine A which is equivalent to that of the control, yet slightly more intense. Appreciation of the color by the naked eye: protocol: The visual test has been carried out 64 days after the start of the vinification. About 50 ml of each wine was poured into three numbered glasses: 0 for wine A, 1 for the control, and 2 for wine B. The visual test was carried out with a panel of 22 persons. The question was: Which wine looks different?

The result: Out of 22 persons, 19 (86%) selected glass 2 (Wine B) as being different, qualifying it as "less coloured". Two persons selected either glass 0 or glass 1 as being different. One person indicated to be unable to see any difference. The crude enzyme preparation, added to Pinot noir in an oenological quantity, immediately induces a discoloration as measured by photospectrometry at A₅₂₀ and as observed after 60 days by a panel of 22 persons, whereas with the purified β-glucosidase no difference was observed. It is concluded that the purified β-glucosidases can safely be used in oenological quantities to enhance the aroma of red wines, without the concommittant loss of color intensity observed when commercially available preparations are being used.

The results of a similar experiment, comparing the effect of the purified anthocyanase (see below) with the purified β-D-glucosidase and a control wine, is depicted in Figures 16 and 17. (Example 7).

Example 5 The effect of the purified β-glucosidase on aroma release in micro vinification using Muscat grapes

Aroma enhancing of wines is mainly due to the hydrolysis of terpenyl glycosides into terpenol. The main terpenyl glycosides in muscat grapes are 6-O-α-L-arabinofuranosyl-β-D-glucopyranosides of geraniol, nerol and linalool (G nata, 1988, Carbohydr. Res. 184, 139-149). The release of terpenol from 6-O-α-L-arabinofuranosyl-β-D-glucopyranosyl-terpenol is obtained through the sequential action of an α-L-arabinofuranosidase, which releases terpenyl glucoside and a β-D-glucopyranosidase, which finally releases the terpenol (Gϋnata et al. , 1988, supra).

preparation of the wines. A muscat wine (residual glucose + fructose inferior to 2 g/1) was produced at the INRA of Colmar (Station de Recherche Vigne et Vin, Laboratoire d'Oenologie, Colmar). To 200 ml wine samples were added (figure 7):

- no enzymes for the control;

- 800 nkat of a purified α-L-arabinofuranosidase (sample A) (Saulnier et al., 1992, Carbohydr. Res. 224, 219 235);

- 800 nkat of the purified α-L-arabinofuranosidase and 680 nkat of the purified β-D-glucosidase (sample B).

The samples were incubated 8 days at 20°C. Extraction of the free aroma fraction. 150 ml of each wine sample was diluted with 450 ml of water, 96 mg 4-nonanol was added as an internal standard to the mixture. Volatile aroma compounds were extracted four times with 25 ml of dichloromethane/pentane (1:2; v/v). The extracts were frozen and filtered on anhydrous sodium sulphate to eliminate residual water and then concentrated to 2 ml (figure 7).

Gas chromatography analysis. A Varian (Palo Alto, CA, USA) gas chromatograph equipped with a retention gap and a DB-Wax capillary column (30 m x 0.32 mm i.d.; 0.5 mm film thickness) (J&W Scientific, Folsom, CA, USA) was used.

Hydrogen was used as carrier gas at 0.95 ml min. Samples (1 ml) were injected into column at 20°C; the injector was heated at 180°C/min to 245°C and 90 min. at 245°C. The Oven temperature was programmed from 60 to 245°C at 3°C/min with initial hold time 3 min at 60°C and final hold time 20 min at 245°C. The detector (FID) temperature was 245°C.

Relative amounts of compounds were reported to the internal standard 4-nonanol.

Identification of the compounds by GC-MS. Samples were analyzed with a Hewlett Packard 5890 (Palo Alto, CA, USA) gas chromatograph coupled with a Hewlett Packard 5989 A mass spectrometer. The chromatographs were fitted with the same column and GC conditions as above but helium was used as carrier gas at 1.35 ml/min. Spectra were recorded in the electron impact (El) mode (70 eV, source temperature 250°C, quadruple detector temperature 120°C). Mass spectra were obtained from m/z 29 to m/z 350 at intervals of 1 sec.

GC-MS analysis of the three samples revealed the presence of some free aromatic molecules such as linalool (peak 2), geraniol (peak 3), 3,7-dimethylocta-l,5-diene-3,7-diol (peak 4), 4-vinylgaiacol (peak 5), 3,7-dimethyloct-l,5-diene-3,8-diol (peak 6) and geranic acid (peak 7) (figure 8A). These aroma compounds are known to be present in muscat wines both as free volatile molecules and as their glycosylated non-volatile precursors. Addition of α-L-arabinofuranosidase alone (sample A, figure 8B) had no influence on the aromatic profile since no significant difference could be observed by GC or GC-MS analyses.

The treatment with both α-L-arabinosidase and the β-D-glucosidase (figure 8C) induced a 15% increase of 3,7-dimethylocta-l,5-diene-3,7-diol and 4-vinyl-gaiacol and a dramatic increase of geraniol (340%), s 3,7-dimethylocta-l,5-diene-3,8-diol (265%) and geranic acid (305%) (Table 7).

Table 7: Effects of the purified β-D-glucosidase from A. niger on some aromatic compounds of muscat wine, diol-3, 7: 3, 7-dimethylocta-l,5-diene-3, 7-diol; diol-3,8: 3, 7-dimethylocta-l, 5-diene-3, 8-diol. 0

Retention time Relative increase

(min) (%) linalool 23.6 2 geraniol 35.4 340 5 diol-3,7 38.8 15

4-vinyl-gaiacol 46.9 13 diol-3,8 50.8 264 geranic ; acid 51.9 305

0

It is concluded that the purified β-D-glucosidase can be used to improve the aroma of wines that contain glucosidic precursors of terpenols and other aromatic alcohols.

s Example 6

Amino acid sequence determination of the purified β-glucosidase

The sequence analysis is performed by Edman degradation (Edman, P., 1956, Acta Chem. Scand 10, 761-768; Use, D. & Edman P., 1963, Aust. J. Chem.16, 41 1-416 with an automated sequenator (Model 477 A, Applied Biosystems (Hewick, 0 R.M., Hunkapiller, M.W., Hood, L.E. & Dreyer, W.J., 1981, J. Biol. Chem. 15, 7990-7997) using protocols, reagents, chemicals and materials from Applied Biosystems (Warrington, U.K. and Foster City, California, U.S.A.). Step-wise released phenylthiohydantoin amino acids are identified with an on-line HPLC (Model 120A, Applied Biosystems) on the basis of their elution times 5 ('Chromatogram Reports'). For this reason, one or more calibrations are performed prior to each sequence analysis. The internal sequences which were determined are represented in SEQIDNOs 1 and 2.

Example 7 Characterization of the purified anthocyanase activity

To verify the nature of the decolorising activity, the enzymatic activity eluting at 120 mM was characterised. (For methodology, see Experimental part).

The initial purification steps are exactly as described for the purified β-glucosidase. The anthocyanase was separated from the β-D-glucosidase on D-Zephyr at pH 6.5, indicating that its isoelectric point is slightly more acidic (Fig. 12). The purification profile is given in Table 8:

Table 8. Purification of the anthocyanase from Aspergillus niger

Homogeneity and Molecular weight. The anthocyanase was purified to homogeneity and SDS/PAGE revealed a homogeneous protein band with an apparent molecular weight of 109 kDa. The fact that the anthocyanase fraction was excluded on Sephadex S-200 HR can be explained by its putative oligomeric nature. General properties. β-D-glucosidase of the purified enzyme was maximum at pH 3 and 70°C and stable between pH 2.0 and 9.0, and 20 to 50°C. A rapid loss of activity was observed after 60 min at pH 10 or 60°C.

Activity on p-NP-glycosides. The purified enzyme was highly specific for pNP-β-D-glucoside since the activities on other p-nitrophenyl-glycosides represented less than 2% of the β-glucosidase activity (Table 9).

Table 9. Activity and relative activity of the purified anthocyanase on p-NP-β-D-glycosides. Activity was measured by incubation of the enzyme 20 min at 40° C in 50 mM sodium acetate pH 4.2, containing 2 mM p-NP-β-D-glycoside.

Substrate Total activity Relative activity

nkat % p-NP-β-D-glucopyranoside 415 100 p-NP-α-D-glucopyranoside 0 0 p-NP-β-D-galactopyranoside 7 2 p-NP-α-L-arabinofuranoside 3 0.7 p-NP-α-D-rhamnopyranoside 0 0 p-NP-β-D-cellobioside 0 0

Kinetic constants on β-D-glycosylated substrates. The isolated anthocyanase was assayed for its activity on three substrates representing the technological targets of β-D-glucosidases. The enzyme induced the release of glucose, and geranyl- β-D-glucoside. The affinity constants K^) were comprised between 0.11 and 0.27 mM (Table 10). However, the activity levels were very different. The purified enzyme presented the highest activity towards malvidin-3-glucoside with a catalytic constant (k^ superior to 800 nkat*mg^"1. The activity was reduced by half on pNP-β-D-glucoside and the activity levels on cellobiose and geranyl-β-D-glucoside were very low, representing less than 1% of the activity on malvidin-3-glucoside (Table 10).

Activity on cyanaidin-, peonidin-, and malvidin-3-glucoside. The purified enzyme was active on the three anthocyanidin glucosides assayed. The highest activity level was observed on malvidin-3-glucoside (815 nkat*mg^"'). The relative activities on cyanidin- and peonidin-3-glucoside were similar and represented respectively 40 and 35 % of the activity measured on malvidin-3-glucoside (Table 10).

Table 10. Kinetic constants of the anthocyanase on various substrates.

Activity was measured by incubation of the enzyme for 20 min at 40°C in 50 mM sodium acetate pH 4.2, containing p-NP-β-D-glucosidase from 0.02 to 0.3 mM, cellobiose from 0.2 to 0.7 mM, malvidin-3-glucoside from 0.05 to 0.3 mM, and geranyl-β-D-glucoside from 0.1 to 0.3 mM.

Inhibition constants. To confirm the identity of the β-D-glucosidase activity measured on p-NP-β-D-glucoside and malvidin-3-glucoside, three known competitive inhibitors of β-D-glucosidase were tested. Glucose, gluconolactone and deoxynojirimycin each demonstrated competitive inhibition of the β-D-glucosidase (measured on p-NP-β-D-glucoside) and anthocyanase (measured on malvidin-3-glucoside) activity of the isolated enzyme. The K_x values were equivalent for both substrates; deoxynojirimycin appeared a very strong inhibitor (Table 11).

Table 11. Inhibition constants of the anthocyanase on pNP-β-D-glucoside and malvidin-3-glucoside.

Inhibitor p-NP- β-D-glucoside malvidin-3-glucoside mM mM

K_{ glucose 0.40 0.28

Afj gluconolactone 0.005 0.003

K_{ deoxynojirimycin 0.001 0.001 These results show the β-D-glucosidase nature of the anthocyanase according to the invention.

Example 8

Evolution of color during vinification of red grapes

The experiment of Example 4 was run with the purified anthocyanase. The purified anthocyanase was added in an amount of 125 nkat β-D-glucosidase activity as measured on p-NP-β-D-glucoside. The results are depicted in Figures 16 and 17. It is clear that the purified anthocyanase has a drastic effect on color intensity when present during vinification. These results suggest a potential application in the making of white or claret wines from red grapes and grape skins, as well as in champagne making. For example, the yield from the pressing of Pinot noir and Pinot meunier grapes can be increased without (undesired color).

Example 9 Amino acid sequence determination of the purified β-glucosidase

Determination of the amino acid sequence of the purified anthocyanase was carried out essentially as described for the purified β-glucanase, above. A partial amino acid sequence was obtained of 19 amino acids, which showed some resemblence to an E. coli periplasmic β-D-glucosidase. Of the partial amino acid sequence depicted in SEQIDNO: 3, Leu-1, Tyr- 18 and Leu- 19 are as yet uncertain. On the basis of these amino acid sequences oligonucleotide sequences can be designed which can be used in a suitable cloning strategy to obtain the cDNA or the gene coding for the anthocyanase from Aspergillus niger, as described in more detail for the β-glucosidase according to the invention. Large amounts of anthocyanase may be obtained by subsequent over-expression of the cDNA or the gene, as the case may be, in any suitable host organism, followed by recovery from the organisms cells, or the periplasm, or the medium wherein the cells are being grown. The following sets of oligomer primers may be synthesised for use in a PCR-based amplification of a DNA fragment coding for the β-glucosidase and the anthocyanase, respectively:

(a) GAA GGCA/C/G/TTAT/CCAA/GGAT/CTA (Glu-Ala-Tyr-Gln-Asp-Tyr) (b) TTT/C ATT/C/ATA T/CCCA C/G/TTGGC/TT (Phe-Ile-Tyr-Pro-Trp-Leu).

Example 10 Evolution of color and hue during the ageing of red wines from the grape variety Pinot noir

Color developments during the ageing

The wines produced using a microvinification experiment of a grape variety Pinot noir, wine T without enzyme addition (control wine), and wine B with addition of the purified anthocyanase as described in Example 8 were stored at 15°C for 15 months.

The red color (A520) and the hue (A420/A520) were followed during the ageing of both samples. The wine treated with the anthocyanase presented a faster evolution of these parameters (Figure 17) since its total absorbance represented less than 25% of the non-treated wine absorbance, while its hue reached a value of 1.3, characteristic of old wines (liao, H., Cai, Y. & Haslam, E. (1992) Polyphenol interactions,. Anthocyanins: Co-pigmentation and colour changes in red wines, J. Sci. Food Agric. 59, 299-305).

Thus, treatments of the must with the anthocyanase induced the production of a wine which presented, after one year of storage, the color characteristics of old wines.

The change from red-purple to red-brown color was attributed to the decrease of monomeric anthocyanins and the increase of anthocyanin polymers (Somers, T.C. (1971) The polymeric nature of wine pigments, Phytochem. 10, 2175- 2186). The polymerization may be catalysed by quercetin (Price, S.F., Breen, P.J., Valladao, M. & Watson, B.T. (1995) Cluster sun exposure and quercetin in Pinot noir grapes and wine, Am. J. Enol. Vitic, 46, 187-194; Wightman, J.D., Price, S.F., Watson, B.T. & Wrolstad, R.E. (1997) Some effects of processing enzymes on anthocyanins and phenolics in Pinot noir and Cabernet sauvignon wines, Am. J.

Enol. Vitic, 48, 39-48). The anthocyanase B hydrolysed quercetin-3-glucoside into quercetin and so contributed probably to the acceleration of anthocyanins polymerization.

Wine tasting

After 15 months of storage, both non-treated and anthocyanase-treated wines were submitted to a panel of four wine experts. They found the color, the hue and even the flavour of the anthocyanase-treated wine as being characteristic of a 2-5 years old Pinot noir wine.

It is concluded that the purified anthocyanase can be used in oenological quantities to accelerate the evolution of wine during ageing and improve their organoleptic characteristics.

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): 2611 XT

(G) TELEPHONE: 31.15.2793644 (H) TELEFAX: 31.15.2793957

A) NAME: Institut National de la Recherche Agronomique

B) STREET: 145 rue de l'Universite

C) CITY: Paris

E) COUNTRY: France

F) POSTAL CODE (ZIP): F-75341

(ii) TITLE OF INVENTION: Beta-glucosidases, methods for obtaining same, compositions containing said beta-glucosidase and use thereof

(iii) NUMBER OF SEQUENCES: 3

(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)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE: (A) ORGANISM: Aspergillus niger

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Glu Ala Tyr Gin Asp Tyr Leu Val Thr Glu Pro Asn Asn Gly 1 5 10

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Phe He Tyr Pro Tφ Leu Xaa Ser Thr Asp Leu Glu Ala Ser Ser Gly 1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 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 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Leu Ser Val Ser Phe Pro His Tyr Val Gly Asp Leu Pro He Tyr Tyr 1 5 10 15

Asp Tyr Leu

Claims

Claims

1. A substantially pure peptide having the following amino acid sequence Glu-Ala-Tyr-Gln-Xaal-Tyr-Leu-Val-Thr-Glu-Pro-Asn-Xaa2-Gly, wherein Xaal and

Xaa2 may be any DNA encodable amino acid.

2. A substantially pure peptide according to claim 1, wherein Xaal is Asp, and/or Xaa2 is Asn.

3. A substantially pure polypeptide having β-glucosidase activity but substantially no anthocyanase activity.

4. The substantially pure polypeptide according to claim 3, wherein said polypeptide comprises a part having an amino acid sequence

Glu-Ala-Tyr-Gln-Xaal-Tyr-Leu-Val-Thr-Glu-Pro-Asn-Xaa2-Gly, wherein Xaal and Xaa2 may be any DNA encodable amino acid.

5. A composition comprising a polypeptide having β-glucosidase activity, characterised in that said composition is low in β-glucosidase activity on an anthocy- anidin-glucoside substrate selected from the group consisting of malvidin-3-glucosi- de, delphinidin-3-glucoside, anthocyanidin-3-glucoside, petunidin-3-glucoside and peonidin-3-glucoside.

6. A composition according to claim 5, characterised in that the said β-glucosidase activity on an anthocyanidin-glucoside substrate selected from the group consisting of malvidin-3-glucoside, delphinidin-3 -glucoside, cyanidin-3-glucoside, petunidin-3-glucoside and peonidin-3-glucoside amounts to 20% or less, preferably 10% or less, of the total β-glucosidase activity in said composition.

7 A composition according to claim 5, wherein said polypeptide having β -g luco sidase activity c omprise s an amino acid sequence

Glu-Ala-Tyr-Gln-Xaal-Tyr-Leu-Val-Thr-Glu-Pro-Asn-Xaa2-Gly, wherein Xaal and Xaa2 may be any DNA encodable amino acid.

8. Use of a substantially pure polypeptide according to claim 4 or a composition according to any one of claims 5 to 7 in a process for releasing aromatic compounds from their glycosidic precursors.

9. Use according to claim 8, wherein said process is a process for making a beverage or a food product for human consumption or a feed product for animal consumption.

10. Use according to claim 8, wherein said process is a process for making a starting material for a beverage a food or feed, or a process for making an ingredient for use in a beverage, food or feed product.

11. Use according to any one of claims 8 to 10, wherein said glycosi- dic-precursor is selected from the group consisting of geraniol-glycosides, 3,7-dimethyl-octa-l,5-diene-3,7-diol, 3,7-dimethylocta-l,5-diene-3,8-diol-glycosides, 4-vinyl-gaiacolglycosides and geranic acid-glycosides.

12. Use according to any one of the preceding claims, wherein said beverage, food or feed product comprises a cyanidin-glycoside selected from the group consisting of malvidin-3-glucoside, delphinidin-3-glucoside, cyanidin-3-glucoside, petunidin-3-glucoside and peonidin-3-glucoside.

13. Use according to any one of the preceding claims, wherein said beverage product is red grape juice, or red wine.

14. Use of a substantially pure peptide according to claim 1 in a process of making a nucleic acid primer or primers and/or a nucleic acid probe which can be used to detect and/or amplify and/or isolate a nucleic acid sequence coding for at least part of a polypeptide having β-glucosidase activity.

15. A composition comprising a polypeptide having β-glucosidase activity, characterised in that the k^ of the said β-glucosidase for malvidin-3-glucoside represents less than 1% of the k_at for cellobiose or p-NP-β-D-glucoside.

5 16. A substantially pure peptide having anthocyanase activity comprising an amino acid sequence:

NH₂ - Leu Ser Val Ser Phe Pro His Tyr Val Gly Asp Leu Pro He Tyr Tyr Asp Tyr Leu - COOH.

ιo 17. Use of a substantially pure peptide according to claim 16 in a process of making a nucleic acid primer or primers and/or a nucleic acid probe which can be used to detect and/or amplify and/or isolate a nucleic acid sequence coding for at least part of a polypeptide having anthocyanase activity.

is

18. An isolated nucleic acid sequence encoding a peptide according to claim 16.

19. Use of a substantially pure polypeptide having anthocyanase activity and which comprises an amino acid sequence according to claim 16, to formulate an enzyme preparation.

20

20. Use of a substantially pure polypeptide having anthocyanase activity and which comprises an amino acid sequence according to claim 16, or an enzyme preparation according to claim 19 in a process for making a food, feed or beverage.

25 21. Use according to claim 20, wherein said beverage is wine, fruit juice or champagne.

22. Use of a substantially pure polypeptide having anthocyanase activity and which comprises an amino acid sequence according to claim 16, or an enzyme 30 preparation according to claim 19 in a process of hydrolysing an anthocyanidin-precursor.

23. Use of a substantially pure polypeptide having anthocyanase activity or a composition enriched in anthocyanase activity in a process of accelerating the ageing of a red wine.

24. Use according to claim 3, wherein the anthocyanase comprises an amino acid sequence as represented in SEQ ID NO: 3.