Enzyme with rhamnogalacturonase activity

Animal feed compositions and methods for treating one or more of soy, pea or rape-seed, or other material derived from Fabales or Cruciferaceae, with an enzyme having rhamnogalacturonase activity, wherein the enzyme having rhamnogalacturonase activity cleaves a rhamnogalacturonan backbone to produce rhamnose as a non-reducing end (RGase II) or cleaves a rhamnogalaturonan backbone to produce galacturonic acid as a non-reducing end.

FIELD OF INVENTION 
The present invention relates an enzyme exhibiting rhamnogalacturonase 
activity, a DNA construct encoding the enzyme, an enzyme preparation 
comprising the enzyme, and the use of a rhamnogalacturonase for 
degradation or modification of plant cell materials. 
BACKGROUND OF THE INVENTION 
Plants contain rhamnogalacturonans, i.e. polysaccharides with more or less 
regularly alternating rhamnose and galacturonic acid residues in the 
backbone. The rhamnogalacturonans may have mono, oligo, and polysaccharide 
side-branches. Rhamnogalacturonans are part of the pectin-polymers, a 
major component of the plant cell walls. 
Most pectin-polymers are composed of smooth regions, i.e. linear 
homogalacturonan, and hairy (ramified) regions. The hairy regions consist 
of a rhamnogalacturonan backbone with side-branches of varying length. The 
side-branches includes monosaccharides like xylose, galactose and 
arabinose, and oligo and polysaccharides like araban, galactan and 
arabinogalactan. Further the rhamnogalacturonan backbone is methylated and 
acetylated. The composition of the very complex structure of the hairy 
regions vary according to the source of the plant cell wall, cf Fry 
(1988), Schols et al. (1990b), O'Neill et al. (1990), Voragen and Schols 
(1992) and Carpita and Gibeaut (1993). 
The enzymatic liquefaction and degradation of plant materials (e.g. fruits, 
vegetables, cereals, oil fruits and seeds) by technical processes involves 
combinations of pectolytic, cellulolytic and proteolytic enzyme 
preparations. However the hairy regions of pectin cause problems in such 
processes, because they are resistant to degradation of most technical 
enzyme preparations. A more extensive degradation of hairy regions is 
desired in many processes in order to improve the liquefaction and 
degradation of the plant material. For instance, an extensive degradation 
is important in processing of clear liquids and in processing of viscous 
plant cell wall containing material where a viscosity reduction is 
otherwise difficult to obtain. Furthermore, a more specific enzymatic 
degradation of the hairy regions is desirable for e.g. production of 
cloudy liquids, purification of pectin and soluble dietary fibres. 
For these processes a degradation of the backbone of pectin hairy regions 
is of major importance. The degradation of the backbone of the hairy 
regions is performed by enzymes designated rhamnogalacturonases (RGases). 
RGases are believed to hydrolyse the bond between rhamnose and 
galacturonic acid. In order to facilitate the activity of RGases it may be 
desirable to reduce the degree of acetylation of the backbone, e.g. by use 
of the enzyme rhamnogalacturonan acetyl esterase (cf Searle-van Leeuwen et 
al., 1992). Furthermore, a reduced degree of branching of parts of the 
hairy regions may be desirable. The reduced degree of branching may be 
obtained by enzymes which attacks the side-branches, like galactanases, 
arabinanases, beta-galactosidases, alpha-arabinosidases and 
beta-xylosidases. 
The isolation and purification of a RG'ase from Aspergillus aculeatus is 
described by Schols et al. (1990a). 
WO 92/19728, the contents of which are incorporated by reference herein, 
discloses partial amino acid sequences of different RGases isolated from 
the Asperglllus sp. A. aculeatus and A. japonicus and from Irpex lacteus. 
EP 570 075 discloses an Aspergillus RGase gene and the construction of 
recombinant Aspergillus strains which overexpress RGase. 
The RGase described by Schols et al., (1990a) has been found to have a 
similar degradation pattern to the A. aculeatus RGase described in WO 
92/19728 and has been found to be immunologically cross-reactive with said 
RGase. RGase of this type is termed RGase II in the following disclosure. 
Furthermore, in an article of Colquhoun (1990), the composition of a 
mixture of oligosaccharides obtained by enzymatic degradation of the 
modified hairy (ramified) regions of apple pectin with RGase II is 
described. It is shown that RGase II hydrolyses in the rhamnogalacturonan 
backbone leaving rhamnose as the non-reducing end in the degradation 
products. 
Furthermore, RGase II type activity isolated from Trametes sanguinea on 
protopectin extracted from sugar beet has been reported recently by 
Sakamoto et al., 1993. 
It has been shown that A. aculeatus RGase II exhibits optimum activity in 
the pH range of 3-4, which is lower than the pH of most plant materials 
and lower than desired pH in most industrial processes. The RGase 
disclosed in EP 570 075 and the Trametes sanguinea RGase mentioned above 
are stated to be used at pH 5.0. 
High activity of RGase II has only been demonstrated on hairy regions from 
a limited number of plants, there is no reports of a significant activity 
of RGase II on hairy regions from soy and beets. 
Especially for the industries dealing with modifications of plant cell 
walls for e.g. human nutrition and for animal feed (e.g. liquefaction of 
fruits, vegetables, cereals, oil fruits and seeds), it is important to 
provide a variety of different RGases (in respect to mode of action, pH 
and temperature range) in order to be able to exploit the desirable 
actions of RGases under widely varying technical process conditions. In 
particular there is a need for rhamnogalacturonase which is active at a 
higher pH than RGase II and which is active on plant materials for which 
RGase II has only limited activity. 
BRIEF DISCLOSURE OF THE INVENTION 
It has now surprisingly been found that a strain of A. aculeatus produces a 
second RGase which has a mode of action entirely different to that of the 
A. aculeatus RGase disclosed in WO 92/19728 and by Schols et al. (1990a). 
Accordingly, in a first aspect the invention relates to an enzyme 
exhibiting RGase activity, which enzyme. 
a) is encoded by the DNA sequence shown in SEQ ID No. 1 or a sequence 
homologous thereto encoding a polypeptide with RGase activity, 
b) has the amino acid sequence shown in SEQ ID No. 2 or an analogous 
sequence thereof, and/or 
c) is reactive with an antibody raised against the enzyme encoded by the 
DNA sequence shown in SEQ ID No. 1, 
d) has a pH optimum above pH 5, and/or 
e) has a relative activity of at least 30% at a pH in the range of 5.5-6.5. 
In the following disclosure said enzyme is termed RGase I. 
The pH optimum is determined as described in the Materials and Methods 
section herein. The relative activity is determined as described in the 
Materials and Methods section herein and is evaluated relative to activity 
at optimum pH. It will be understood that the pH optimum or relative 
activity as defined above is to be determined on an enzyme purified to 
homogeneity. 
In a further aspect the invention relates to an enzyme exhibiting RGase 
activity, which enzyme is encoded by a DNA sequence comprising at least 
one of the following partial sequences 
______________________________________ 
(a) CGTCGC TTCTGTCGTT 
(b) CGTGGCCTTC ACGGCCCAGGT 
(c) CGCCCACGCG GCCTTTGGCA 
(d) TCACCACCAG CTCCAGCGCC 
(e) TATGTCATCG ACACCAACGC 
(f) GCCAAACCAG CTGAAGTTCA 
(g) CCGTCAGCCG CAGCAGCTGC 
(h) ACATTACCTC CATCATCCAC 
(i) TATGGCACGG AGCTGCAGTA 
(j) CTCCAGCCAG GGCAGTCACA 
(k) TTGGGTCGGG TCTGGGCTCT 
______________________________________ 
In a still further aspect the invention relates to an enzyme exhibiting 
RGase activity, which is encoded by a DNA sequence comprising the 
following partial DNA sequence. 
CGTCGC TTCTGTCGTT CGTGGCCTTC ACGGCCCAGGT CGCCCACGCG GCCTTTGGCA TCACCACCAG 
CTCCAGCGCC TATGTCATCG ACACCAACGC GCCAAACCAG CTGAAGTTCA CCGTCAGCCG 
CAGCAGCTGC GACATTACCTC CATCATCCAC TATGGCACGG AGCTGCAGTA CTCCAGCCAG 
GGCAGTCACA TTGGGTCGGG TCTGGGCTCT 
or a sequence homologous thereto encoding a polypeptide with RGase 
activity. 
In the present context the term "RGase activity" is intended to indicate 
that the enzyme exhibits depolymerization activity of pectin hairy regions 
by attacking the rhamnogalacturonan backbone. The depolymerization of the 
pectin hairy region can be demonstrated by gel filtration chromatography 
as described below. The pectin hairy regions may be prepared from apples 
by purification and deacetylation as described in Schols et al (1990a,b). 
The degradation of possible polygalacturonic parts present in the material 
is ensured by further degradation with Pectinex 3X (obtainable from Novo 
Nordisk) and recovery of the deacetylated hairy regions by 
ultrafiltration. 
In the present context, the term "homologue" is intended to indicate a 
polypeptide encoded by DNA which hybridizes to the same probe as the DNA 
coding for the RGase enzyme under certain specified conditions (such as 
presoaking in 5.times.SSC and prehybridizing for 1 h at -40.degree. C. in 
a solution of 5.times.SSC, 5.times.Denhardt's solution, 50 mM sodium 
phosphate, pH 6.8, and 50 .mu.g of denatured sonicated calf thymus DNA, 
followed by hybridization in the same solution supplemented with 50 .mu.Ci 
32-P-dCTP labelled probe for 18 h at -40.degree. C. followed by washing 
three times in 2.times.SSC, 0.2% SDS at 40.degree. C. for 30 minutes). 
More specifically, the term is intended to refer to a DNA sequence which 
is at least 70% homologous to the sequence shown above encoding the RGase 
1 of the invention, such as at least 75%, at least 80%, at least 85%, at 
least 90% or even at least 95% homologous to any of the sequences shown 
above. The term is intended to include modifications of the DNA sequence 
shown above, such as nucleotide substitutions which do not give rise to 
another amino acid sequence of the RGase but which correspond to the codon 
usage of the host organism into which the DNA construct is introduced or 
nucleotide substitutions which do give rise to a different amino acid 
sequence and therefore, possibly, a different protein structure which 
might give rise to a RGase mutant with different properties than the 
native enzyme. Other examples of possible modifications are insertion of 
one or more codons into the sequence, addition of one or more codons at 
either end of the sequence, or deletion of one or more codons at either 
end or within the sequence. 
In the present context, the term "analogous sequence" is intended to 
indicate an amino acid sequence differing from that of SEQ ID No. 2 
respectively, by one or more amino acid residues. The analogous sequence 
may be one resulting from modification of the amino acid sequence shown in 
the SEQ ID, e.g. involving 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 the enzyme 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. The 
modification of the amino acid sequence may suitably be performed by 
modifying the DNA sequence encoding the enzyme, e.g. by site-directed or 
by random mutagenesis or a combination of these techniques in accordance 
with well-known procedures. Alternatively, the analogous sequence may be 
one of an enzyme derived from another origin than the RGase corresponding 
to SEQ ID No. 2. The analogous sequence will normally exhibit a degree of 
homology (in terms of identity) of at least 70%, such as at least 75%, 
80%, 85%, 90% or even 95% with the amino acid sequence shown in SEQ ID No. 
2. 
In a further aspect the invention relates to a DNA construct encoding the 
RGase of the invention, an expression vector comprising the DNA construct 
and a host cell comprising the vector or the DNA construct. 
In final aspects, the invention relates to the use of an enzyme exhibiting 
RGase activity for reduction of the viscosity of a plant cell wall 
material, for extraction of high molecular weight material from a plant 
cell wall material or for degradation or modification of a plant cell wall 
material at a pH in the range of 4-8, as well as to methods for obtaining 
such effects by use of an enzyme exhibiting RGase activity. 
As far as the present inventors are aware the use of RGase for these 
purposes has neither been disclosed nor suggested in any prior art 
reference. 
DETAILED DISCLOSURE OF THE INVENTION 
It has surprisingly been found that the RGase comprising the amino acid 
sequence shown in SEQ ID No. 2 cleaves a rhamnogalacturonan backbone in 
another manner than the prior art RGases. More specifically, it has been 
found that the RGase cleaves a rhamnogalacturonan backbone in such a 
manner that galacturonic acids are left as the non-reducing ends in the 
degradation products. As far as the present inventors are aware no prior 
disclosure exists of an RGase having this type of cleavage capabilities. 
Furthermore, it has been found the RGase of the invention exhibits activity 
on hairy regions from a soy bean material. As far as the present inventors 
are aware RGase activity on soy bean material has never been reported. In 
addition it has been found that RGase I of the invention exhibits activity 
on saponified hairy regions from a sugar beet material. 
On the basis of the above observations it is contemplated that RGases 
disclosed herein are members of an entirely new class of RGases. 
Accordingly, in a further aspect the invention relates to a RGase enzyme 
which. 
i) cleaves a rhamnogalacturonan backbone in such a manner that galacturonic 
acids are left as the non-reducing ends, 
ii) exhibits activity on hairy regions from a soy bean material, and/or 
iii) exhibits activity on saponified hairy regions from a sugar beet 
material. 
The enzyme of the invention is preferably derivable from a strain of 
Aspergillus sp. in particular A. aculeatus or A. japonicus, a strain of 
Irpex sp., e.g. I. lacteus, or a strain of Trichoderma sp., Neurospora 
sp., Penicillium sp., Trametes sp. or Polyporus sp. 
In the present context, the term "derivable from" is intended not only to 
indicate a RGase produced by a strain of the above mentioned fungi, but 
also a RGase encoded by a DNA sequence isolated from strain of A. 
aculeatus and produced in a host organism transformed with said DNA 
sequence. 
In particular the enzyme of the invention may be encoded by a DNA sequence 
isolated from a DNA library of A. aculeatus, CBS 101.43. 
The DNA construct of the invention may be isolated by a general method 
involving. 
cloning, in suitable vectors, a DNA library from Aspergillus aculeatus, 
transforming suitable yeast 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, and 
screening for positive clones by determining any RGase activity of the 
enzyme produced by such clones, the RGase activity being determined by use 
of coloured crosslinked substrates containing the rhamnogalacturonan 
backbone of the pectin hairy regions such as AZCL-arabinan, AZCL-galactan 
or a coloured and crosslinked MHR-substrate as described below in the 
section entitled "Materials and Methods". 
The AZCL-arabinan and AZCL-galactan substrates can be used as screening 
substrates for RGases since the present inventors discovered that these 
arabinan and galactan substrates are hairy regions in which the galactan 
and arabinan are linked as side branches to a rhamnogalacturonan backbone. 
Enzymatic hydrolysis of the backbone leads to a depolymerization of the 
AZCL-substrate and consequently to a release of the colour. Activity by 
galactanases and arabinanases, respectively, will also lead to a colour 
release from such AZCL-substrates. The RGase of the invention was 
initially isolated on both AZCL-galactan and AZCL-arabinan and coloured 
cross-linked hairy regions from apples. In the priority establishing 
application this enzyme was designated as "Carbohydrase" due to the 
activity on the different types of carbohydrate substrates. The subsequent 
studies of the enzyme revealed that it was a RGase, and this term is, 
accordingly, used throughout the present application. 
A more detailed description of this screening method is given in Example 1 
below. 
The DNA sequence coding for the RGase enzyme may for instance be isolated 
by screening a cDNA library of Aspergillus aculeatus, e.g strain CBS 
101.43, publicly available from the Centraalbureau voor Schimmelcultures, 
Delft, NL, and selecting for clones expressing RGase activity. The 
appropriate DNA sequence may then be isolated from the clone by standard 
procedures, e.g. as described in Example 1. 
The DNA sequence may subsequently 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 RGase 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 RGase, 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, N.Y., 1989). 
The host cell which is transformed with the DNA sequence 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 
niger. 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. 
In a still further aspect, the present invention relates to a method of 
producing an RGase enzyme, wherein a suitable host cell transformed with a 
DNA sequence encoding the enzyme is cultured under conditions permitting 
the production of the enzyme, and the resulting enzyme is recovered from 
the culture. 
The medium used to culture the transformed host cells may be any 
conventional medium suitable for growing the host cells in question. The 
expressed RGase may conveniently be secreted into the culture medium and 
may be recovered therefrom by well-known procedures including separating 
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 chromatographic procedures such as ion exchange 
chromatography, affinity chromatography, or the like. 
The thus purified RGase may be employed for immunization of animals for the 
production of antibodies. More specifically, antiserum against the RGase 
of the invention may be raised by immunizing rabbits (or other rodents) 
according to the procedure described by N. Axelsen et al. in: A Manual of 
Ouantitative Immunoelectrophoresis, Blackwell Scientific Publications, 
1973, Chapter 23, or A. Johnstone and R. Thorpe, Immunochemistry in 
Practice, Blackwell Scientific Publications, 1982 (more specifically pp. 
27-31). Purified immunoglobulins may be obtained from the antisera, for 
example by salt precipitation ((NH.sub.4).sub.2 SO.sub.4), followed by 
dialysis and ion exchange chromatography, e.g. on DEAE-Sephadex. 
Immunochemical characterization of proteins may be done either by 
Outcherlony double-diffusion analysis (O. Ouchterlony in: Handbook of 
Experimental Immunology (D. M. Weir, Ed.), Blackwell Scientific 
Publications, 1967, pp. 655-706), by crossed immunoelectrophoresis (N. 
Axelsen et al., supra, Chapters 3 and 4), or by rocket 
immunoelectrophoresis (N. Axelsen et al., Chapter 2,). 
The Enzyme Preparation of the Invention 
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 exhibiting RGase I activity as 
described above. In this manner a boosting of the cell wall degrading 
ability of the enzyme preparation can be obtained. 
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 preparation comprising multiple plant 
cell wall degrading enzymes such as Pectinex.RTM., Pectinex Ultra SP.RTM., 
Celluclast or Celluzyme (all available from Novo Nordisk A/S). In the 
present context, the term "enriched" is intended to indicate that the 
RGase I 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 exhibiting 
RGase I 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 RGase I of 
the invention, contain one or more other plant cell wall degrading 
enzymes, for instance those with proteolytic, cellulytic, xylanolytic or 
pectinolytic activities such as, xylanase, arabinanase, RGase, e.g. RGase 
II, pectin acetyl esterase, galactanase, polygalacturonase, pectin lyase, 
pectate lyase, glucanase, rhamnogalacturonan acetyl esterase or pectin 
methylesterase. The preparation may further contain one or more enzymes 
exhibiting exo-activity on the same substrates as the above-mentioned 
endo-enzymes, like .alpha.-arabinosidase, .beta.-galactosidase and 
.beta.-xylosidase. 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 Aspergillus oryzae, 
Penicillium or Trichoderma. 
Uses of RGase 
In the following various uses of an enzyme of the invention or of RGase in 
general are described. 
An important use of the enzyme or enzyme preparation of the invention or 
any enzyme exhibiting RGase activity in the pH range of 4-8 is in the 
degradation or modification of plant cell wall containing material which 
is to be performed at a pH in the range of 4-8, preferably 4.5-7.5, such 
as 5.0-7.5, 5.2-7.3 or 5.5-7 and most preferably in the range of 5.5-6.5. 
It has surprisingly been found that the enzyme according to the invention 
is active under conditions where the RGase II described in the prior art 
has no or little activity. The activity at these pH values is advantagous 
for processing of many types fruits and vegetable where the pH may be to 
high for optimal use of RGase II. This may apply, e.g. to the processing 
of rape seeds, carrots, tomatoes, potatoes, olives, soy and sugar beets. 
The enzyme and enzyme preparation of the invention is preferably used at a 
temperature in the range of 15-65.degree. C., preferably 20-60.degree. C., 
more preferably 25-55.degree. C. and most preferably in the range of 
35-40.degree. C. 
Another important use of the enzyme or the enzyme preparation of the 
invention or any enzyme with RGase activity is in the degradation of plant 
cell wall containing material, in particular plant cell wall material 
originating from plants belonging to Fabales (Leguminales), preferably 
soya, peas, bean, locust bean and guar, or to the family Cruciferaceae, 
such as rape seed or cabbage. It has surprisingly been demonstrated that 
the enzyme according to the invention has a significant activity on cell 
wall material including hairy regions of these types of plants, and it is 
contemplated that any RGase may exhibit activity on such plants. The 
activity may be obtained without any deacetylation of the hairy regions. 
The use of RGases on cell wall containing materials from the members of the 
order Fabales or the family Cruciferaceae is advantageous in that an 
increased digestibility of, e.g., soya, pea and rape seed may be obtained. 
Furthermore, processing of these materials may be facilitated by use of 
RGase. For instance, the use of RGase may facilitate the purification of 
soya protein and soya isolate or facilitate the processing of soy material 
to be used for animal feed or human food. Furthermore, the use is 
advantageous for the release of high molecular weight material from this 
type of plant material (such as soy beans, peas or rapeseeds). For 
instance, RGase may be used to release galactan-rich material from soy 
beans. This material can be used as soluble fibres to be added to 
different types of food in order to improve the nutritional value thereof. 
Furthermore, the RGase of the invention have been found to exhibit 
acitivity on plants belonging to the order Chenopodiaceae, such as beets, 
sugar beets, red beets and spinach. 
The use of RGases on cell wall containing material from plant belonging to 
the Chenopodiaceae is advantagous for processing of sugar beets e.g. 
liquefaction of sugar beets for production of ethanol and recovery of 
galacturonic acid, or for processing of sugar beet pulp for recovery of 
pectin, recovery of galacturonic acid, or to improve the pressability of 
the beet pulp. Further the RGases may be used to improve the recovery of 
the colour from red beets. The RGase may be used alone or in combination 
with other enzymes, such as a rhamnogalacturonan acetyl esterase. 
Another important use of the enzyme and enzyme preparation of the invention 
or any enzyme with RGase activity is in reducing the viscosity of a plant 
material. For instance, the viscosity reduction may be used to facilitate 
processing of the plant material or the separation of the plant material 
into different components. The viscosity reduction may also be used to 
improve the digestibility of a plant material. Specific examples include 
improving the purification of protein from e.g. soy beans, peas or 
rapeseeds, and improving the digestibility of soy based animal feed. It 
has surprisingly been demonstrated that the enzyme according to the 
invention can reduce the viscosity in jet cooked soy without addition of 
any other polysaccharide degrading enzymes. 
By use of the method of the invention viscosity reductions of at least 20%, 
such as at least 30% and preferably at least 50% may be obtained. 
Another important use of the enzyme or enzyme preparation of the invention 
is for extracting high molecular weight molecules from a plant cell wall 
material. In the present context, the term "high molecular weight 
molecules" is intended to indicate molecules having a degree of 
polymerization (DP) of at least 50 as defined herein. The enzyme is 
especially useful for extraction of pectin material which would otherwise 
be difficult to extract. For instance, the enzyme or enzyme preparation 
may be used to extract galactan and arabinan containing oligomers and 
polysaccharides, e.g. from soy fibres as described in the following 
examples. For this purpose the RGase of the invention may be used alone or 
in combination with another enzyme, such as with rhamnogalacturonan acetyl 
esterase. 
By use of a RGase for such extraction it is possible to obtain high 
molecular weight polysaccharides having a DP of at least such as at least 
500 or at least 1000. 
Furthermore, the enzyme or enzyme preparation can be used to facilitate the 
extraction of pectin from, e.g., citrus, apples, beet or sunflower 
material. For this purpose it may be advantageous to use the enzyme alone 
or substantially free from polygalacturonase, pectin lyase or other 
enzymes which may depolymerize the pectin to be produced. 
From the above disclosure, it will be apparent that it may be advantageous 
to use the RGase of the invention in combination with another plant cell 
wall degrading enzyme, e.g. of the type disclosed above. 
Methods of the Invention 
In further aspects the invention relates to methods of using a RGase, 
preferably in the form of an enzyme or enzyme preparation of the 
invention, for the above described uses. In the following these methods 
are summerized. It will be understood that the information provided in the 
above section ("Use of RGase) will also be applicable for the methods 
described in the present section. 
The invention relates to a method of reducing the viscosity of a plant 
material, which method comprises treating the plant material with a enzyme 
or enzyme preparation of the invention under suitable conditions for the 
viscosity to be reduced. The pH and temperature under which the treatment 
is performed is typically as defined above. 
By use of the method of the invention viscosity reductions of at least 20%, 
such as at least 30% and preferably at least 50% may be obtained. 
The invention relates to a method of producing high molecular weight 
molecules having a DP of at least 50 from a plant material, which method 
comprises treating the plant material with an enzyme or enzyme preparation 
according to the invention under suitable conditions for the viscosity to 
be reduced. The pH and temperature under which the treatment is performed 
is typically as defined above. 
Any of the plant materials specified above may be treated by the methods of 
the invention. 
In a particularly important aspect the invention relates to a soy treatment 
process. 
The production of soy isolates includes: 
aqueous extraction for defatted soy flakes in mildly alkaline media, 
separation of the soluble protein from undissolved material, 
precipitation of protein by acid, 
separation of protein from soluble carbohydrates, 
neutralization of the precipitated protein, 
drying of protein, cf Circle et al., 1978. 
It is characteristic for this process that the first extraction step is 
troublesome due to the water binding ability of the carbohydrate fraction 
which results in a high viscosity. This limits the yield and the 
production capacity of the separation equipment. It is contemplated that 
the first extraction step may be considerably improved by use of an RGase 
of the invention whereby a higher yield and a higher capacity of the 
entire process can be obtained. 
Accordingly, in a further important aspect the invention relates to a 
method of producing a soy isolate, which method comprises treating a 
suspension of a defatted soy flour with a sufficient amount of an RGase to 
reducing the viscosity of the suspension, and isolating a soy isolate from 
the resulting suspension. 
In this manner the suspension will be significantly easier to handle during 
processing for soy protein isolation. The processing of the suspension 
typically includes the steps of 
i) extracting protein by aqueous extraction at near neutral or slightly 
alkaline pH, 
ii) separating protein from undissolved material, 
iii) precipitating protein by acid precipitation, 
iv) separating precipitated protein from soluble material, and 
v) neutralizing and drying the protein. 
Feed 
The present invention relates to a feed comprising RGase, in particular in 
the form of an enzyme or enzyme preparation of the invention. The RGase is 
contemplated to reduce the viscosity of the feed by modifying components 
of the feed, i.e. in vitro, or in vivo. The RGase is particularly suited 
for addition to animal feed compositions containing soy, pea or rapeseed, 
or other material derived from Fabales or Cruciferaceae.

The invention is described in further detail in the following examples 
which are not in any way intended to limit the scope of the invention as 
claimed. 
EXAMPLES 
Materials and Methods 
Donor organism: mRNA was isolated from Aspergillus aculeatus, CBS 101.43, 
grown in a soy-containing fermentation medium with agitation to ensure 
sufficient aeration. Mycelia were harvested after 3-5 days' growth, 
immediately frozen in liquid nitrogen and stored at -80.degree. C. 
Yeast strains: The Saccharomyces cerevisiae strain used was yNG231 (MAT 
alpha, leu2, ura3-52, his4-539, pep4-delta 1, cir+) or JG169 (MAT.alpha.; 
ura 3-52; leu 2-3, 112; his 3-D200; pep 4-113; prc1::HIS3; prb1:: LEU2; 
cir+). 
Construction of an expression plasmid: The commercially available plasmid 
pYES II (Invitrogen) was cut with SpeI, filled in with Klenow DNA 
polymerase+dNTP and cut with ClaI. The DNA was size fractionated on an 
agarose gel, and a fragment of about 2000 bp was purified by 
electroelution. The same plasmid was cut with ClaI/PvuII, and a fragment 
of about 3400 bp was purified by electroelution. The two fragments were 
ligated to a blunt-ended SphI/EcoRI fragment containing the yeast TPI 
promoter. This fragment was isolated from a plasmid in which the TPI 
promoter from S. cerevisiae (cf. T. Albers and G. Kawasaki, J. Mol. Appl. 
Genet. 1, 1982, pp. 419-434) was slightly modified: an internal SphI site 
was removed by deleting the four bp constituting the core of this site. 
Furthermore, redundant sequences upstream of the promoter were removed by 
Ball exonuclease treatment followed by addition of a SphI linker. Finally, 
an EcoRI linker was added at position -10. After these modifications, the 
promoter is included in a SphIEcoRI fragment. Its efficiency compared to 
the original promoter appears to be unaffected by the modifications. The 
resuiting plasmid pYHD17 is shown in FIG. 1. 
Preparation of RNase-free glassware, tips and solutions: All glassware used 
in RNA isolations was baked at +220.degree. C. for at least 12 h. 
Eppendorf tubes, pipet tips and plastic columns were treated in 0.1% 
diethylpyrocarbonate (DEPC) in EtOH for 12 h, and autoclaved. All buffers 
and water (except Tris-containing buffers) were treated with 0.1% DEPC for 
12 h at 37.degree. C., and autoclaved. 
Extraction of total RNA: The total RNA was prepared by extraction with 
guanidinium thiocyanate followed by ultracentrifugation through a 5.7 M 
CsCl cushion (Chirgwin et al., 1979) using the following modifications. 
The frozen mycelia were ground in liquid N.sub.2 to fine powder with a 
mortar and a pestle, followed by grinding in a precooled coffee mill, and 
immediately suspended in 5 vols of RNA extraction buffer (4 M GuSCN, 0.5% 
Nalaurylsarcosine, 25 mM Na-citrate, pH 7.0, 0.1 M 
.beta.-mercaptoethanol). The mixture was stirred for 30 min. at RT.degree. 
and centrifuged (30 min., 5000 rpm, RT.degree., Heraeus Megafuge 1.0 R) to 
pellet the cell debris. The supernatant was collected, carefully layered 
onto a 5.7 M CsCl cushion (5.7 M CsCl, 0.1 M EDTA, pH 7.5, 0.1% DEPC; 
autoclaved prior to use) using 26.5 ml supernatant per 12.0 ml CsCl 
cushion, and centrifuged to obtain the total RNA (Beckman, SW 28 rotor, 25 
000 rpm, RT.degree., 24h). After centrifugation the supernatant was 
carefully removed and the bottom of the tube containing the RNA pellet was 
cut off and rinsed with 70% EtOH. The total RNA pellet was transferred 
into an Eppendorf tube, suspended in 500 .mu.l TE, pH 7.6 (if difficult, 
heat occasionally for 5 min at 65.degree. C.), phenol extracted and 
precipitated with ethanol for 12 h at -20.degree. C. (2.5 vols EtOH, 0.1 
vol 3M NaAc, pH 5.2). The RNA was collected by centrifugation, washed in 
70% EtOH, and resuspended in a minimum volume of DEPC-DIW. The RNA 
concentration was determined by measuring OD.sub.260/280. 
Isolation of poly(A) .sup.+ RNA: The poly(A) .sup.+ RNAs were isolated by 
oligo(dT)-cellulose affinity chromatography (Aviv & Leder, 1972). 
Typically, 0.2 g of oligo(dT) cellulose (Boehringer Mannheim) was 
preswollen in 10 ml of 1.times.column loading buffer (20 mM-Tris-Cl, pH 
7.6, 0.5 M NaCl, 1 mM EDTA, 0.1% SDS), loaded onto a DEPC-treated, plugged 
plastic column (Poly Prep Chromatography Column, Bio Rad), and 
equilibrated with 20 ml 1.times.loading buffer. The total RNA was heated 
at 65.degree. C. for 8 min., quenched on ice for 5 min, and after addition 
of 1 vol 2.times.column loading buffer to the RNA sample loaded onto the 
column. The eluate was collected and reloaded 2-3 times by heating the 
sample as above and quenching on ice prior to each loading. The oligo(dT) 
column was washed with 10 vols of 1.times.loading buffer, then with 3 vols 
of medium salt buffer (20 mM Tris-Cl, pH 7.6, 0.1 M NaCl, 1 mM EDTA, 0.1% 
SDS), followed by elution of the poly(A) .sup.+ RNA with 3 vols of elution 
buffer (10 mM Tris-Cl, pH 7.6, 1 mM EDTA, 0.05% SDS) preheated to 
+65.degree. C., by collecting 500 .mu.l fractions. The OD.sub.260 was read 
for each collected fraction, and the mRNA containing fractions were pooled 
and ethanol precipitated at -20.degree. C. for 12h. The poly(A) .sup.+ RNA 
was collected by centrifugation, resuspended in DEPC-DIW and stored in 
5-10 .mu.g aliquots at -80.degree. C. 
Northern blot analysis: The poly(A).sup.+ RNAs (5 .mu.g/sample) from 
various mycelia were electrophoresed in 1.2 agarose-2.2 M formaldehyde 
gels (Sambrook et al., 1989) and blotted to nylon membranes (Hybond-N, 
Amersham) with 10.times.SSC (Sambrook et al., 1989) as transfer buffer. 
Three random-primed (Feinberg & Vogelstein, 1983) .sup.32 P-labeled cDNA 
probes were used in individual hybridizations: 1) a 1.3 kb Not I-Spe I 
fragment for polygalacturonase I from A. aculeatus (described in 
PCT/DK93/00445), 2) a 1.3 kb Not I-Spe I fragment encoding endoglucanase I 
from A. aculeatus (described in DK 0419/92) and 3) a 1.2 kb Eag I fragment 
for galactanase I from A. aculeatus (described in WO 92/13945). Northern 
hybridizations were carried out in 5.times.SSC (Sambrook et al., 1989), 
5.times.Denhardt's solution (Sambrook et al., 1989), 0.5% SDS (w/v) and 
100 .mu.g/ml denatured salmon sperm DNA with a probe concentration of ca. 
2 ng/ml for 16 h at 65.degree. C. followed by washes in 5.times.SSC at 
65.degree. C. (2.times.15 min), 2.times.SSC, 0.5% SDS (1.times.30 min), 
0.2.times.SSC, 0.5% SDS (1.times.30 min), and 5.times.SSC (2.times.15 
min). After autoradiography at -80.degree. C. for 12 h, the probe #1 was 
removed from the filter according to the manufacturer's instructions and 
rehybridized with probe #2, and eventually with probe #3. The RNA ladder 
from Bethesda Research Laboratories was used as a size marker. 
cDNA Synthesis: 
First strand synthesis: Double-stranded cDNA was synthesized from 5 .mu.g 
of A. aculeatus poly (A).sup.+ RNA by the RNase H method (Gubler & 
Hoffman 1983, Sambrook et al., 1989) using the hairpin modification. The 
poly(A).sup.+ RNA (5 .mu.g in 5 .mu.l of DEPC-treated water) was heated 
at 70.degree. C. for 8 min., quenched on ice, and combined in a final 
volume of 50 .mu.l with reverse transcriptase buffer (50 mM Tris-Cl, pH 
8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, Bethesda Research Laboratories) 
containing 1 mM each dNTP (Pharmacia), 40 units of human placental 
ribonuclease inhibitor (RNasin, Promega), 10 .mu.g of oligo(dT).sub.12-18 
primer (Pharmacia) and 1000 units of SuperScript II RNase H- reverse 
transcriptase (Bethesda Research Laboratories). First-strand cDNA was 
synthesized by incubating the reaction mixture at 45.degree. C. for 1 h. 
Second strand synthesis: After synthesis 30 .mu.l of 10 mM Tris-Cl, pH 7.5, 
1 mM EDTA was added, and the mRNA:cDNA hybrids were ethanol precipitated 
for 12 h at -20.degree. C. by addition of 40 .mu.g glycogen carrier 
(Boehringer Mannheim) 0.2 vols 10 M NH.sub.4 Ac and 2.5 vols 96% EtOH. The 
hybrids were recovered by centrifugation, washed in 70% EtOH, air dried 
and resuspended in 250 .mu.l of second strand buffer (20 mM Tris-Cl, pH 
7.4, 90 mM KCl, 4.6 mM MgCl2, 10 mM (NH.sub.4).sub.2 SO.sub.4, 16 .mu.M 
.beta.NAD.sup.+) containing 100 .mu.M each dNTP, 44 units of E. coli DNA 
polymerase I (Amersham), 6.25 units of RNase H (Bethesda Research 
Laboratories) and 10.5 units of E. coli DNA ligase (New England Biolabs). 
Second strand cDNA synthesis was performed by incubating the reaction tube 
at 16.degree. C. for 3 h, and the reaction was stopped by addition of EDTA 
to 20 mM final concentration followed by phenol extraction. 
Mung bean nuclease treatment: The double-stranded (ds) cDNA was ethanol 
precipitated at -20.degree. C. for 12 h by addition of 2 vols of 96% EtOH, 
0.1 vol 3 M NaAc, pH 5.2, recovered by centrifugation, washed in 70% EtOH, 
dried (SpeedVac), and resuspended in 30 .mu.l of Mung bean nuclease buffer 
(30 mM NaAc, pH 4.6, 300 mM NaCl, 1 mM ZnSO4, 0.35 mM DTT, 2% glycerol) 
containing 36 units of Mung bean nuclease (Bethesda Research 
Laboratories). The single-stranded hair-pin DNA was clipped by incubating 
the reaction at 30.degree. C. for 30 min, followed by addition of 70 .mu.l 
10 mM Tris-Cl, pH 7.5, 1 mM EDTA, phenol extraction, and ethanol 
precipitation with 2 vols of 96% EtOH and 0.1 vol 3M NaAc, pH 5.2 at 
-20.degree. C. for 12 h. 
Blunt-ending with T4 DNA polymerase: The ds cDNA was blunt-ended with T4 
DNA polymerase in 50 .mu.l of T4 DNA polymerase buffer (20 mM 
Tris-acetate, pH 7.9, 10 mM MgAc, 50 mM KAc, 1 mM DTT) containing 0.5 mM 
each dNTP and 7.5 units of T4 DNA polymerase (Invitrogen) by incubating 
the reaction mixture at +37.degree. C. for 15 min. The reaction was 
stopped by addition of EDTA to 20 mM final concentration, followed by 
phenol extraction and ethanol precipitation. 
Adaptor ligation and size selection: After the fill-in reaction the cDNA 
was ligated to non-palindromic BstX I adaptors (1 .mu.g/.mu.l, Invitrogen) 
in 30 .mu.l of ligation buffer (50 mM Tris-Cl, pH 7.8, 10 mM MgCl2, 10 mM 
DTT, 1 mM ATP, 25 .mu.g/ml bovine serum albumin) containing 600 pmol BstX 
I adaptors and 5 units of T4 ligase (Invitrogen) by incubating the 
reaction mix at +16.degree. C. for 12 h. The reaction was stopped by 
heating at +70.degree. C. for 5 min, and the adapted cDNA was 
size-fractionated by agarose gel electrophoresis (0.8% HSB-agarose, FMC) 
to separate unligated adaptors and small cDNAs. The cDNA was size-selected 
with a cut-off at 0.7 kb, and the cDNA was electroeluted from the agarose 
gel in 10 mM Tris-Cl, pH 7.5, 1 mM EDTA for 1 h at 100 volts, phenol 
extracted and ethanol precipitated at -20.degree. C. for 12 h as above. 
Construction of cDNA libraries: The adapted, ds cDNA was recovered by 
centrifugation, washed in 70% EtOH and resuspended in 25 ml DIW. Prior to 
large-scale library ligation, four test ligations were carried out in 10 
.mu.l of ligation buffer (same as above) each containing 1 .mu.l ds cDNA 
(reaction tubes #1-#3), 2 units of T4 ligase (Invitrogen) and 50 ng (tube 
#1), 100 ng (tube #2) and 200 ng (tubes #3 and #4) Bst XI cleaved yeast 
expression vector either pYES 2.0 vector Invitrogen or yHD13). The 
ligation reactions were performed by incubation at +16.degree. C. for 12 
h, heated at 70.degree. C. for 5 min, and 1 .mu.l of each ligation 
electroporated (200 .OMEGA., 2.5 kV, 25 .mu.F) to 40 .mu.l competent E. 
coli 1061 cells (OD600=0.9 in 1 liter LB-broth, washed twice in cold DIW, 
once in 20 ml of 10% glycerol, resuspended in 2 ml 10% glycerol). After 
addition of 1 ml SOC to each transformation mix, the cells were grown at 
+37.degree. C. for 1 h , 50 .mu.l plated on LB+ampicillin plates (100 
.mu.g/ml) and grown at +37.degree. C. for 12h. 
Using the optimal conditions a large-scale ligation was set up in 40 .mu.l 
of ligation buffer containing 9 units of T4 ligase, and the reaction was 
incubated at +16.degree. C. for 12 h. The ligation reaction was stopped by 
heating at 70.degree. C. for 5 min, ethanol precipitated at -20.degree. C. 
for 12 h, recovered by centrifugation and resuspended in 10 .mu.l DIW. One 
.mu.l aliquots were transformed into electrocompetent E. coli 1061 cells 
using the same electroporation conditions as above, and the transformed 
cells were titered and the library plated on LB+ampicillin plates with 
5000-7000 c.f.u./plate. To each plate was added 3 ml of medium. The 
bacteria were scraped off, 1 ml glycerol was added and stored at 
-80.degree. C. as pools. The remaining 2 ml were used for DNA isolation. 
If the amount of DNA was insufficient to give the required number of yeast 
transformants, large scale DNA was prepared from 500 ml medium (TB) 
inoculated with 50 .mu.l of -80.degree. C. bacterial stock propagated 
overnight. 
Construction of yeast libraries: To ensure that all the bacterial clones 
were tested in yeast, a number of yeast transformants 5 times larger than 
the number of bacterial clones in the original pools was set as the limit. 
One .mu.l aliquots of purified plasmid DNA (100 ng/.mu.l) from individual 
pools were electroporated (200 .OMEGA., 1.5 kV, 25 .mu.F) into 40 .mu.l 
competent S. cerevisiae JG 169 cells (OD600=1.5 in 500 ml YPD, washed 
twice in cold DIW, once in cold 1 M sorbitol, resuspended in 0.5 ml 1 M 
sorbitol, Becker & Guarante, 1991). After addition of 1 ml 1M cold 
sorbitol, 80 .mu.l aliquots were plated on SC+glucose - uracil to give 
250-400 c.f.u./plate and incubated at 30.degree. C. for 3-5 days. 
Construction of an Aspergillus expression vector: the vector pHD414 is a 
derivative of the plasmid p775 (described in EP 238 023). In contrast to 
this plasmid, pHD 414 has a string of unique restriction sites between the 
promoter and the terminator. The plasmid was constructed by removal of an 
approximately 200 bp long fragment (containing undesirable RE sites) at 
the 3' end of the terminator, and subsequent removal of an approximately 
250 bp long fragment at the 5' end of the promoter, also containing 
undesirable sites. The 200 bp region was removed by cleavage with NarI 
(positioned in the pUC vector) and XbaI (just 3' to the terminator), 
subsequent filling in the generated ends with Klenow DNA polymerase +dNTP, 
purification of the vector fragment on gel and religation of the vector 
fragment. This plasmid was called pHD413. pHD413 was cut with StuI 
(positioned in the 5' of the promoter) and PvuII (in the pUC vector), 
fractionated on gel and religated. The plasmid pHD 414 is shown in FIG. 2. 
Transformation of Aspergillus oryzae or Aspergillus niger (general 
procedure) 
100 ml of YPD (Sherman et al., Methods in Yeast Genetics, Cold Spring 
Harbor Laboratory, 1981) is inoculated with spores of A. oryzae or A. 
niger and incubated with shaking at 37.degree. C. for about 2 days. The 
mycelium is harvested by filtration through miracloth and washed with 200 
ml of 0.6 M MgSO.sub.4. The mycelium is suspended in 15 ml of 1.2 M 
MgSO.sub.4. 10 mM NaH.sub.2 PO.sub.4, pH=5.8. The suspension is cooled on 
ice and 1 ml of buffer containing 120 mg of Novozym.RTM. 234, batch 1687 
is added. After 5 minutes 1 ml of 12 mg/ml BSA (Sigma type H25) is added 
and incubation with gentle agitation continued for 1.5-2.5 hours at 
37.degree. C. until a large number of protoplasts is visible in a sample 
inspected under the microscope. 
The suspension is filtered through miracloth, the filtrate transferred to a 
sterile tube and overlayered with 5 ml of 0.6 M sorbitol, 100 mM Tris-HCl, 
pH=7.0. Centrifugation is performed for 15 minutes at 100 g and the 
protoplasts are collected from the top of the MgSO.sub.4 cushion. 2 
volumes of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH=7.5. 10 mM CaCl.sub.2) 
are added to the protoplast suspension and the mixture is centrifugated 
for 5 minutes at 1000 g. The protoplast pellet is resuspended in 3 ml of 
STC and repelleted. This is repeated. Finally the protoplasts are 
resuspended in 0.2-1 ml of STC. 
100 .mu.l of protoplast suspension is mixed with 5-25 .mu.g of the 
appropriate DNA in 10 .mu.l of STC. Protoplasts are mixed with p3SR2 (an 
A. nidulans amdS gene carrying plasmid). The mixture is left at room 
temperature for 25 minutes. 0.2 ml of 60% PEG 4000 (BDH 29576). 10 mM 
CaCl.sub.2 and 10 mM Tris-HCl, pH=7.5 is added and carefully mixed (twice) 
and finally 0.85 ml of the same solution is added and carefully mixed. The 
mixture is left at room temperature for 25 minutes, spun at 2500 g for 15 
minutes and the pellet is resuspended in 2 ml of 1.2 M sorbitol. After one 
more sedimentation the protoplasts are spread on the appropriate plates. 
Protoplasts are spread on minimal plates (Cove Biochem.Biophys.Acta 113 
(1966) 51-56) containing 1.0 M sucrose, pH=7.0, 10 mM acetamide as 
nitrogen source and 20 mM CsCl to inhibit background growth. After 
incubation for 4-7 days at 37.degree. C. spores are picked and spread for 
single colonies. This procedure is repeated and spores of a single colony 
after the second reisolation is stored as a defined transformant. 
Media: 
YPD: 10 g yeast extract, 20 g peptone, H.sub.2 O to 810 ml. Autoclaved, 90 
ml 20% glucose (sterile filtered) added. 
10.times.Basal salt: 66.8 g yeast nitrogen base, 100 g succinic acid, 60 g 
NaOH, H.sub.2 O ad 1000 ml, sterile filtered. 
SC-URA: 90 ml 10.times.Basal salt, 22.5 ml 20% casamino acids, 9 ml 1% 
tryptophan, H.sub.2 O ad 806 ml, autoclaved, 3.6 ml 5% threonine and 90 ml 
20% glucose or 20% galactose added. 
SC-H broth: 7.5 g/l yeast nitrogen base without amino acids, 11.3 g/l 
succinic acid, 6.8 g/l NaOH, 5.6 g/l casamino acids without vitamins, 0.1 
g/l tryptophan. Autoclaved for 20 min. at 121.degree. C. After 
autoclaving, 10 ml of a 30% galactose solution, 5 ml of a 30% glucose 
solution and 0.4 ml of a 5% threonine solution were added per 100 ml 
medium. 
SC-H agar: 7.5 g/l yeast nitrogen base without amino acids, 11.3 g/l 
succinic acid, 6.8 g/l NaOH, 5.6 g/l casamino acids without vitamins, 0.1 
g/l tryptophan, and 20 g/l agar (Bacto). Autoclaved for 20 min. at 
121.degree. C. After autoclaving, 55 ml of a 22% galactose solution and 
1.8 ml of a 5% threonine solution were added per 450 ml agar. 
YNB-1 agar: 3.3 g/l KH.sub.2 PO.sub.4, 16.7 g/l agar, pH adjusted to 7. 
Autoclaved for 20 min. at 121.degree. C. After autoclaving, 25 ml of a 
13.6% yeast nitrogen base without amino acids, 25 ml of a 40% glucose 
solution, 1.5 ml of a 1% L-leucine solution and 1.5 ml of a 1% histidine 
solution were added per 450 ml agar. 
YNB-1 broth: Composition as YNB-1 agar, but without the agar. 
FG-4-Agar: 35 g/L agar, 30 g/L Soy bean meal, 15 g/L maltodextrin (Glucidex 
6), 5 g/L Bacto pepton, pH 7. Autoclaved 40 min at 121.degree. C. 
FG-4 medium: 30 g/L Soy bean meal, 15 g/L maltodextrin (Glucidex 6), 5 g/L 
Bacto peptone. Autoclaved 40 min at 121.degree. C. 
MDU-2 medium: 45 g/L maltose, 1 g/L MgSO.sub.4 -7 H.sub.2 O, 1 g/L NaCl, 
2g/L K.sub.2 SO.sub.4, 12 g/L KH.sub.2 PO.sub.4, 0.1 ml/L Pluronic 61 L, 
0.5 ml/L Trace metal solution. pH 5.0. Autoclaved 20 min at 121.degree. C. 
15 ml/L 50% sterile filtered urea is added after autoclaving. 
AZCL galactan from lupin: available from Megazyme, Australia. 
AZCL arabinan from sugar beet: available from Megazyme, Australia. 
Dyeing and Crosslinking of RG-Substrate 
Hairy regions from apples (MHR) were extracted from apples according to the 
method of Schols et al. (1990b). In order to remove arabinan sidechains 
from this rhamnogalacturonan 25 g of MHR was dissolved in 500 ml of water 
and 1.9 ml concentrated TFA was added. After 1 hour at 100.degree. C. the 
solution was dialysed against distilled water. The dialysed material was 
dyed by a modification of the method described by Call and Emeis, 1983 (J. 
Food-Biochem, 7(1): 43-52) by adding NaOH to pH 7.0, 5 g Cibacron C blau 
and 7.5 g Cibacron C gelb to the content of the dialysis bag and adding 25 
g of Na.sub.2 SO.sub.4 over a period of 10 minutes at 50.degree. C. After 
30 minutes 3.75 g of Na.sub.3 PO.sub.4 was added and pH was adjusted to 
11. pH was adjusted to 7 after 1 hour and 2.5 1 of ethanol was added. The 
precipitate was recovered by centrifugation and was redissolved and 
reprecipitated three times. To crosslink the material 0.5 g of the dyed 
dehaired rhamnogalacturonan was dissolved in 50 ml of water, pH was 
adjusted to 12 and 2.5 ml Divinylsulphone and 200 ml ethanol was added. 
After 1 hour at room temperature the mixture was neutralised with HCl, 
centrifuged and the precipitate washed in boiling water 2 times. Purified 
A. aculeatus RGase II obtained as described in WO 92/19728 and pure 
arabinanase and galactanase from MegaZyme were used to test the 
specificity of the assay. 
The purified A. aculeatus RGase II dissolved the crosslinked 
rhamnogalacturonan with a concomitant release of green colour. The 
arabinanase and galactanase did not dissolve the substrate. 
Fed Batch Fermentation 
The medium used for fed-batch fermentation of RGase I by A. oryzae 
comprised maltodextrin as a carbon source, urea as a nitrogen source and 
yeast extract. 
The fed batch fermentation was performed by innoculating a shake flask 
culture of the A. oryzae host cells in question into a medium comprising 
3.5% of the carbon source and 0.5% of the nitrogen source. After 24 hours 
of cultivation at pH 5.0 and 34.degree. C. the continuous supply of 
additional carbon and nitrogen sources were initiated. The carbon source 
was kept as the limiting factor and it was secured that oxygen was present 
in excess. The fed batch cultivation was continued for 4 days, after which 
the enzymes could be recovered. 
Characterization of Enzymes 
For pH optimum and temperature optimum determination, RGase activity was 
assayed on AZCL-galactan (from MegaZyme). 0.4% suspensions of 
AZCL-galactan were mixed 1:1 with 0.1M buffer (preferably acetate buffer, 
but for pH optimum citrate/trisodiumphosphate buffer systems were used) 
and a suitable amount of enzyme was added. Incubations were carried out in 
Eppendorf.RTM. thermomixers at 30.degree. C. (except for temperature 
optimum) for 15 minutes before the enzyme was inactivated at 95.degree. C. 
for 20 minutes. Centrifugation was carried out and the release of blue 
colour into the supernatant was measured in microtiter plates at 620 nm. 
The relative activity is defined as the activity divided by the activity 
at optimal pH or optimal temperature. 
Size Exclusion Chromatography (SEC) 
MHR was saponified by leaving a 2% solution at pH 12 for 1h at 50.degree. 
C. The saponified MHR (MHR-S) was recovered by precipitation in ethanol. A 
1% solution of MHR-S in 0.1M acetate buffer of optimal pH was added 10 
.mu.l of enzyme solution and incubation was carried out in thermomixers at 
30.degree. C. for 1, 2, 4 and 24 hours before heat-inactivation of enzyme. 
25 .mu.l of enzyme treated material was injected into three SEC-columns 
connected in series (TSK PW G4000, G3500 & G2500 including a guard column) 
and was eluted by 0.8 ml/min 0.4M sodium acetate buffer pH 3.0 supplied by 
a Dionex HPLC system. Eluting saccharides were detected by a Shimadzu RI 
detektor and collection of data was commenced 15 minutes after injection. 
Data were processed by Dionex software. Dextrans from Serva were used as 
molecular weight standards. 
High Pressure Anion Exchange Chromatography (HPAEC) 
The MHR-S digests were also analysed by anion-exchange chromatography using 
a Dionex HPLC system (Dionex Corporation, Sunnyvale, Calif.) equipped with 
a CarboPac column. This system was used for detection of saccharides 
as prescribed by the Dionex Corporation (Dionex Technical note TN 20). 
Eluents were A: 0.1M NaOH and B: 1M Sodium acetate in 0.1M NaOH. 25 .mu.l 
of sample was injected and saccharides were eluted at 1 ml/min with a 
gradient of 15% B to 50% B within 40 minutes. Data were processed by 
Dionex software. 
Degradation of Soy Polysaccharides 
Soy remanens was prepared by jetcooking of soy flour at 115.degree. C. for 
4 minutes, then hydrolysing the protein with Alcalase.RTM. (Novo Nordisk 
A/S), recovering of the residue and then repeating the Alcalase.RTM. 
treatment and recovery of residue. The so obtained residue of soy 
polysaccharides is essentially free from protein and was spraydried. 
To 1 ml of a 1% suspension of soy polysaccharide in 0.1M acetate buffer pH 
5.0 is added a suitable amount of enzyme and the mixture is incubated for 
0, 1, 2, 4, and 24 hours before heat inactivation and analysis by Size 
Exclusion Chromatography. 
The enzymes added were RGase I described herein, an rhamnogalacturonan 
acetyl esterase, and a combination of these enzymes. The 
rhamnogalacturonan acetyl esterase used was the A. aculeatus 
rhamnogalacturonan acetyl esterase described in WO 15 93/20190. 
Example 1 
Cloning of RGase I 
A library from A. aculeatus consisting of approx. 1.5.times.10.sup.6 
individual clones in 150 pools was constructed. 
DNA was isolated from 20 individual clones from the library and subjected 
to analysis for cDNA insertion. The insertion frequency was found to be 
&gt;90% and the average insert size was approximately 1400 bp. 
DNA from some of the pools was transformed into yeast, and 50-100 plates 
containing 200-500 yeast colonies were obtained from each pool. After 3-5 
days of growth, the agar plates were replica plated onto several sets of 
agar plates. One set of plates containing 0.1% AZCL galactan, potato or 
lupin (Megazyme), one set containing 0.2% dyed and crosslinked 
MHR-substrate and one set containing 0.1% AZCL-arabinan were then 
incubated for 3-5 days at 30.degree. C. for detection of activity. 
Positive arabinanase and galactanase colonies were identified as colonies 
surrounded by a blue halo. 
Positive RGase colonies were identified where the grains of insoluble 
MHR-substrate were dissolved. 
The screening of yeast colonies yielded four clones possessing RGase 
activity. Upon DNA sequencing of the inserts it became evident that all 
four clones represented the same enzyme (RG I). From the DNA sequence it 
also became evident that this sequence did not correspond to the amino 
acid sequence of the purified RGase II disclosed in Wo 92/19728). 
It was very surprisingly observed that the four rhamnogalacturonase 
positive colonies also could be detected on AZCL-galactan and 
AZCL-arabinan. The arabinanase and galactanase colonies on the other hand 
did not give positive reaction on the crosslinked rhamnogalacturonan, 
which verifies the results obtained in the testing with pure arabinanase 
and galactanase. Thus, AZCL-galactan and AZCL-arabinan can be used as 
unspecific substrates for RGases (and are concomitantly shown not to be 
specific for arabinanases or galactanases either), whereas the dyed and 
crosslinked dehaired rhamnogalacturonan is highly specific for RGases. 
Cells from enzyme-positive colonies were spread for single colony isolation 
on agar, and an enzyme-producing single colony was selected for each of 
the positive colonies identified. 
Characterization of positive clones: The positive clones were obtained as 
single colonies, the cDNA inserts were amplified directly from the yeast 
colony using biotinylated polylinker primers, purified by magnetic beads 
(Dynabead M-280, Dynal) system and characterized individually by 
sequencing the 5'-end of each cDNA clone using the chain-termination 
method (Sanger et al., 1977) and the Sequenase system (United States 
Biochemical). The DNA sequence of the enzyme gene is shown in SEQ ID No. 1 
and the amino acid sequence deduced therefrom in SEQ ID No. 2. The DNA and 
amino acid sequence is further shown in Table 1 hereinafter. A comparison 
of the DNA and amino acid sequence of SEQ ID No. 2 with that of RGase II 
did not reveal any homology. Furthermore, no homologous sequences could be 
detected by a search in the EMBL/GenBank databases performed on the basis 
of SEQ ID Nos. 1 and 2. 
Isolation of a cDNA gene for expression in Aspergillus: In order to avoid 
PCR errors in the gene to be cloned, the cDNA was isolated from the yeast 
plasmid by standard procedures as described below. 
One or more of the positive colonies were inoculated into 20 ml YNB-1 broth 
in a 50 ml glass test tube. The tube was shaken for 2 days at 30.degree. 
C. The cells were harvested by centrifugation for 10 min. at 3000 rpm. 
The cells were resuspended in 1 ml 0.9 M sorbitol, 0.1 M EDTA, pH 7.5. The 
pellet was transferred to an Eppendorf tube, and spun for 30 seconds at 
full speed. The cells were resuspended in 0.4 ml 0.9 M sorbitol, 0.1 M 
EDTA, 14 mM .beta.-mercaptoethanol. 100 .mu.l 2 mg/ml Zymolase was added, 
and the suspension was incubated at 37.degree. C. for 30 minutes and spun 
for 30 seconds. The pellet (spheroplasts) was resuspended in 0.4 ml TE. 90 
.mu.l of (1.5 ml 0.5 M EDTA pH 8.0, 0.6 ml 2 M Tris-Cl pH 8.0, 0.6 ml 10% 
SDS) was added, and the suspension was incubated at 65.degree. C. for 30 
minutes. 80 .mu.l 5 M KOAc was added, and the suspension was incubated on 
ice for at least 60 minutes and spun for 15 minutes at full speed. The 
supernatant was transferred to a fresh tube which was filled with EtOH 
(room temp.) followed by thorough but gentle mixing and spinning for 30 
seconds. The pellet was washed with cold 70% ETOH, spun for 30 seconds and 
dried at room temperature. The pellet was resuspended in 50 .mu.l TE and 
spun for 15 minutes. The supernatant was transferred to a fresh tube. 2.5 
.mu.l 10 mg/ml RNase was added, followed by incubation at 37.degree. C. 
for 30 minutes and addition of 500 .mu.l isopropanol with gentle mixing. 
The mixture was spun for 30 seconds, and the supernatant was removed. The 
pellet was rinsed with cold 96% EtOH and dried at room temperature. The 
DNA was dissolved in 50 .mu.l water to a final concentration of 
approximately 100 .mu.l/ml. 
The DNA was transformed into E. coli by standard procedures. Two E. coli 
colonies were isolated from each of the transformations and analysed with 
the restriction enzymes HindIII and XbaI which excised the DNA insert. DNA 
from one of these clones was retransformed into yeast strain JG169. 
The DNA sequences of several of the positive clones where partially 
determined. The DNA sequence encoding RGase I of the invention is shown in 
SEQ ID No. 1. The deduced amino acid sequence is shown in SEQ ID No. 2. 
Example 2 
Expression of Rhamnogalacturonase I (RGase I) 
In order to express the genes in Aspergillus, cDNA was isolated from one or 
more representatives of each family by digestion with HindIII/XbaI or 
other appropriate restriction enzymes, size fractionation on a gel and 
purification and subsequently ligated to pHD414, resulting in the plasmid 
pGal-II. After amplification in E. coli, the plasmids were transformed 
into A. oryzae or A. niger according to the general procedure described 
above. 
Test of A. oryzae Transformants 
Each of the transformants was inoculated in the center of a Petri dish with 
FG-4 agar. After 5 days of incubation at 30.degree. C. 4 mm diameter plugs 
were removed from the center of the colonies by a corkscrew. The plugs 
were embedded in a galactan overlayer gel, containing 0.1% AZCL galactan 
and 1% agarose in a buffer with an appropriate pH, and incubated overnight 
at 40.degree. C. The RGase activity was identified as described above. 
Some of the transformants had halos which were significantly larger than 
the Aspergillus oryzae background. This demonstrates efficient expression 
of RGase in Asperzgilus oryzae. The 8 transformants with the highest RGase 
activity were selected and inoculated and maintained on YPG-agar. 
Each of the 8 selected transformants were inoculated from YPG-agar slants 
on 500 ml shake flask with FG-4 and MDU-2 media. After 3-5 days of 
fermentation with sufficient agitation to ensure good aeration, the 
culture broths were centrifuged for 10 minutes at 2000 g and the 
supernatants were analyzed. 
A volume of 15 .mu.l of each supernatant was applied to 4 mm diameter holes 
punched out in a 0.1% AZCL galactan overlayer gel (25 ml in a 13 cm 
diameter Petri dish). The RGase activity was identified by the formation 
of a blue halo on incubation. 
Fed Batch Fermentation 
Subsequently, RGase I was produced by fed batch fermentation of A. oryzae 
expressing the enzyme using the procedure described above. 
Example 3 
Purification of RGase I 
The culture supernatant from fermentation of Aspergillus oryzae or A. niger 
expressing the recombinant enzyme is centrifuged at 5000.times.g and 
filtered through a 0.2 .mu.m filter to remove the mycelia. 35-50 ml of the 
filtered supernatant containing 30-70 mg of the recombinant enzyme is 
ultrafiltrated in an Amicon 200 ml ultrafiltration device with a 10 kDa 
membrane to achieve 10 fold concentration. This concentrate is diluted 100 
times in 20 mM Tris pH 8.0 in two successive rounds of ultrafiltration in 
the same device, and the final volume is adjusted to approximately 40 ml. 
This ultrafiltrated sample is loaded at 2 ml/min on a Pharmacia HR16/10 
Fast Flow Q Sepharose anion exchanger equilibrated in 20 mM Tris pH 8.0. 
After the sample has been applied, the column is washed with two column 
volumes 20 mM Tris pH 8.0, and bound proteins are eluted with a linear 
increasing NaCl gradient from 0 to 0.5 M NaCl in 20 mM Tris pH 8.0. RGase 
I elutes at approximately 0.3 M NaCl, and fractions containing RGase I 
activity are pooled and concentrated by ultrafiltration. RGase I in this 
fraction was purified to more than 99% homogeneity. 
Example 4 
Characterization of RGase I and II 
After transformation, expression and purification as described above, RGase 
I was characterized. For comparison, some characterizing data for A. 
aculeatus RGase II (obtained as described in WO 92/19728) are included. 
AZCL-galactan can be used for detection of RGase activity since it was 
discovered that this substrate is not a pure galactan but is composed of 
fragments of rhamnoglacturonan backbone with galactan sidechains attached 
to it. The MW and pI for RG I can be seen in the table below and the pH 
optimum and temperature optimum from FIG. 3 and FIG. 4, respectively. 
______________________________________ 
RG I 
______________________________________ 
Mw 59.2 
pI 5.1 
pH optimum 6.0 
pH stability &gt;6.0 
temp. optimum 50.degree. C. 
temp. stability &lt;60.degree. C. 
______________________________________ 
It is seen that RG I is most active about pH 6.0 whereas RG II is most 
active around pH 3.5. This signifies the different application 
possibilities of the two enzymes, RGase I being much more active in the 
neutral pH range. 
Both enzymes were found to be more active in acetate buffer than in 
phosphate or citrate buffer and therefore all experiments except the pH 
optimum were carried out in acetate buffer of the optimal pH for the 
enzyme. Both enzymes became partly inactivated after 1 hour at 60.degree. 
C. 
The molecular weight of RGase I (59.2 kD) is lower than the molecular 
weight of RGase II (62 kD), and polyclonal antibodies raised against RGase 
II do not cross-react with RGase I. The glycosylation differs 
significantly between the two RGases. RGase II (but not RGase I) reacts 
with GNA lectin which is specific for terminal .beta.-1,3, .beta.-1,2 or 
.beta.-1,6 bound mannose in protein attached glycan structures. 
The SEC degradation patterns obtained on MHR-S with RG I and RG II can be 
seen from FIGS. 5 and 6. The smallest oligosaccharide obtained can be 
estimated to have a DP of approximately DP 6-10. From studies on the 
purified RG II from A. aculeatus it is known that RG II hydrolyses between 
galacturonic acid and rhamnose leaving rhamnose as the non-reducing 
residue (Colquhoun et al. (1990)). The SEC analysis shows that the 
degradation products of the two RGases are of the same molecular weight 
after 24 hours (see FIGS. 5 and 6). The two enzymes are dosed in order to 
give the same activity on AZCL-galactan. However, it can be seen that on 
MHR-S RG II is more active than RGI, which shows that the two enzymes have 
very different substrate specificity. When the degradation products are 
analysed by anion exchange chromatography different chromatograms are 
obtained with the two enzymes. The RG I oligomer peaks elute much later 
than the RG II oligomers (see FIGS. 7 and 8). This is not due to the RG I 
oligomers being larger, since the SEC analysis showed that the oligomers 
have the same size. It is known that oligomers with a deoxyhexose 
(rhamnose or fucose which elute very early from the column) at the 
non-reducing end tend to elute much earlier than even smaller oligomers 
with no deoxyhexose attached (McDougall & Fry (1991)). Thus the results 
obtained with the RGI degradation products shows that RGI hydrolyses at 
the other side of the rhamnose leaving galacturonic acid as the 
non-reducing end. This mode of action of a RGase has never been reported 
before and therefore the activity possessed by RG I must be considered to 
be a completely new type of enzyme activity. 
Example 5 
Extraction of Soluble Fibres from Soy 
From the SEC of the enzyme treated soy polysaccharides (obtained as 
described in the Materials and Methods section-above) the peak area can be 
obtained, cf FIG. 9 which shows the degradation obtained by RGase I. When 
compared to the area obtained with a 1% dextran solution it can be 
calculated that RG I can solubilize about 25% of the soy polysaccharides 
and the combination of RG I and the A. aculeatus rhamnogalacturonan acetyl 
esterase (RGAE) (obtained as described in Wo 93/20190) about 50%. This 
solubilized material has a surprisingly high molecular weight exceeding DP 
500 (calculated from the dextfan standards). The high molecular weight of 
the solubilized polysaccharides and monosaccharide analysis show that the 
liberated material is pieces of rhamnogalacturonan with sidechains 
attached. Monosaccharide analysis has shown that these sidechains are 
predominately galactans and secondarily arabinans. Thus, the RG I can be 
used for extraction of hairy regions from soy which maintain a 
surprisingly high molecular weight. RGase II was found to be inferior to 
RGase I for these purposes. Similar results have been obtained with rape 
seed polysaccharides. 
Example 6 
Viscosity Reduction of Soy Flour by RGase 1 
Soy flour treated with 1 AU/kg DS Alcalase.RTM. (Novo Nordisk A/S) for 2 
hours at pH 6.4, 50.degree. C. and 23% DS was jet cooked for 4 min. at 
115.degree. C. and pH 5.0. The resulting slurry was spray dried. 
2.times.60 g of spray dried jet cooked soy were suspended in 180 and 190 g 
of deionized water, respectively. The slurries were homogenized in a 
Warring Blender (the power switch in position 4) for 2 min. and then 
transferred to 2.times.250ml beakers. pH was adjusted to 5.0 and the 
samples were heated to 50.degree. C. on a water bath. When the temperature 
reached 50.degree. C. in the samples, 10 ml of an enzyme solution of RGase 
1 containing 170 mg emzyme was added to the low volume sample. The slurry 
was mixed well with a glas rod. The viscosity in the samples were measured 
after 24 hours of incubation on a Brookfield viscometer, model RVDVII, 
with spindle 91 (T-A), at 50 rpm. The viscosity in the enzyme sample was 
only 48% of that in the sample without enzyme, due to the action of RGase 
I. Thus, it has been shown that a substantial viscosity reduction may be 
obtained by use of only one carbohydrate degrading enzyme. 
Example 7 
Purification of Saponified Beet Pectic Hairy Regions 
Sugar beet fibres were produced from whole beets. The beets where washed, 
mashed, and pressed at 60-70.degree. C., water was added to the filter 
cake, before it was repressed. The press-cake was dried and designated 
beet fibres. 
150 g beet fibre were incubated in 3 L 0.05 M sodium succinate pH 5 with 2 
w/w % Pectinex Ultra SP (obtainable from Novo Nordisk A/S) for 24 hours at 
40.degree. C., the incubation was stopped by heating at 100.degree. C. for 
30 min, and centrifugated for 60 minutes at 10.000 g. The pellet was 
washed 3 times with distilled water and the supernatants were pooled and 
concentrated with Ultrafiltration (Nephross Andante HF, Organon Technika, 
cut off 5.000). The solution was dialysed 24 hours against distilled water 
at room temperature. The retentate was freeze dried. The retentate was 
designated beet pectic modified hairy regions. 
The sugar composition of the beet pectic modified hairy regions was 
determined to be 4% rhamnose, 48% arabinose, 1% xylose, 11% galactose, 1% 
glucose and 34% anhydrous uronic acid. The neutral sugars were determined 
by G.L.C. after pretreatment (1 h, 30.degree. C.) with aqueous 72% H.sub.2 
SO.sub.4 followed by hydrolysis with 1 M H.sub.2 SO.sub.4 (3 h, 
100.degree. C.) and conversion of the products into alditol acetates 
(Englyst & Cummings, Analyst 109 (1984) 937-942). The aditol acetates were 
analysed on a glass column (3 m.times.2 mm i.d.), packed with Chrom WAW 
80-100 mesh coated with 3% OV275 in a Carlo Erba Fractovap 2300 GC. The 
uronic acid were determined by the calorimetric assay described by A. E. 
R. Ahmed et al., 1977, Food Biochemistry 1: 361-365. 
The beet pectic hairy regions was saponified as described by Schols et al. 
(1990a). 
RGase I, corresponding to 473 .mu.g pure enzyme protein pr/ml, obtained as 
described in Examples 2 and 3, and an equivalent activity of RGase II 
(corresponding to 44 .mu.g/ml pure enzyme protein pr ml) obtained as 
described in WO 92/19728 were added to 4 mg/ml saponified beet pectic 
modified hairy regions obtained as described above in separate 
experiments. The degradation patterns were analyzed by HPSEC after 24 
hours incubation at 40.degree. C. in 50 mM sodium acetate pH 5.0 
(preserved by NaN.sub.3). 
The HPSEC was performed as described in H. A. Schols et al. 1990 
(Carbohydrat. Res. 206: 105-115) on a SP800 HPLC (Spectra Physics) 
equipped with three BioGel TSK colmns (each 300.times.7.5 mm) in series 
(40XL, 30XL, and 20XL, Bio-Rad Labs) in combination with a TSK XL guard 
column (40.times.6 mm) and eluted at 30.degree. C. with 0.4 M acetic 
acid/sodium acetate (pH 3.0) at 0.8 ml/min. The eluate was monitored using 
a Shodex SE-61 Refractive Index detector. 
The RGase II had only insignificant activity on the saponified beet pectic 
hairy regions, where the RGase I gave a significant degradation of the 
saponified beet pectic hairy regions as showed on the HPSEC 
chromatogramme, cf FIG. 10. 
REFERENCES 
P. Albersheim et al., Pure & Appl. Chem., Vol. 53, pp. 79-88, 1981. 
Aviv, H. & Leder, P. 1972. Proc. Natl. Acad. Sci. U. S. A. 69: 1408-1412. 
Axelsen N. et al., Blackwell Scientific Publications, 1973, Chapter 23 
Becker, D. M. & Guarante, L. 1991. Methods Enzymol. 194: 182-187. 
N. C. Carpita and D. M. Gebeaut, The Plant Journal, Vol. 3, No. 1, pp. 
1-30, 1993. 
Circle et al., 1978, in Smith, A. K. Editor: Soybeans: Chemistry and 
Technology, Vol 1, Proteins. 
Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. 1979. 
Biochemistry 18: 5294-5299. 
Colquhoun et al., 1990, Carbohydrate Research 206, pp. 131-144. 
S. C. Fry, in The growing plant cell wall: chemical and metabolic analysis, 
Longman Scientific & Technical, 1988. 
Gubler, U. & Hoffman, B. J. 1983. Gene 25: 263-269. 
Johnstone A. and Thorpe R., Blackwell Scientific Publications, 1982 (more 
specifically pp. 27-31). 
McDougall & Fry, 1991, Carbohyarate Research 219, pp. 123-132. 
M. O'Neill et al., Methods in Plant Biochemistry, Vol. 2, pp. 415, 1990. 
O. Ouchterlony, Handbook of Experimental Immunology (D. M. Weir, Ed.), 
Blackwell Scientific Publications, 1967, pp. 655-706) 
T. Sakamoto et al., Biosci. Biotech. & Biochem., Vol. 57, No. 11, pp. 
1837-1837, 1993. 
Sambrook, J., Fritsch, E. F. & Maniatis, T. 1989. Molecular Cloning: A 
Laboratory Manual. Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. 
Sanger, F., Nicklen, S. & Coulson, A. R. 1977. Proc. Natl. Acad. Sci. U. S. 
A. 74: 5463-5467. 
Schols et al., Carbohydrate Research 206 (1990a) 105-115. 
Schols et al., Carbohydrate Research 206 (1990b) 117-129. 
Searle-van Leeuwen et al., "Rhamnogalacturonan acetyl esterase: a novel 
enzyme from Aspergillus aculeatus, specific for the deacetylation of hairy 
(ramified) regions of pectin", Appl. Microbiol. Biotechnol., 38: p. 
347-349, 1992 
A. G. J. Voragen et al., Proceedings of the International Symposium on 
Plant Polymeric Carbohydrates, Berlin, Jul. 1-3, 1992, (F. Meuser, ed.), 
Royal Society of Chemistry, Cambridge, U.K. 
TABLE 1 
__________________________________________________________________________ 
SEQUENCE LISTING 
Nucleotide sequence of the rhg1 gene, 
and the deduced primary structure of RGase 
I from A. aculeatus. The proposed 
signal sequence is 
underlined, and the NH.sub.2 -terminal amino 
acid sequence of the purified 
rhamnogalacturonase from A. japonicus 
is shown aligned with the homologous 
region in the A. aculeatus enzyme. 
__________________________________________________________________________ 
60 
* * * * * * 
CGGACAATGCTCAAAGCGTCGCTTCTGTCGTTCGTGGCCTTCACGGCCCAGGTCGCCCAC 
M L K A S L L S F V A F T A O V A H 
120 
* * * * * * 
GCGGCCTTTGGCATCACCACCAGCTCCAGCGCCTATGTCATCGACACCAACGCGCCAAAC 
A A F G I T T S S S A Y V I D T N A P N 
A F G I T T S S S A Y V I D T D A P N 
180 
* * * * * * 
CAGCTGAAGTTCACCGTCAGCCGCAGCAGCTGCGACATTACCTCCATCATCCACTATGGC 
Q L K F T V S R S S C D I T S I I H Y G 
Q L K ? T V S R 
240 
* * * * * * 
ACGGAGCTGCAGTACTCCAGCCAGGGCAGTCACATTGGGTCGGGTCTGGGCTCTGCGACG 
T E L Q Y S S Q G S H I G S G L G S A T 
300 
* * * * * * 
GTGACCGCCACGCAGTCCGGGGACTATATCAAGGTGACCTGTGTGACGGACACCTTGACG 
V T A T Q S G D Y I K V T C V T D T L T 
360 
* * * * * * 
CAGTACATGGTGGTGCATAATGGGGACCCAATCATTCACATGGCGACATATATCACTGCC 
Q Y M V V H N G D P I I H M A T Y I T A 
420 
* * * * * * 
GAGCCGTCAATCGGCGAGCTGCGGTTCATCGCTCGACTGAATTCGGACCTGCTACCGACG 
E P S I G E L R F I A R L N S D L L P T 
480 
* * * * * * 
AGGAGCCGTTTGGCGACGTTTCCACCACCGCTGACGGGACTGCCATTGAGGGATCAGATG 
R S R L A T F P P P L T G L P L R D Q M 
540 
* * * * * * 
TGTTTTTGGTCGGCAGTGAAACCCGCAGCAAGTTCTACTAGAGCGAGCGATTTATCGACG 
C F W S A V K P A A S S T R A S D L S T 
600 
* * * * * * 
ATCAGCGACACTGCATTGCCGGGGATGCCCACCGCCGTTTGCATGATCTTGAATCAATAC 
I S D T A L P G M P T A V C M I L N Q Y 
660 
* * * * * * 
GAAAGCTCCTCCGGAGGTCCTTTCCACCGGGATATCAACTCGAACAACGGAGGGAGCTAC 
E S S S G G P F H R D I N S N N G G S Y 
720 
* * * * * * 
AACGCCCTCTACTGGTACATGAACTCCGGCCACGTTCAAACCGAGTCCTACCGGATGGGT 
N A L Y W Y M N S G H V Q T E S Y R M G 
780 
* * * * * * 
CTCCACGGCCCATACTCGATGTACTTTAGTCGCAGCGGTACCCCCAGCACCAGCATCGAT 
L H G P Y S M Y F S R S G T P S T S I D 
840 
* * * * * * 
ACCTCATTCTTCGCCGACCTTGACATCAAAGGCTATGTTGCCGCCTCAGGCCGAGGCAAA 
T S F F A D L D I K G Y V A A S G R G K 
900 
* * * * * * 
GTGGCCGGCACGGCATCCGGAGCAGACTCGAGCATGGATTGGGTGGTTCACTGGTACAAC 
V A G T A S G A D S S M D W V V H W Y N 
960 
* * * * * * 
GATGCGGCACAGTACTGGACTTATACCAGCTCCAGCGGCAGCTTCACCTCGCCCGCCATG 
D A A Q Y W T Y T S S S G S F T S P A M 
1020 
* * * * * * 
AAGCCCGGAACGTACACCATGGTCTATTACCAAGGCGAGTACGCGGTCGCCACGAGCTCG 
K P G T Y T M V Y Y Q G E Y A V A T S S 
1080 
* * * * * * 
GTCACCGTGTCCGCCGGATCAACCACAACGAAGAACATTTCGGGGTCCGTGAAGACCGGC 
V T V S A G S T T T K N I S G S V K T G 
1140 
* * * * * * 
ACTACCATTTTCAAGATTGGTGAATGGGACGGACAACCGACCGCGTTCCGCAACGCAGCC 
T T I F K I G E W D G Q P T A F R N A A 
1200 
* * * * * * 
AACCACGTCCGCATGCACCCCTCCGACTCGCGCATGCCCTCCTGGGGTCCACTGACCTAT 
N H V R M H P S D S R M P S W G P L T Y 
1260 
* * * * * * 
ACGGTTGGCAGTTCCGCTCTGACTGACTTCCCAATGGCCGTGTTCAAAAGCGTCAACAAC 
T V G S S A L T D F P M A V F K S V N N 
1320 
* * * * * * 
CCGGTCACCATCAAATTCACCGCCACATCCGCGCAGACCGGCGCAGCGACCCTGCGAATC 
P V T I K F T A T S A Q T G A A T L R I 
1380 
* * * * * * 
GGGACGACCTTGTCGTTTGCCGGTGGACGACCCCAGGCGACGATCAACAGCTACACAGGA 
G T T L S F A G G R P Q A T I N S Y T G 
1440 
* * * * * * 
AGCGCACCAGCCGCGCCGACAAACCTGGACTCTCGGGGCGTGACCCGCGGTGCGTACCGG 
S A P A A P T N L D S R G V T R G A Y R 
1500 
* * * * * * 
GGATTGGGCGAGGTGTATGATGTGTCCATCCCGTCGGGGACGATCGTCGCGGGAACAAAT 
G L G E V Y D V S I P S G T I V A G T N 
1560 
* * * * * * 
ACAATTACGATCAACGTGATCTCTGGCAGTTCGGGGGATACGTATTTGAGTCCGAACTTT 
T I T I N V I S G S S G D T Y L S P N F 
1620 
* * * * * * 
ATCTTTGATTGTGTGGAGTTGTTCCAGTAGCTGATTGTTTCTCGGGCTGTATGGTGCAGC 
I F D C V E L F Q * 
1680 
* * * * * * 
CGGGAGTAGATAGCTGTACTGGACAGTTCTAGTCGTATGTGGAGGAAAGACCTAAGATCA 
1740 
* * * * * * 
ACTGAATTCATGACCTACTGTCATTTTCTGTTGAAGTATTGTTCTGCTTGAATAAAGGTA 
* * * 
TGTGGTTCAGCCTGGCAAAAAAAAAAAAAAAAAAAA 
SEQ ID No. 1 
CGGACAATGCTCAAAGCGTCGCTTCTGTCGTTCGTGGCCTTCACGGCCCAGGTCGCCCAC 
GCGGCCTTTGGCATCACCACCAGCTCCAGCGCCTATGTCATCGACACCAACGCGCCAAAC 
CAGCTGAAGTTCACCGTCAGCCGCAGCAGCTGCGACATTACCTCCATCATCCACTATGGC 
ACGGAGCTGCAGTACTCCAGCCAGGGCAGTCACATTGGGTCGGGTCTGGGCTCTGCGACG 
GTGACCGCCACGCAGTCCGGGGACTATATCAAGGTGACCTGTGTGACGGACACCTTGACG 
CAGTACATGGTGGTGCATAATGGGGACCCAATCATTCACATGGCGACATATATCACTGCC 
GAGCCGTCAATCGGCGAGCTGCGGTTCATCGCTCGACTGAATTCGGACCTGCTACCGACG 
AGGAGCCGTTTGGCGACGTTTCCACCACCGCTGACGGGACTGCCATTGAGGGATCAGATG 
TGTTTTTGGTCGGCAGTGAAACCCGCAGCAAGTTCTACTAGAGCGAGCGATTTATCGACG 
ATCAGCGACACTGCATTGCCGGGGATGCCCACCGCCGTTTGCATGATCTTGAATCAATAC 
GAAAGCTCCTCCGGAGGTCCTTTCCACCGGGATATCAACTCGAACAACGGAGGGAGCTAC 
AACGCCCTCTACTGGTACATGAACTCCGGCCACGTTCAAACCGAGTCCTACCGGATGGGT 
CTCCACGGCCCATACTCGATGTACTTTAGTCGCAGCGGTACCCCCAGCACCAGCATCGAT 
ACCTCATTCTTCGCCGACCTTGACATCAAAGGCTATGTTGCCGCCTCAGGCCGAGGCAAA 
GTGGCCGGCACGGCATCCGGAGCAGACTCGAGCATGGATTGGGTGGTTCACTGGTACAAC 
GATGCGGCACAGTACTGGACTTATACCAGCTCCAGCGGCAGCTTCACCTCGCCCGCCATG 
AAGCCCGGAACGTACACCATGGTCTATTACCAAGGCGAGTACGCGGTCGCCACGAGCTCG 
GTCACCGTGTCCGCCGGATCAACCACAACGAAGAACATTTCGGGGTCCGTGAAGACCGGC 
ACTACCATTTTCAAGATTGGTGAATGGGACGGACAACCGACCGCGTTCCGCAACGCAGCC 
AACCACGTCCGCATGCACCCCTCCGACTCGCGCATGCCCTCCTGGGGTCCACTGACCTAT 
ACGGTTGGCAGTTCCGCTCTGACTGACTTCCCAATGGCCGTGTTCAAAAGCGTCAACAAC 
CCGGTCACCATCAAATTCACCGCCACATCCGCGCAGACCGGCGCAGCGACCCTGCGAATC 
GGGACGACCTTGTCGTTTGCCGGTGGACGACCCCAGGCGACGATCAACAGCTACACAGGA 
AGCGCACCAGCCGCGCCGACAAACCTGGACTCTCGGGGCGTGACCCGCGGTGCGTACCGG 
GGATTGGGCGAGGTGTATGATGTGTCCATCCCGTCGGGGACGATCGTCGCGGGAACAAAT 
ACAATTACGATCAACGTGATCTCTGGCAGTTCGGGGGATACGTATTTGAGTCCGAACTTT 
ATCTTTGATTGTGTGGAGTTGTTCCAGTAGCTGATTGTTTCTCGGGCTGTATGGTGCAGC 
CGGGAGTAGATAGCTGTACTGGACAGTTCTAGTCGTATGTGGAGGAAAGACCTAAGATCA 
ACTGAATTCATGACCTACTGTCATTTTCTGTTGAAGTATTGTTCTGCTTGAATAAAGGTA 
TGTGGTTCAGCCTGGCAAAAAAAAAAAAAAAAAAAA 
SEQ ID No. 2 
M L K A S L L S F V A F T A Q V A H 
A A F G I T T S S S A Y V I D T N A P N 
Q L K F T V S R S S C D I T S I I H Y G 
T E L Q Y S S Q G S H I G S G L G S A T 
V T A T Q S G D Y I K V T C V T D T L T 
Q Y M V V H N G D P I I H M A T Y I T A 
E P S I G E L R F I A R L N S D L L P T 
R S R L A T F P P P L T G L P L R D Q M 
C F W S A V K P A A S S T R A S D L S T 
I S D T A L P G M P T A V C M I L N Q Y 
E S S S G G P F H R D I N S N N G G S Y 
N A L Y W Y M N S G H V Q T E S Y R M G 
L H G P Y S M Y F S R S G T P S T S I D 
T S F F A D L D I K G Y V A A S G R G K 
V A G T A S G A D S S M D W V V H W Y N 
D A A Q Y W T Y T S S S G S F T S P A M 
K P G T Y T M V Y Y Q G E Y A V A T S S 
V T V S A G S T T T K N I S G S V K T G 
T T I F K I G E W D G Q P T A F R N A A 
N H V R M H P S D S R M P S W G P L T Y 
T V G S S A L T D F P M A V F K S V N N 
P V T I K F T A T S A Q T G A A T L R I 
G T T L S F A G G R P Q A T I N S Y T G 
S A P A A P T N L D S R G V T R G A Y R 
G L G E V Y D V S I P S G T I V A G T N 
T I T I N V I S G S S G D T Y L S P N F 
I F D C V E L F Q 
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