WO1996006163A1

WO1996006163A1 - Novel rhamnogalacturonan rhamnosidases

Info

Publication number: WO1996006163A1
Application number: PCT/DK1995/000337
Authority: WO
Inventors: Margien Mutter; Henk Arie Schols; Gerrit Beldman; Alphons Gerard Joseph Voragen
Original assignee: Novo Nordisk A/S
Priority date: 1994-08-18
Filing date: 1995-08-17
Publication date: 1996-02-29
Also published as: AU3253395A

Abstract

The present invention relates to hitherto unknown class of enzymes with rhamnogalacturonan rhamnosidase (RG-rhamnosidase) activity as well as the use of such enzymes for the degradation or modification of plant cell wall materials.

Description

NOVEL RHAMNOGALACTURONAN RHAMNOSIDASES

FIELD OF THE INVENTION

The present invention relates to a hitherto unknown class of enzymes with rhamnogalacturonan rhamnosidase (RG-rhamnosidase) 5 activity as well as the use of such enzymes for the degradation or modification of plant cell wall materials.

BACKGROUND OF THE INVENTION

In the processing of plant material, e.g. fruits and vegetables, endogenous enzymes are used as processing aids to

10 improve yield and quality of the end product (Pilnik, . , A.G.J. Voragen. , "Effect of enzyme treatment on the quality of processed fruits and vegetables", in: Jen J.J. , "Quality factors of fruits and vegetables, chemistry and technology", ACS Symp. Ser. 405, American Chemical Society, Washington DC,

15 250-269, 1989) .

Because of the complex nature of plant materials a variety of different enzymes are necessary for the modification or degradation of such materials. Such enzymes include pectinases, cellulases and hemicellulases. Although a variety of these 20 enzymes are known plant parts remain which cannot be degraded by any known enzyme.

Only recently enzymes have been identified, which are capable of degrading the so-called modified hairy regions (MHR) from apple and other plant cell walls. MHR isolated from apple cell

25 walls consists of an acetylated rhamnogalacturonan backbone, which consists of repeating units of the disaccharide α-l,2-L- Rha-α-l,4-D-GalA. (Schols, H.A. , M.A. Posthu us, A.G.J. Voragen, "Structural features of hairy regions of pectins isolated from apple juice produced by the liquefaction 0 process", Carbohydrate Research, 206, 117-129, 1990) . The hydroxyl at the C-4 position of the rhamnose serves as attachment point for side chains composed of neutral sugars, mainly arabinan, galactan and arbinogalactan.

Rhamnogalacturonases (RGases) have been isolated from the fungal species Aspercrillus aculeatus. These enzymes are capable of cleaving the backbone of MHR (Schols et al., 1990 and WO 92/19728) isolated from the liquefaction juice from different fruit and vegetable sources.

RGase liberates specific rhamnogalacturonan oligomers (RG oligomers) from the hairy regions backbone. These oligomers have the basic structure α-L-Rha-1,4-α-D-Gal-A-l, 2-L-Rha-l, 4-α- D-GalA (Colquhoun et al., 1990, Schols et al., 1994) . A β-Gal unit is 4-linked to approximately half of the terminal Rha residues and to half of the (1,2) -linked Rha residues. Furthermore, Searle-van Leeuwen et al. (1992, and WO 93/20190) disclose a novel acetylesterase that is specific for hairy regions of pectins.

Rhamnohydrolases belong to a group of enzymes capable of hydrolyzing rhamnosyl linkages. These enzymes have been found to be active towards the synthetic substrate pnp-α-L- rhamnopyranohydrolase, naringin and/or hesperidin (Romero et al., 1985, Hsieh and Tsen, 1991, Chase, 1974, Ono et al. , 1974). Bushway et al. (1988) and Bushway et al. (1990) described rhamnohydrolase activities that were able to liberate the Rha units from both alpha-solanine and alpha-charconine (both being glycoalkaloids present in potato. To date, however, no rhamnohydrolase has been described which is active towards MHR.

No enzyme or enzyme preparation has been described which can hydrolyze rhamnose groups from RG oligomers, and the object of the invention is to provide an enzyme capable of such hydrolysis. BRIEF DISCLOSURE OF THE INVENTION

The present inventors have surprisingly identified a novel enzyme which is capable of releasing rhamnose groups from RG- oligomers. This enzyme is termed rhamnogalacturonan-alpha- rhamnosidase or RG-rhamnosidase in the following disclosure.

Accordingly, in a first important aspect the invention relates to an enzyme with RG-rhamnosidase activity.

In the present context the term "RG-rhamnosidase activity" is intended to indicate that the enzyme is capable of releasing rhamnose from RG-oligomers. A suitable assay for determining RG-rhamnosidase activity is described in the Materials and Methods section below.

Initially, the present inventors identified the enzyme of the invention as a very minor component of the commercially available enzyme preparation Pectinex Ultra SP, available from Novo Nordisk A/S) , which is produced by a strain of the fungal species Aspergillus aculeatus. The enzyme was characterized by its capability of releasing rhamnose groups from RG-oligomers.

It is believed that the enzyme of the invention either alone or in combination with other enzymes may be used for obtaining a more extensive degradation of plant cell wall tissue than hitherto possible.

Accordingly, in a further aspect the invention relates to an enzyme preparation which has been enriched in the RG- rhamnosidase of the invention. The enzyme preparation may be used for any purpose, in which degradation of RG-oligomers is desirable, e.g. for the production of rhamnose from rhamnogalacturonan containing material, and, when used together with other enzymes active on rhamnogalacturonans, for the production of galacturonic acid from the rhamnogalacturonan containing material. Further, the RG-rhamnosidase may be use to reduce the size of rhamnogalacturonan oligomers.

Alternatively, the invention may be used to provide nove products, wherein the proportion of the RG-rhamnosidase i decreased in relation to the proportion in the origina product.

In still further aspects the invention relates to the use o the enzyme or enzyme preparation of the invention fo degradation or modification of plant cell wall components, e.g. for the above stated purposes.

DETAILED DISCLOSURE OF THE INVENTION

In one embodiment the enzyme of the invention is specific fo rhamnose α-1,4-linked to galacturonic acid in RG-oligomers o polymers. Furthermore, the RG-rhamnosidase may be hindered b galactose side-chains attached to rhamnose, in that the RG rhamnosidase has been found to have a higher activity on RG oligomers from which the galactose sidegroups have been partl removed. The enzyme seems equally active towards low and hig molecular weight substrates.

Furthermore, it has surprisingly been found that the RG rhamnosidase of the invention is highly specific for th rhamnogalactoronan oligomers obtained from MHR, but that i does not show any acitivity towards p-nitrophenol rhamnopuranoside. Also, in contrast to prior art rhamnosidases it exhibits specificity on RG-oligomers.

It is preferred that the enzyme of the invention has a specifi activity of at least 5, preferably at least 20, and mos preferably at least 40 rhamnogalacturonan rhamnosidase units (RGRU)/g. An assay for determining RGRU/g is described in the Materials and Methods section. Furthermore, it is preferred that the specificity of the enzyme of the invention is so that the ratio between RG-rhamnosidase activity and the p-nitro-phenyl-rhamnopyranosde (pNP-rha) activity is at least 5, preferably at least 10 and most preferably at least 25.

Enzymes with a specific kind of activity are normally found to be produced by a variety of different organisms. In accordance herewith, it is contemplated that enzymes with RG-rhamnosidase activity are produced by a variety of different organims, in particular microorganisms. Thus, the enzyme of the invention is obtainable from a microbial organism such as a bacterium or a fungus.

In the present context the term "obtainable" is intended not only to indicate that the enzyme is produced by a strain of the organism in question, but also that it is produced in a host organism transformed with a DNA sequence encoding the enzyme.

Examples of fungi from which the enzyme is obtainable strains of Asperqillus sp.. in particular a strain of A. aculeatus, A. awamori. A. orvzae, A. iaponicus or A. niger. strains of Trichoderma sp. , in particular a strain of T. harzianu or T. reesie. strains of Fusarium sp.. in particular a strain of F. oxysporum, strains of Humicola sp. , such as H. insolens or H. lanuqinosa, strains of Irpex sp. such as I. lacteus. or strains of Streptomvces sp.

In one embodiment the enzyme of the invention has the following N-terminal amino acid sequence: AQYKLQGX.GX_jLWYX_jF, in which X₁ , X₂ and X₃ may be different or identical and selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, gluta ic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

Preferably, X, is asparagine (N) or aspartic acid (D) . It will be understood that homologues of the enzyme identified by the above N-terminal sequence are to be considered to be within the present invention. In the present context the term "homologue" is intended to indicate an enzyme with RG- rhamnosidase activity which has an N-terminal amino acid sequence differing in one or more amino acid residues from the sequence shown above, without substantially impairing the characteristic activity of the enzyme. For instance, the homologue may be a naturally occurring or genetically engineered variant of the enzyme with the above N-terminal sequence, e.g. prepared by suitably modifying the DNA sequence encoding the amino acid sequence, resulting in the addition of one or more amino acid residues to either or both the N- and C- terminal end of the sequence, substitution of one or more amino acid residues at one or more different sites in the amino acid sequence, deletion of one or more amino acid residues at either or both ends of or at one or more sites in the amino acid sequence, or insertion of one or more amino acid residues at one or more sites in the amino acid sequence.

Preferably, the N-terminal amino acid sequence of the homologous enzyme is at least 70% homologous, such as at least 80%, 90% or 95% homologous with the N-terminal amino acid sequence shown above.

In one embodiment the enzyme of the invention has a pH-optimum in the range of 2.5-7.0, preferably 3.0-6.0 and most preferably of about 4.0 (the pH optimum is determined by incubating the enzyme with 0.1% w/v of deGal-RGoligo (prepared as described in the Materials and methods) for 30 min at 40°C using Mcllvaine buffers (a mixture of 0.1 M citric acid and 0.2 M sodium hydrogenphosphate) ; a temperature optimum between 10 and 75°C, preferably between 40 and 70°C, and most preferably of about

60°C (the temperature optimum is determined by incubating the enzyme with the above mentioned substrate in 50 mM sodium acetate buffer pH 5) ; and/or a molecular weight in the range of between 40 and 120 kDa, more preferably of about 84 kDa, as determined by SDS-polyacrylamide gel electrophoresis on a 10- 15% polyacrylamide Phast gel (Pharmacia) , standardised by a low molecular weight kit (Pharmacia) .

The enzyme of the invention is preferably substantially pure, i.e. greater than 90% pure, and more preferably more than 95% pure as determined by SDS-PAGE under the conditions specified in the examples hereinafter.

While the RG-rhamnosidase described herein was isolated by conventional methods from the commercially available enzyme preparation (Pectinex Ultra SP) as further described in the examples hereinafter, it is contemplated that it may be produced by use of other methods. For instance, the enzyme may be recovered from the fermentation broth of an organism expressing the enzyme (e.g. as described in WO 92/19728) or from a recombinant organism transformed with a DNA sequence encoding the enzyme.

The DNA sequence encoding the enzyme may be isolated from the organism in question by conventional methods, including expression cloning or PCR.

When using expression cloning the DNA sequence encoding the enzyme of the invention may be isolated by a general method involving

- cloning, in suitable vectors, a DNA library from the organism in question known to or contemplated to produce the enzyme, transforming suitable host cells with said vectors, culturing the host cells under suitable conditions to express any enzyme of interest encoded by a clone in the DNA library, - screening for positive clones by determining any RG- rhamnosidase activity of the enzyme produced by such clones, and isolating the enzyme encoding DNA from such clones. Yeast cells have been found to be particularly useful as host cells for expression cloning purposes. A general method in which yeast cells are used is disclosed in WO 93/11249 which is hereby incorporated by reference.

5 A DNA sequence coding for the enzyme may for instance be isolated by screening a cDNA library of a any of the above mentioned organisms preferred examples of which include strains of Aspergillus, such as A. niger, A. awamori. A. oryzae, A. iaponicus or A. aculeatus. e.g A. aculeatus strain CBS 101.43,

10 publicly available from the Centraalbureau voor Schim elcultures, Delft, NL, and selecting for clones ex¬ pressing the appropriate enzyme activity (i.e. RG-rhamnosidase activity defined as the ability of the enzyme to release rhamnose from RG-oligomers as described in the materials and is methods section hereinafter) . The appropriate DNA sequence may then be isolated from the clone by standard procedures, e.g. as described in Sambrook et al., 1989.

Alternatively, a DNA sequence encoding a RG-rhamnosidase of the invention may, in accordance with well-known procedures,

20 conveniently be isolated by screening a cDNA or genomic library of a suitable organism, and selecting for clones hybridizing with a DNA probe prepared on the basis of an amino acid sequence of said enzyme, such as the one described above, or for clones expressing an enzyme which is immunologically cross-

25 reactive with an antibody raised against a purified RG- rhamnosidase of the invention. The DNA hybridization may be carried out under conditions known in the art, e.g. under the following conditions: presoaking in 5xSSC and prehybridizing for 1 h at ~40°C in a solution of 5xSSC, 5xDenhardt's solution,

3050 mM sodium phosphate, pH 6.8, and 50 μg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 50 μCi 32-P-dCTP labelled probe for 18 h at ^"40°C followed by washing three times in 2xSSC, 0.2% SDS at 40°C for 30 minutes. The appropriate DNA sequence selected by either of the above methods may then be isolated from the clone by standard procedures.

A DNA sequence encoding the enzyme of the invention may be used either for an overproduction of RG-rhamnosidase, if inserted in the microorganism species, from which the parent DNA molecule originated, or for production of RG-rhamnosidase without accompanying closely related enzymes, if inserted in a host microorganism, which in its not-transformed condition does not produce any enzymes closely related to RG-rhamnosidase.

For this purpose the DNA sequence may be inserted into a recombinant expression vector. This may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

In the vector, the DNA sequence encoding the enzyme of the invention should be operably connected to a suitable promoter and terminator sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. The procedures used to ligate the DNA sequences coding for the enzyme of the invention, the promoter and the terminator, respectively, and to insert them into suitable vectors are well known to persons skilled in the art (cf., for instance, Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY, 1989) . The host cell which is transformed with the DNA sequence encoding the enzyme of the invention is preferably a eukaryotic cell, in particular a fungal cell such as a yeast or filamentous fungal cell. In particular, the cell may belong to a species of Aspergillus. most preferably Aspergillus oryzae or Aspergillus niqer. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. The use of Aspergillus as a host microorganism is described in EP 238 023 (of Novo Nordisk A/S) , the contents of which are hereby incorporated by reference. The host cell may also be a yeast cell, e.g. a strain of Saccharomyces, in particular Saccharomyces cerevisiae.

The enzyme of the invention is conveniently produced by culturing a cell of an organism capable of producing the enzyme under conditions permitting the production of the enzyme, and recovering the resulting enzyme from the culture. The organims producing the enzyme may either be one inherently producing the enzyme or one (i.e. a host cell) transformed with a DNA sequence encoding the enzyme.

The medium used to culture the cells (e.g. transformed host cells) may be any conventional medium suitable for growing the host cells in question. The expressed RG-rhamnosidase enzyme may conveniently be secreted into the culture medium and may be recovered therefrom by well-known procedures including separat¬ ing the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chro- matographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

In a still further aspect, the present invention relates to an enzyme preparation useful for the degradation of plant cell wall components, said preparation being enriched in an enzyme of the invention having RG-rhamnosidase activity. The enzyme preparation having been enriched with an enzyme of the invention may e.g. be an enzyme preparation comprising multiple enzymatic activities, in particular an enzyme pre¬ paration comprising multiple plant cell wall degrading enzymes such as Pectinex^®, Pectinex Ultra SP^®, Gamanase, Celluclast or Celluzy e (all available from Novo Nordisk A/S) . In the present context, the term "enriched" is intended to indicate that the RG-rhamnosidase activity of the enzyme preparation has been increased, e.g. with an enrichment factor of at least 1.1, conveniently due to addition of an enzyme of the invention prepared by the method described above.

Alternatively, the enzyme preparation enriched in an enzyme with RG-rhamnosidase activity may be one which comprises an enzyme of the invention as the major enzymatic component, e.g. a mono-component enzyme preparation.

The enzyme preparation may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry preparation. For instance, the enzyme preparation may be in the form of a granulate or a microgranulate. The enzyme to be included in the preparation may be stabilized in accordance with methods known in the art.

The enzyme preparation of the invention may, in addition to a RG-rhamnosidase of the invention, contain one or more other plant cell wall degrading enzymes, for instance those with cellulytic, mannanolytic, xylanolytic or pectinolytic activities such as xylanase, α-galactosidase, 3-galactosidase, -galacturonisidase, /3-mannosidase, xylan acetyl esterase, mannan acetyl esterase, arabinanase, rhamnogalacturonase, rhamnogalacturonan acetylesterase, pectin acetyl esterase, galactanase, polygalacturonase, pectin lyase, pectate lyase, mannanase, glucanase or pectin methylesterase. The additional enzyme(s) may be producible by means of a microorganism belonging to the genus Aspergillus, preferably Aspergillus niger, Aspergillus aculeatus, Aspergillus awamori or Aspergil¬ lus orvzae. or Trichoderma.

In particular, since the RG-rhamnosidase of the invention seems to be hindered by 3-galactoside groups the enzyme preparation may contain an enzyme capable of removing /3-galactoside groups from RG-oligomers to be decomposed, e.g. a 3-galactosidase.

The enzyme or enzyme preparation of the invention may be used for the modification or degradation of plant cell wall materials, and in particular as an agent for degradation or modification of optionally acetylated rhamnogalacturonans and RG-oligomers.

More particularly, the enzyme or enzyme preparation of the invention may be used for the production of rhamnose from rhamnogalacturonan containing material and for the production of galacturonic acid from the rhamnogalacturonan containing material. Furthermore, the enzyme or enzyme preparation may be used to reduce the size of rhamnogalacturonan oligomers.

Furthermore, the enzyme or enzyme preparation of the invention may be used for the synthesis of rhamnose-containing substances such as oligo- or polysaccharides. This may be accomplished by transferring rhamnose (in the form of free rhamnose or as a rhamnose-containing molecule, e.g. a RG-oligomer) to a receptor molecule such as a monosaccharide, e.g. galacturonic acid, or an oligosaccharide, e.g. a RG-oligomer or a polysaccharide.

In a preferred embodiment the RG-rhamnosidase of the invention is used together with enzymes capable of degrading the rhamnogalacturonan structure. Such enzymes are capable of catalyzing full or partial hydrolysis of sidebranches of the rhamnogalacturonan and specific examples include galactanases, beta-galactosidases, arabinases, alpha-arabinosidases and beta- xylosidases. Also, enzymes which attack deacetylated and partially deacetylated ramified or not ramified rhamnogalacturonans may be used. Examples of such enzymes include rhamnogalacturonisidases and enzymes capable of removing acetyl and methyl groups from rhamnogalacturonan structures, e.g. rhamnogalacturonan acetyl esterases.

MATERIALS AND METHODS

Materials

Pectinex Ultra SP (KRN 048) available from Novo Nordisk A/S.

Substrates for RG-rhamnosidase

Modified hairy regions (MHR) were isolated from apple juice according to Schols et al., 1990. Saponified MHR (MHR-S) was obtained by saponification of MHR was performed according to Schols et al. (1990a) using Rapidase C600 (available from Gist- Brocades, the Netherlands) .

RGa, RGb and RGc (degradation products of MHR-S) were prepared by degrading MHR-S (1% w/v, 24h, 40 °C in 50 mM ammonium acetate buffer pH 4.8) on large scale by the enzyme rhamno- galacturonase (RGase) (110 μg) from Aspergillus aculeatus as purified by Schols et al. (1990b). The degradation products were separated on a column (900 x 22 mm) of Sephadex G50 (fine; separation range for dextrans: 200-10,000 Da, Pharmacia) , according to Schols et al. (1990b) using a volatile ammonium acetate buffer (0.05 M pH 4.8) at a flowrate of 30 mL/h. Fractions (5 mL) were assayed by a colorimetric method for uronic acids (Ahmed and Lanovitch, 1977) and total neutral sugars (Tollier and Robin (1979) . The neutral sugar values were corrected for the contribution of the uronic acids in the orcinol assay. Fractions were analysed by high performance size exclusion chromatography (HPSEC) using a series of Biogel TSK 40+30+20 XL columns in combination with a TSK guard column (6x40 mm) at 30°C using 0.4M sodium acetate pH 3.0 as eluent at a flowrate of 0.8 mL/min. Polysaccharides, oligo eric and monomeric sugars were detected by a Shodex SE-61 Rl detector (Showa Denko K.K., Tokyo, Japan) . Fractions were also analysed by HPAEC as described by de Ruiter et al. (1992) in the section entitled Enzymatic activity assays. Fractions were pooled as RGa (higher molecular weight degradation material) , RGc (low molecular weight degradation material containing the rhamnogalacturonan (RG) oligomers) and RGb a small fraction in between. Pools were lyophilized several times to remove the buffer.

A fraction containing predominantly rhamnogalacturonan (RG) oligomer 4 Schols et al. (1994) was obtained by chromatography of the RGase digest of MHR-S on two Fractogel TSK HW-40 (S) columns (600 x 26 mm) in series using a flow rate of 2.5 mL/min and 0.1 M sodium acetate pH 3.0 at 60°C. Fractions were screened on HPAEC as described above and fractions containing RG oligomer 4 were pooled.

Sugar composition analysis of modified hairy regions and other rhamnogalacturonan substrates was performed with methanolysis and subsequent hydrolysis with trifluoracetic acid as described by de Ruiter et al. (1992) using the same HPLC system for detection and quantification, the results are given in the table below:

15

Sugar MHR-S^a RGa^b RGc^c RG4^d (mol%)

Rha 16 9 29 31

Ara 20 23 10 5

Xyl 11 19 2 3

Gal 18 13 27 24

Glc 2 3 3 4

UA 33 34 29 34

saponified Modified Hairy Regions high molecular weight degradation material obtained from incubation of MHR-S with RGase rhamnogalacturonan (RG) oligomers as released from

MHR-S by RGase

RG oligomer 4

The rhamnogalacturonan substrates were incubated for 24h at 40°C and pH 5 in 0.05 M sodium acetate with sufficient amounts of β-galactosidase purified from Aspergillus niger to remove the majority of the galactose from the substrate in 6h, resulting in "treated" substrates, designated "tr" (e.g "tr.RGc") for the treated low molecular fraction 40%, 12%, 84%, and 95%, respectively, of the galactose were removed by the galactosidase from MHR-S, RGa, RGc and RG4 , respectively.

RG-rhamnosidase activity

The activity of the RG-rhamnosidase is expressed in RGRU, where one RGRU is the activity which in 50 mM sodium acetate buffer pH 5 releases 1 micromole rhamnose per min at 30°C from 300 micromoles/1 rhamnogalacturonan oligomers obtained and purified from apple hairy regions and treated with a beta-galactosidase as described above and which consists of at least 60% oligomers with DP-4 monosaccharides, and with a composition of at least 35% galacturonic acid, at least 35% rhamnose and with less than 5% galactose.

The activity on p-nitro-phenyl-rhamnopyranoside (pNP-rha) is expressed in pNPRU, where one pNPRU is is the activity which in 50 mM Sodium acetate buffer pH 5 releases 1 micromole rhamnose per min at 30 C from 0.02% w/v pNP-rha.

EXAMPLE 1

Purification of RG-rhamnosidase

All purification steps were carried out at 4 °C

Pectinex Ultra SP 60 ml was desalted on a Bio-Gel P10 column into 10 mM sodium acetate buffer pH 5.0. Desalted protein (Mort et al. (1991)) was applied onto a DEAE Bio-Gel A column (3.5 x 18.5cm) . After washing the column with 10 mM sodium acetate buffer pH 5.0, bound proteins were eluted by increasing the salt concentration in the elution buffer (50 mM sodium acetate, pH 5.0) from 0 to 0.5 M sodium chloride over 150 min.

Fractions were screened on pnp-Rha and on tr.RGc prepared as described in example 1. Of the desalted protein the majority was bound to the anion exchanger at pH 5.0. Both bound and unbound fractions contained rhamnosidase activity on pnp-Rha as well as on tr.RGc. The RG-rhamnosidase in the unbound fraction (84 mg) was purified further. The fraction was ultrafiltrated into 20 mM sodium acetate, pH 4.25, on an Amicon 200 ml ultrafiltration device. This material was loaded on a cation exchanger MonoS HR 5/5. After washing the column, the protein was eluted by linearly increasing the salt concentration in the buffer from 0 to 0.2 M sodium chloride over 50 min. Screening of column fractions revealed that para-nitrophenyl-alpha- rhamnopyronosid (pnp-RHA) and tr.RGc degrading activity were eluted in two separate peaks. Further purification of the factions active on tr.RGc was performed by FPLC on Biogel HTP (hydroxy apartite column (10 x 0.5 cm). The pooled fractions from the anion exchanger containing tr.RGc degrading activity was ultra-filtrated into 10 mM NaP04, Ph 7.0, applied to the column at a flow of 0.5 ml/ in and bound proteins were eluted by a linear increasing NaP04 gradient from 10-200 mM. Finally, the RG-rhamnosidase containing fractions were purified on a 30 x 0.5 cm Superose 12/HR gele equillibrated in 0.15 M Na Acetate pH 5.0 at a flow rate of 0.5 ml/min.

SDS-PAGE shows only one band in the fractions with tr.RGc degrading activity from the gel filtration column. The preparation does however contain some other enzyme activities which are specifically inactivated by the sodium phosphate- buffer at pH 7.0 used in the HTP-separation.

The RG-rhamnosidase containing fraction was used for N-terminal sequencing

EXAMPLE 2

Characterization of RG-rhamnosidase

Standard incubations with the RG-rhamnosidase enzyme took place for 30 minutes in 50 mM sodium acetate pH 5.0 at 40°C. Activities were calculated from the release of rhamnose as determined by HPAEC, using a Dionex Bio-LC system (Sunnyvale, USA) equipped with a Dionex CarboPac PA-100 (4 x 250 mm) and a Dionex PED detector in the pulsed amperometric detection (PAD) mode, as described by de Ruiter et al. (1992) . Isocratic elution at a flow rate of 1 mL/min was performed with 100 mM NaOH during 5 min at 20°C. Rhamnogalacturonan oligomers (obtained as described in example 1) and degradation products were analysed with the same system using a gradient of NaAc in 100 mM NaOH as follows: 0-5 min, 0 mM; 5-35 min, 0-430 mM; 35-40 min, 430-1000 mM; 40-45 min, 1000 mM; 45-60 min, 0 mM. Release of p- nitrophenol from p-nitrophenyl-α-L-rhamnopyranoside was measured spectrophotometrically at 405 nm and enzymatic activity calculated using the molar extinction coefficient of 13,700 M^c ^"1. Enzyme activities are expressed as units: one unit (U) corresponds with the release of 1 μmol rhamnose per minute.

The result of the characterization is presented the the table below:

⁸ Reaction mixture: substrate solution with an initial available rhamnose content of at least 300 μM (for RG substrates based on treated substrates) in 0.05 M sodium acetate buffer pH 5.0; protein content for 24 h incubation 1.77 μg/mL for RG-rhamnosidase and for

30 min incubation respectively 0.18 μg/mL; 40 °C; ^b mixture of rhamnogalacturonan (RG) oligomers as released from saponified Modified Hairy regions by RGase ^c treated with β-galactosidase ^d RG oligomer 4 (figure 4) ^e Modified Hairy Regions saponified Modified Hairy Regions

⁹ high molecular weight degradation material obtained from incubation of MHR-S with RGase no treatment with β-galactosidase

The activities of the purified enzyme is thereby determined to 52.6 RGRU/mg and 0 pNPRU/mg.

N-terminal sequencing was performed on purified RG-rhamnosidase by means of automated sequencing (Applied Biosystems 473A protein sequences) .

REFERENCES

Schols et al., Carbohydr. Res. , 206 117-129, 1990a

Schols et al. Carbohvdr. Res. 206 105-115, 1990b

Tollier and Robin, 1979, J. Ann. Technol. Agric 28: 1-15

de Ruiter et al., 1992; Anal. Biochem. 207: 176-185

Schols, H.A. and Voragen A.G.J., 1994: Carbohydrate Res. Vol 256: 97-111

Mort et al.; 1991; Carbohydrat. Res. 215: 219-227

Sambrook et al. , Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY, 1989) .

Ahmed A.R., Labavitch J.M. (1977): A simplified method for accurate determination of cell wall uronide content. J. Food Biochem 1: 361-365 (mml26) .

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Claims

1. An enzyme exhibiting rhamnogalacturonan rhamnosidase (RG- rhamnosidase) activity.

2. The enzyme according claim 1, which has a specific activity 5 of at least 5, preferably at least 20, and most preferably at least 40 RG-rhamnosidase units (RGRU)/mg.

3. The enzyme according to claim 1, which has a RGRU/NPRU ratio of at least 5, preferably at least 10 and most preferably at least 25.

104. The enzyme according to any of claims 1-3 which has the following characteristics:

- a molecular weight in the range of 40-120 kDa

- a pH optimum in the range of 2.5-7.0

- a temperature optimum in the range of 10-75°C.

15 5. The enzyme according to claim 1, which has the following N- terminal amino acid sequence AQYKLQGX^X_jLWYX_jF, in which X₁ , X₂ and X₃ may be different or identical and selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, gluta ine, glutamic acid, glycine, histidine,

20 isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

6. The enzyme according to any of claims 1-5 which is of microbial origin.

7. The enzyme according to claim 6, which is obtainable from a 25 fungus.

8. The enzyme according to claim 7, which is obtainable from a strain of an Aspergillus sp.

9. The enzyme according to claim 8, which is obtainable from a strain of A. aculeatus. in particular the A. aculeatus strain CBS 101.43.

10. An enzyme preparation useful for degradation or modification of plant cell wall materials, said preparation being enriched in an enzyme exhibiting RG-rhamnosidase activity according to any of claims 1-9.

11. The preparation according to claim 10, which additionally comprises a RGase, a rhamnogalactanase, a rhamnogalacturonan acetylesterase, a _/3-galactosidase or an arabinase.

12. Use of an enzyme according to any of claims 1-9 or an enzyme preparation according to claim 10 or 11 for the degradation or modification of a plant cell wall material.

13. The use according to claim 12, in which the plant cell material comprises MHR.