EP0583313A1 - Rhamnogalacturonase, corresponding dna sequence, rhamnogalacturonase containing enzyme preparation and use of the enzyme preparation - Google Patents

Rhamnogalacturonase, corresponding dna sequence, rhamnogalacturonase containing enzyme preparation and use of the enzyme preparation

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Publication number
EP0583313A1
EP0583313A1 EP92909677A EP92909677A EP0583313A1 EP 0583313 A1 EP0583313 A1 EP 0583313A1 EP 92909677 A EP92909677 A EP 92909677A EP 92909677 A EP92909677 A EP 92909677A EP 0583313 A1 EP0583313 A1 EP 0583313A1
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EP
European Patent Office
Prior art keywords
ala
rgase
gly
ser
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP92909677A
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German (de)
French (fr)
Inventor
Kurt Dörreich
Henrik Dalboge
Jan M Ller Mikkelsen
Flemming Mark Christensen
Torben Halkier
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Novozymes AS
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Novo Nordisk AS
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Priority to EP92909677A priority Critical patent/EP0583313A1/en
Priority to EP00118463A priority patent/EP1067180A1/en
Publication of EP0583313A1 publication Critical patent/EP0583313A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01171—Rhamnogalacturonan hydrolase (3.2.1.171), i.e. rhamnogalacturonase

Definitions

  • the invention comprises a rhamnogalacturonase (in the following usually abbreviated RGase), a corresponding DNA sequence, an RGase containing enzyme preparation and a use of the enzyme preparation.
  • RGase rhamnogalacturonase
  • the invention relates to genetic engineering and provides partial amino acid sequences of an RGase.
  • These partial amino acid sequences can be used for construction of DNA probes which can be used for screening a genomic library from organisms expressing such enzyme, or a cDNA library, thereby obtaining DNA sequences, which can be used either for an overproduction of RGase, if inserted in the microorganism species, from which the parent DNA molecule originated, or for production of RGase 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 RGase.
  • Plant cell walls comprising rhamnogalacturonans are of complex nature.
  • Pectins are proposed to consist of highly carboxyl-methylated linear homogalacturonan regions which alternate with "hairy" (ramified) regions that comprise highly branched rhamnogalacturonans mainly.
  • linear homogalacturonan regions are very well known and characterized, the structure of the so-called hairy regions is still not fully characterized, and thus is the subject of many investigations. But besides the scientific interest it is very important to be able to degrade these hairy regions for technical reasons.
  • the enzymatic liquefaction of plant material like e.g. fruits, vegetables, cereals, oil fruits and seeds by technical processes involves combinations of pectolytic, cellulolytic and proteolytic enzyme preparations. This enzymatic treatment solubilizes the hairy regions and other pectic fragments, which originate from the insoluble cell wall protopectin.
  • the linear homogalacturonan regions are very well known and characterized, the structure of the so-called hairy regions is still not fully characterized, and thus is
  • RGase is described in the poster "Rhamnogalacturonase; a novel enzyme degrading the highly branched rhamnogalacturonan regions in apple pectic substances" from Wageningen Agricultural University, Department of Food Science, Biotechnion, Bomenweg 2, 6703 HD Wageningen, The Netherlands. From this poster Is appears that a rhamnogalacturonase, the origin of which is not described, is well suited for degradation of the backbone of a modified "hairy region” (MHR) in plant cell walls. Also, it is described that this enzyme might play a role in the degradation of plant cell wall tissue, particularly in combination with other enzymes. However, it is not specified which other enzymes.
  • SUBSTITUTE SHEET composition of a mixture of oligosaccharides obtained by enzymatic degradation of the modified hairy (ramified) regions of apple pectin with a RGase is described.
  • RGase belongs to the prior art, and has been purified, i.e. the A. aculeatus RGase described by Schols et al., this RGase being the RGase appearing in all the previosly indicated references. Also, this RGase has only been partly characterized and has not been characterized in regard to amino acid sequence, the corresponding RGase produced gene has not been cloned, and thus, the prior art RGase has not been available as a cheap, industrially useable product.
  • the purpose of the invention is the provision of an RGase, which covers embodiments exhibiting varying characteristics corresponding to the different industrial conditions, under which rhamnogalacturonans have to be degraded, and of an RGase, which can be produced in better yield, and thus cheaper, than hitherto possible, and in higher purity. Also, it is the purpose of the invention to provide novel products, wherein the proportion of the RGase is either increased or decreased in relation to the proportion in the original product.
  • amino acid sequence preferably an N-terminal amino acid sequence with a homology thereto of at least 70%, preferably at least 80%, more preferably at least
  • RGases with an advantageously high ability to degrade rhmanogalacturonans under the varying conditions appearing in industry.
  • amino acid sequence preferably an N-terminal amino acid sequence with a homology thereto of at least 70%, preferably at least 80%, more preferably at least 90%.
  • SUBSTITUTE SHEET A preferred embodiment of the RGase according to the invention is characterized by the following partial amino acid sequences
  • a preferred embodiment of the RGase according to the invention is characterized by being obtainable by means of Aspergillus aculeatus, CBS 101.43.
  • a preferred embodiment of the RGase according to the invention is characterized by being obtainable by means of A. japonicus ATCC 20236.
  • a preferred embodiment of the RGase according to the invention is characterized by being obtainable by means of Irpex lacteus ATCC 20157.
  • the invention comprises a recombinant DNA sequence, which comprises a DNA sequence coding for a polypeptide having RGase activity, or a DNA sequence having substantial sequence homology to such RGase coding sequence, preferably a homology of at least 70%, more preferably at least 80%, and most preferably at least 90%.
  • a preferred embodiment of the recombinant DNA sequence according to the invention comprises a DNA sequence selected from a) the A. aculeatus, A. japonicus or Irpex lacteus RGase DNA insert in any appropriate plasmid b) a DNA sequence which hybridizes to the coding region for the mature RGase DNA comprised by the DNA insert of a) and which comprises a structural gene for a polypeptide with RGase activity, and optionally a promoter, a coding region for a signal or leader peptide and/or transcriptional terminator c) a derivative of a DNA sequence defined in a) or b), or d) a DNA sequence which codes for a mature RGase or a signal peptide or a leader peptide thereof and which is degenerate within the meaning of the genetic code with respect to a DNA sequence of a) or b).
  • a preferred embodiment of the recombinant DNA sequence according to the invention is characterized by the fact that the RGase activity originates from the RGase producible by means of Aspergillus aculeatus CBS 101.43 with the partial amino acid sequence according to the invention.
  • a preferred embodiment of the recombinant DNA sequence according to the invention is characterized by the fact that the RGase activity originates from the RGase producible by means of Aspergillus japonicus ATCC 20236 with the partial amino acid sequence according to the invention.
  • a preferred embodiment of the recombinant DNA sequence according to the invention is characterized by the fact that the RGase acticity originates from the RGase producible by means of Irpex lacteus ATCC 20157 with the partial amino acid sequence according to the invention.
  • the invention comprises a vector, which comprises the recombinant DNA sequence according to the invention.
  • a preferred embodiment of the vector according to the invention is characterized by the fact that the promoter is the Aspergillus oryzae takaamylase promoter.
  • the invention comprises a transformed host, which is characterized by the fact that it contains the vector according to the invention.
  • a preferred embodiment of the transformed host according to the invention is characterized by the fact that the transformed host is an Aspergillus strain.
  • a preferred embodiment of the transformed host according to the invention is characterized by the fact that the transformed host is a strain belonging to the species Aspergillus aculeatus, Aspergillus niger, Aspergillus oryzae or Aspergillus awamori.
  • a preferred embodiment of the transformed host according to the invention is characterized by the fact that the transformed host is a microorganism, which in its non-transformed condition does not produce RGase or only produces RGase in insignificant amounts, preferably Bacillus sp., E. coli or S. cerevisiae.
  • the invention comprises a method for production of an RGase, which is characterized by the fact that a transformed host according to the invention is used for the production.
  • the invention comprises an RGase which is produced by means of the method according to the invention.
  • the invention comprises an enzyme preparation comprising the
  • RGase which is characterized by the fact that it contains another plant cell wall degradation or modification agent, preferably a pectinase and/or cellulase and/or hemicellulase usable for degradation or modification of plant cell walls enriched with the RGase, preferably with an enrichment factor of at least
  • a preferred embodiment of the enzyme preparation according to the invention is characterized by the fact that the other plant cell wall degradation or modification agent is producible by means of a microorganism belonging to the genus Aspergillus, preferably Aspergillus niger, Aspergillus aculeatus, Aspergillus awamori or Aspergillus oryzae.
  • the invention comprises a use of the RGase according to the invention as an agent for degradation or modification of plant cell walls and/or plant cell wall components.
  • the invention comprises a use of the enzyme preparation according to the invention as an agent for degradation or modification of plant cell walls and/or plant cell wall components.
  • the enzyme preparation according to the invention as an agent for degradation or modification of plant cell walls and/or plant cell wall components.
  • strain Aspergillus aculeatus CBS 101.43 as a gene donor was fermented in a pilot plant scale in the following way.
  • the strain CBS 101.43 was cultivated on an E-agar slant (37°C). The spores from the slant were suspended in sterilized skim-milk, and the suspension was lyophilized in vials. The contents of one lyophilized vial was transferred to the Fernbach flask. The flask was then incubated for 13 days at 30°C.
  • a substrate with the following composition was prepared in a 500 litre seed fermenter:
  • Tap water was added to a total volume of around 240 litres. pH was adjusted to around 5.5 before addition of CaCO .
  • the substrate was sterilized in the seed fermenter for 1 hour at 121°C. Final volume before inoculation was around 300 litres.
  • the Fernbach flask spore suspension was transferred to the seed fermenter. Seed fermentation conditions were:
  • SUBSTITUTE SHEET Fermenter type Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.3.
  • a substrate with the following composition was prepared in a 2500 litre main fermenter:
  • Fermenter type Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.7.
  • pectin solution was added aseptically to the main fermenter at a constant rate of around
  • the concentrate was filtered on a Seitz filter sheet (type supra 100) with 0.25% Hyflo Super-Cell as a filter aid (in the following table referred to as filtration I).
  • the filtrate was precipitated with 561 g of (NH 4 ) 2 S0 4 /l at a pH of 5.5, and 4% Hyflo Super-Cell diatomaceous earth is added as a filter aid.
  • the precipitate and the filter aid are separated by filtration on a frame filter.
  • the filter cake is dissolved in water, and insoluble parts are separated by filtration on a frame filter.
  • the filtrate is check filtered on a Seitz filter sheet (type supra 100) with 0.25% Hyflo Super-Cell as a filter aid (in the following table referred to as filtration II).
  • the filtrate is diafiltered on an ultrafiltration apparatus. After diafiltration the liquid is concentrated to a dry matter content of 12.7% (in the following table referred to as dry matter content in concentrate).
  • the RGase was isolated from the above indicated Aspergillus aculeatus enzyme preparation broth in the manner described in Table 1 (Figs. 1 - 4).
  • Filtron Minisette filter area 3500 cm 2 , membrane NMWL 10,000 20mM TRIS, pH 5.0; 5 x volume
  • I 6 IEC: PROTEIN PAC DEAE-8HR, Fig. 3
  • HAC HYDROXYLAPATITE BIOGEL HT
  • Fig. 4 columnumn: 4.9 x 11.0 cm, flow 25 ml/min
  • eluent Na-phosphate buffer, pH 7.6, increasing gradient in molarity: 10mM-linear-200mM-step-500mM
  • IEC is ion exchange chromatography. The rhamnogalacturonase fraction was pooled from 0.04 - 0.08 M NaCl.
  • ad 5 pH adaption in order to prepare for step 6.
  • HAC hydroxylapatite chromatography.
  • the rhamnogalacturonase fraction was pooled from 130 mM - 160 mM NaH 2 P0 4 .
  • IEC is ion exchange chromatography.
  • the rhamnogalacturonase fraction was pooled from 55 mM - 65 mM NaCl.
  • the Aspergillus aculeatus RGase was further characterized, as follows.
  • Figs. 5 and 6 show the pH activity and pH stability, respectively.
  • the pH-optimum is around pH 5.0.
  • the stability is good between pH 3 and 6.5 (> 80% residual activity), when treated for 1 hour at room temperature.
  • the activity decreases slightly in the more acidic range; at pH 2.5 still around 70% of activity is found.
  • Figs. 7 and 8 shows the temperature activity dependency and the temperature stability dependency, respectively.
  • the temperature optimum is around 40°C, and the temperature activity range is relatively broad.
  • the activity in the low temperature range is very remarkable: Around 50% activity at 10°C, and around 40% activity at 5°C.
  • the RGase activity unit which is the same for A. aculeatus RGase, A. japonicus RGase and irpex lacteus RGase, is defined as follows. 5 1 unit of RGase is the amount of enzyme which at pH 5, 30°C and in 1 minute releases 1 ⁇ mole of molecules from Saponified Modified Hairy Regions (MHR- S) from apples as substrate.
  • This MHR-S substrate was made according to the method described in
  • the release of molecules is calculated from the change in distribution of molecular weights determined with High Performance Gel Permeation
  • HPGPC Gel Permeation Chromatography
  • the strain Aspergillus japonicus ATCC 20236 as a gene donor was fermented in a pilot plant scale in the following way.
  • An agar substrate with the following composition was prepared in a
  • HEET pH was adjusted to between 5.30 and 5.35. Then 40 g of Difco agar was added, and the mixture was autoclaved for 20 minutes at 120°C (the substrate is named E-agar).
  • the strain ATCC 20236 was cultivated on an E-agar slant (30°C). The spores from the slant were suspended in sterilized skim milk, and the suspension was lyophilized in vials. The contents of one lyophilized vial was transferred to the
  • a substrate with the following composition was prepared in a 500 litre seed fermenter: CaC0 3 1.2 kg
  • Tap water was added to a total volume of around 240 litres. pH was adjusted to around 5.5 before addition of CaC0 3 . The substrate was sterilized in the seed fermenter for 1 hour at 121°C. The final volume before inoculation was around 300 litres.
  • the Fernbach flask spore suspension was transferred to the seed fermenter. Seed fermentation conditions were:
  • Fermenter type Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.3.
  • a substrate with the following composition was prepared in a 2500 litre main fermenter:
  • Fermenter type Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.7.
  • pectin solution was added aseptically to the main fermenter at a constant rate of around
  • the pectin solution with the following composition was prepared in a 500 litre dosing tank:
  • Genu pectin was of the citrus type NF from the Copenhagen pectin factory Ltd. Tap water was added to a total volume of around 325 litres. The substrate was sterilized in the dosing tank for 1 hour at 121°C. The final volume before start of dosage was around 360 litres. When this portion ran out, another similar portion was made. 5 After around 151 fermentation hours the fermentation process was stopped. The resulting culture broth with a volume of approximately 1850 litres was cooled to around 5°C, and the enzymes were recovered according to the following method.
  • the culture broth was drum filtered on a vacuum drum filter (Dorr Oliver), o which was precoated with Hyflo Super-Cell diatomaceous earth (filter aid) .
  • the filtrate was concentrated by evaporation to around 15% of the volume of the culture broth.
  • the concentrate was filtered on a Seitz filter sheet (type supra 100) with 0.25% Hyflo Super-Cell as a filter aid.
  • the filtrate was precipitated with (NH 4 ) 2 S0 4 at a pH of 5.5, and 4% Hyflo Super-Cell diatomaceous earth is added as a filter aid.
  • the precipitate and the filter aid are separated by filtration on a frame filter.
  • the filter cake is dissolved in water, and insoluble parts are separated by filtration on a frame filter.
  • the filtrate is check filtered on a Seitz filter sheet (type supra 100) with 0.25% Hyflo Super-Cell as a filter aid.
  • the filtrate is diafiltered on an ultrafiltration apparatus. After diafiltration the liquid is concentrated.
  • the RGase was isolated from the above indicated Aspergillus japonicus enzyme preparation in the manner described in Table 2 (Figs. 9 - 11).
  • Buffer exchange in order to prepare for step 2, removal of small particles and about 50% of the colour, dilution to maximum 15 mg protein/ml (otherwise the sample will not bind to the column in step 2).
  • IEC is ion exchange chromatography.
  • the rhamnogalacturonase fraction was pooled from 0.05 to 0.06 M NaCl.
  • HIC hydrophobic interaction chromatography.
  • the rhamnogalacturonase fraction was pooled from 1.18 to 1.41 M (NH 4 ) 2 S0 4 .
  • IEC is ion exchange chromatography.
  • the rhamnogalacturonase fraction was pooled from 0.014 to 0.024 M NaCl.
  • amino acid sequence i.e. the N-terminal amino acid sequence of the rhamnogalacturonase from Aspergillus japonicus ATCC 20236.
  • Figs. 12 and 13 show the pH activity and pH stability, respectively.
  • the pH-optimum is around pH 6.5 - 7.0. Especially remarkable is the activity in the neutral and alkaline range: bewteen pH 5.5 and 12 the activity is > 80% of the maximum activity.
  • the stability is good between pH 5.5 and 12, when treated for 1 hour at room temperature, whereas at lower pH the stability decreases significantly.
  • Figs. 14 and 15 show the temperature activity dependency and the temperature stability dependency, respectively.
  • the temperature optimum is around 40°C, and the temperature activity range is relatively broad: between 20 and 60°C the activity is > 80% of the maximum activity.
  • strain Irpex lacteus ATCC 20157 as a gene donor was fermented in a pilot plant scale in the following way.
  • the strain ATCC 20157 was cultivated on an E-agar slant (37°C).
  • the spores from the slant were suspended in sterilized skim milk, and the suspension was lyophilized in vials.
  • the contents of one lyophilized vial was transferred to the Fernbach flask. The flask was then incubated for 18 days at 37°C.
  • a substrate with the following composition was prepared in a 500 litre seed fermenter:
  • Tap water was added to a total volume of around 240 litres. pH was adjusted to around 5.5 before addition of CaC0 .
  • the substrate was sterilized in the seed fermenter for 1 hour at 121°C. The final volume before inoculation was around 300 litres.
  • the Fernbach flask spore suspension was transferred to the seed fermenter.
  • the seed fermentation conditions were: Fermenter type: Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.3.
  • a substrate with the following composition was prepared in a 2500 litre main fermenter:
  • Tap water was added to a total volume of around 1200 litres.
  • the toasted soy meal was suspended in water.
  • the pH was adjusted to 6.2 before the substrate was sterilized in the main fermenter for 1 1 / 2 hours at 123°C.
  • the final volume before inoculation was around 1550 litres. Then 150 litres of seed culture was added.
  • Fermenter type Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.7.
  • the RGase was isolated from the above indicated I ⁇ ex lacteus enzyme preparation broth in the manner described in Table 3 (Figs. 16 - 18).
  • HIC hydrophobic interaction chromatography.
  • the rhamnogalacturonase fraction was pooled from 1.07 m to 1.16 M (NH 4 ) 2 S0 4 .
  • IEC is ion exchange chromatography.
  • the rhamnogalacturonase fraction that did not bind to the column was pooled and used for step 6.
  • IEC is ion exchange chromatography.
  • the rhamnogalacturonase fraction was pooled from 0.18 M to 0.22 M NaCl.
  • the Irpex lacteus RGase was further characterized, as follows.
  • Figs. 19 and 20 show the pH activity and pH stability, respectively.
  • the pH-optimum is around pH 5.5.
  • the stability is good between pH 3 and 11 (residual activity > 80%), when treated for 1 hour at room temperature.
  • Remarkable is the activity in the neutral and alkaline pH-range: At pH 7 still more than 50% of the activity is found, and at pH 8
  • Figs. 21 and 22 show the temperature activity dependency and the temperature stability dependency, respectively.
  • the temperature optimum is around 40°C, and the temperature activity range is relatively broad: at 10 - 17°C the activity is > 80% of the maximum activity, and even at 80°C more than half of the activity is still present.
  • Recombinant DNA molecules according to the invention are constructed 15 and identified in the following manner.
  • RNA is extracted from homogenized A. aculeatus mycelium, collected at the time for maximum activity of the RGase, using methods as described by Boel et al. (EMBO J., 3: 1097-1102, 1984) and Chirgwin et al. (Biochemistry (Wash), 18: 20 5294-5299, 1979).
  • Poly(A)-containing RNA is obtained by two cycles of affinity chromatography on oligo(dT)-cellulose as described by Aviv and Leder (PNAS, USA 69:1408-1412, 1972).
  • cDNA is synthesized with the use of a cDNA synthesis kit from Invitrogen according to the manufacturer's description. Identification of A. aculeatus RGase specific cDNA recombinants by use of synthetic oli ⁇ odeoxyribonucleotides
  • a mixture of synthetic oligodeoxyribonucleotides corresponding to a part of the determined amino acid sequence is synthesized on an Applied Biosystems. Inc. DNA synthesizer and purified by polyacylamide gel electrophoresis. Approximately 150.000 E. coli recombinants from the A. aculeatus cDNA library is transferred to Whatman 540 paper filters. The colonies are lysed and immobilized as described by Gergen et al. (Nucleic Acids Res. 7, 2115-2135, 1979). The filters are hybridized with the ⁇ ⁇ ⁇ ⁇ P-labelled RGase specific oligo mixture as described by Boel et al. (EMBO J., 3, 1097-1102, 1984).
  • Hybridization and washing of the filters are done at a temperature 10°C below the calculated Tm, followed by autoradiography for 24 hours with an intensifier screen. Following autoradiography, the filters are washed at increasing temperatures followed by autoradiography for 24 hours with an intensifier screen.
  • Miniprep plasmid DNA is isolated from hybridizing colonies by standard procedures (Birnboim and Doly Nucleic Acids Res. 7, 1513-1523, 1979), and the DNA sequence of the cDNA insert is established by the Sanger dideoxy procedure.
  • the RGase cDNA fragment is exised from the vector by cleavage with Hindlll/Xbal (or other appropriate enzymes) and is purified by agarose gel electrophoresis electroeluted and made ready for ligation reactions.
  • the cDNA fragment is ligated to Hindlll/Xbal digested ⁇ HD414 to generate pHD RGase in which the cDNA is under transcriptional control of the TAKA promotor from Aspergillus oryzae and the AMG terminator from Aspergillus niger.
  • the cDNA library was split in 50 pools each containing approximately 3000 different cDNA clones. DNA was isolated from the pools and transformed into an appropriate yeast strain. Approximately 20.000 yeast clones (10 plates) were obtained from each of the original pools, in order to ensure that all clones were represented in the yeast library. The yeast clones were replica plated onto minimal agar plates containing galactose. Nitrocellulose filters were placed on top of the yeast colonies followed by incubation of the plates for 2 days at 30°C. The nitrocellulose filters were peeled off and incubated with a monosDecific antibodv raised against the RGase, using standard immunological procedures. Positive clones were purified twice and rescreened using the same antibody preparations. DNA was isolated from the positive yeast clones, and transformed into E. coll MC1061 in order to get higher quantities of DNA. DNA was isolated and analyzed by use of restriction enzymes.
  • the cDNA was exised from the yeast/E coli vector using Hindlll/Xbal, purified on gel and inserted into the Aspergillus expression vector pHD414 as described in this specification.
  • pHD414 (Fig.23) is a derivative of the plasmid p775 (described in EP 238023). In contrast to this plasmid, pHD414 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 Narl (positioned in the pUC vector) and Xbal Oust 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 Stul (positioned in the 5'end of the promoter) and Pvull (in the pUC vector), fractionated on gel and religated, resulting in pHD414.
  • Fig. 23 is a map of plasmid pHD414, wherein "AMG Terminator” indicates the A. niger glucoamylase terminator, and 'TAKA Promoter" indicates the A. oryzae TAKA amyiase promoter.
  • protoplast suspension 100 ⁇ l of protoplast suspension is mixed with 5-25 ⁇ g of the appropriate DNA in 10 ⁇ l of STC.
  • Protoplasts from the argB strains are mixed with pSal43 DNA (an A. nidulans argB gene carrying plasmid) and protoplasts from the argB + strains are mixed with p3SR2 (an A. nidulans amdS gene carrying plasmid).
  • the mixture is left at room temperature for 25 minutes.
  • 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 from the argB strains transformed with pSal43 are spread on minimal plates (Cove Biochem.Biophys.Acta 113 (1966) 51-56) with glucose and urea as carbon and nitrogen sources, respectively, and containing 1.2 M sorbitol for osmotic stabilization.
  • pHD RGase is transformed into A. oryzae IFO 4177 by cotransformation with p3SR2 containing the amdS gene from A. nidulans as described with a mixture of equal amounts of pHD RGase and p3SR2 (approximately 5 ⁇ g of each).
  • 5 Transformants which can use acetamide as sole nitrogen source are reisolated twice. After growth on YPD (Sherman et al. 1981) for three days culture supernatants are analysed by SDS-PAGE. The gels are stained with coomassie brilliant blue R.
  • the best transformants are selected for further studies and grown in a 2 liter Kieler fermentor on 4% soy bean meal and supplied with glucose during growth.
  • the culture is heavily agitated during fermentation.
  • the recombinant product is isolated from the culture broth by removal of the cells by centrifugation, ultrafiltration of the supernatant and freeze drying.
  • Transformants are selected on minimal plates (Cove Biochem.Biophys.Acta 113
  • the transformants are cultured for seven days in YPD (Sherman et al., 1981) at 30°C.
  • the culture supernatants are analyzed by SDS-PAGE. Most of the transformants produced RGase in their supernatants.
  • the RGase gene is isolated from pHD RGase and introduced into the yeast expression vector pYHD5 in which the cDNA is under transcriptional control of the Gal 1-10 promoter and the ⁇ -factor terminator.
  • a URA3 mutant yeast strain is transformed with the yeast expression plasmid by the Li/salt procedure.
  • Transformants are selected on minimal a ⁇ ar olates without uracil.
  • the transformants are replica plated to minimal agar plate without uracil, but supplemented with galactose (in order to induce the promoter) and tested for expression of RGase by use of antibodies and by measurement of the enzyme activity.
  • RGase cDNA is excised from pHD RGase using Hindlll/Xbal.
  • the fragment is treated with Klenow DNA polymerase and dNTP in order to make blunt ended DNA molecules and purified on gel.
  • the fragment is cloned into the vector pHD282 in the Pvull site (Dalboege et al., Gene, 79, 325-332, 1989). and in a subsequent mutationstep using standard site directed mutagenesis techniques, fused directly in frame to the OmpA signal peptide in pHD282.
  • the OmpA-RGase chimeric gene is transferred to the expression vector pHD 234 as a Clal/BamHI fragment and transferred into E. coli MC1061 (Casadaban and Cohen, J. Mal.Biol., 138, 179-207, 1980) to generate recombinant clones.
  • the bacteria samples are analyzed by SDS-PAGE and activity measurements.
  • the RGase according to the invention can be used as a plant cell wall degrading enzyme, thus including the applications shown on page 35 of GB 2115820A.
  • Pectins have gelation and stabilisation properties, which make them useful for the food industry. They are commercially extracted from waste materials of the food industry, e.g. citrus peels, apple pomace or sugar-beet pulp.
  • pectins Most often the extraction with acids (sulphuric acid or nitric acid) is used for the production of pectins.
  • acids sulphuric acid or nitric acid
  • pectins At a pH around 2 and at an elevated temperature the pectins are extracted from plant material and precipitated with alcohol after precipitation.
  • This acid extraction has several disadvantages: water pollution, corrosion, filtering problems due to desintegration of the plant cell walls, partial break down of the wanted pectin polymers (the degree of polymerisation is one of the most important parameters of a commercial pectin).
  • an extraction of pectins with enzymes, which do not decompose native pectin polymers would be of great advantage.
  • Industrial apple pomace for the pectin production was used to compare the amount of pectin extractable either by chemicals or RGases.
  • the amount of starch is determined with the test kit from Boehringer Mannheim (order no. 207748).
  • the amount of obtained pectin is calculated by determination of the amount of AIS in % obtained from the dry matter substance from the pomace and subtracting the amount of starch in the AIS.
  • Citrofiber DF50 (from Citrosuco Paulisto S/A, Matao, Brazil) is a commercially available dietary fiber product, derived from orange juice pulp. It is a by-product from citrus juice processing containing the juice vesicle membranes and segment walls from oranges. This product consists of cellulolytic and non-cellulolutic polysaccharides such as pectins and hemicellufoses.
  • Fig. 24 shows that RGase is one of the key activities for the liquefaction of this citrofiber.
  • RGase alone can increase the soluble part from around 15% to 25- 30% in respect to the RGase used.
  • the functionality of Pectinex ® Ultra SP-L, a multi-enzyme complex for liquefaction containing RGase (Aspergillus aculeatus) in certain amounts, could even be improved by boosting the RGase activity.
  • By doubling of the amount of RGase in Pectinex ® Ultra SP-L an increase of 5 - 10% of the soluble solids (in respect to treatment time) was obtained.
  • Besides the higher degree of liquefaction a shortening of the processing time is possible. This again proves the importance of RGase for liquefaction of plant cell walls.
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus aculeatus
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus aculeatus
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus aculeatus
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus aculeatus
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus aculeatus
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus aculeatus
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus aculeatus
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus aculeatus
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus aculeatus
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus aculeatus
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus aculeatus
  • ORGANISM A. japonicus
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

Abstract

Sont décrites des séquences aminoacides partielles pour une rhamnogalacturonase (RGase) obtenue au moyen de Aspergillus aculeatus, Aspergillus japonicus and Irpex lacteus. De même, est décrite une séquence d'ADN recombiné correspondante, un vecteur correspondant et un hôte transformé correspondant. La RGase produit grâce à un hôte transformé peut servir d'agent de dégradation ou de modification des parois d'une cellule végétale.Partial amino acid sequences are described for a rhamnogalacturonase (RGase) obtained using Aspergillus aculeatus, Aspergillus japonicus and Irpex lacteus. Likewise, a corresponding recombinant DNA sequence, a corresponding vector and a corresponding transformed host are described. The RGase produced by a transformed host can serve as an agent for degrading or modifying the walls of a plant cell.

Description

RHAMNOGALACTURONASE, CORRESPONDING DNA SEQUENCE,
RHAMNOGALACTURONASE CONTAINING ENZYME PREPARATION
AND USE OF THE ENZYME PREPARATION
The invention comprises a rhamnogalacturonase (in the following usually abbreviated RGase), a corresponding DNA sequence, an RGase containing enzyme preparation and a use of the enzyme preparation.
Thus, the invention relates to genetic engineering and provides partial amino acid sequences of an RGase. These partial amino acid sequences can be used for construction of DNA probes which can be used for screening a genomic library from organisms expressing such enzyme, or a cDNA library, thereby obtaining DNA sequences, which can be used either for an overproduction of RGase, if inserted in the microorganism species, from which the parent DNA molecule originated, or for production of RGase 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 RGase.
Plant cell walls comprising rhamnogalacturonans are of complex nature.
Many publications deal with the polysaccharides serving as building blocks, of which these cell walls consist, and their importance with respect to the growing, ripening and processing of fruits and vegetables. Especially pectins have been studied frequently, because they are among the most important components in this respect.
Pectins are proposed to consist of highly carboxyl-methylated linear homogalacturonan regions which alternate with "hairy" (ramified) regions that comprise highly branched rhamnogalacturonans mainly. Whereas the linear homogalacturonan regions are very well known and characterized, the structure of the so-called hairy regions is still not fully characterized, and thus is the subject of many investigations. But besides the scientific interest it is very important to be able to degrade these hairy regions for technical reasons. The enzymatic liquefaction of plant material like e.g. fruits, vegetables, cereals, oil fruits and seeds by technical processes involves combinations of pectolytic, cellulolytic and proteolytic enzyme preparations. This enzymatic treatment solubilizes the hairy regions and other pectic fragments, which originate from the insoluble cell wall protopectin. On one hand the
SUBSTITUTE SHEET solubilization of these polysaccharides is wanted, e.g. for the production of cloudy liquids and soluble dietary fiber containing solutions. On the other hand these polysaccharides cause problems during the processing of the clear liquids, because they are resistant to complete degradation of most technical enzyme preparations. Only one enzyme preparation (from Aspergillus aculeatus) has so far been described, which can degrade the rhamnogalacturonan backbone of the hairy regions. Therefore, it is of great importance for scientific (studies of the structures of these complex polysaccharides) and technical (liquefaction of plant material) reasons to obtain more knowledge about enzymes that can degrade these hairy regions. 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 of great importance to provide a great 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.
RGase is described in the poster "Rhamnogalacturonase; a novel enzyme degrading the highly branched rhamnogalacturonan regions in apple pectic substances" from Wageningen Agricultural University, Department of Food Science, Biotechnion, Bomenweg 2, 6703 HD Wageningen, The Netherlands. From this poster Is appears that a rhamnogalacturonase, the origin of which is not described, is well suited for degradation of the backbone of a modified "hairy region" (MHR) in plant cell walls. Also, it is described that this enzyme might play a role in the degradation of plant cell wall tissue, particularly in combination with other enzymes. However, it is not specified which other enzymes. Also, the isolation and purification of RGase from Aspergillus aculeatus is described by Schols et al. in Carbohydrate Research 206 (1990) 105-115, "Rhamnogalacturonase: a novel enzyme, that degrades the hairy regions of pectins". From page 11 , line 1 it appears that RGase has been purified to a high degree of purity as it moved as a single band in SDS-polyacrylamide gel electrophoresis. Furthermore, in an article of Colquhoun in Carbohydrate Research 206
(1990) 131-144, "Identification by n.m.r. spectroscopy of oligosaccharides obtained by treatment of the hairy regions of apple pectin with rhamnogalacturonase" the
SUBSTITUTE SHEET composition of a mixture of oligosaccharides obtained by enzymatic degradation of the modified hairy (ramified) regions of apple pectin with a RGase is described.
The extraction of apple pectins by RGase is further described in the poster "Extraction of apple pectins by rhamnogalacturonase, a new pectolytic enzyme" by C.M.G.C. Renard et al., Laboratoire de Biochimie et Technologie des Glucides, INRA, Nantes (France).
To the best of applicant's knowledge, only a single species of RGase belongs to the prior art, and has been purified, i.e. the A. aculeatus RGase described by Schols et al., this RGase being the RGase appearing in all the previosly indicated references. Also, this RGase has only been partly characterized and has not been characterized in regard to amino acid sequence, the corresponding RGase produced gene has not been cloned, and thus, the prior art RGase has not been available as a cheap, industrially useable product.
Thus, in consideration of what has been indicated previously, there is a great need for the provision of a variety of RGases with different properties corresponding to the conditions, under which rhamnogalacturonans have to be degraded industrially, e.g. different specificity, pH optimum and temperature optimum. Secondly, there is a need for cheap and pure RGases which can be used industrially in an economically sound manner. Thus, the purpose of the invention is the provision of an RGase, which covers embodiments exhibiting varying characteristics corresponding to the different industrial conditions, under which rhamnogalacturonans have to be degraded, and of an RGase, which can be produced in better yield, and thus cheaper, than hitherto possible, and in higher purity. Also, it is the purpose of the invention to provide novel products, wherein the proportion of the RGase is either increased or decreased in relation to the proportion in the original product.
Accordingly the RGase according to the invention is characterized by the following amino acid sequences:
1 5 10 15 Gly-Ala-Val-Gln-Gly-Phe-Gly-Tyr-Val-Tyr-His-Ala-Glu-Gly-Thr
Tyr-Gly-Ala-Arg (SEQ ID NO : 1)
SUBSTITUTE SHEET 1 5 10 15
Ser-Xaa-Asn-Ile-Leu-Ser-Tyr-Gly-Ala-Val-Ala-Asp-Xaa-Ser-Thr-
20 25
Asp-Val-Gly-Pro-Ala-Ile-Thr-Ser-Ala-Xaa-Ala-Ala-Arg-Lys (SEQ ID NO: 2)
Ser-Arg-Asn-Ile (SEQ ID NO: 3)
1 5 10
Ser-Ala-Tyr-Gly-Ser-Gly-Tyr-Xaa-Leu-Lys (SEQ ID NO: 4)
1 5
Thr-Leu-Glu-Asp-Ile-Ala-Ile (SEQ ID NO: 5)
1 5 10 15
Gly-Leu-Xaa-Ala-Xaa-Ile-Pro-Ile-Pro-Xaa-Ile-Pro-Pro-Xaa-Phe-
P e (SEQ ID NO: 6)
1 5
Ser-Leu-Asp-Ile-Asp-Gly-Tyr (SEQ ID NO: 7)
1 5 10
Ser-Val-His-Asp-Ile-Ile-Leu-Val-Asp-Ala-Pro-Ala-Phe (SEQ ID NO: 8)
1 5
Ala-Ala-Asp-Leu-Ala- (SEQ ID NO: 9)
Gly-Ser-Asn-Ile (SEQ ID NO: 10)
1 5 Tyr-Pro-Gly-Leu-Thr-Pro-Tyr (SEQ ID NO: 11)
1 5 10
Asn-Val-Tyr-Thr-Trp-Ser-Ser-Asn-Gln-Met-Tyr-Met-Ile-Lys (SEQ ID NO : 12)
SUBSTITUTE SHEET 1 5 10 15
Ala-Phe-Gly-Ile-Thr-Thr-Ser-Ser-Ser-Ala-Tyr-Val-Ile-Asp-Thr-
20 25
Asp-Ala-Pro-Asn-Gln-Leu-Lys-Xaa-Thr-Val-Ser-Arg (SEQ ID NO: 13 )
1 5 10
Asn-Val-Asn-Leu-Phe-Ile-Thr-Asp-Gly-Ala-Arg (SEQ ID NO: 14 )
1 5
Ala-Pro-Asp-Gly-Pro-Ala- (SEQ ID NO: 15)
or a partial amino acid sequence, preferably an N-terminal amino acid sequence with a homology thereto of at least 70%, preferably at least 80%, more preferably at least
90%. To the best of applicants knowledge this class of RGases is a new class of
RGases with an advantageously high ability to degrade rhmanogalacturonans under the varying conditions appearing in industry.
A preferred embodiment of the RGase according to the invention is characterized by the following partial amino acid sequences:
1 5 10 15
Gly-Ala-Val-Gln-Gly-Phe-Gly-Tyr-Val-Tyr-His-Ala-Glu-Gly-Thr
Tyr-Gly-Ala-Arg (SEQ ID NO: 1)
1 5 10 15 Ser-Xaa-Asn-Ile-Leu-Ser-Tyr-Gly-Ala-Val-Ala-Asp-Xaa-Ser-Thr-
20 25
Asp-Val-Gly-Pro-Ala-Ile-Thr-Ser-Ala-Xaa-Ala-Ala-Arg-Lys (SEQ ID NO: 2 )
Ser-Arg-Asn-Ile (SEQ ID NO : 3 )
1 5 10
Ser-Ala-Tyr-Gly-Ser-Gly-Tyr-Xaa-Leu-Lys (SEQ ID NO: 4 )
SUBSTITUTE SHEET 1 5
Thr-Leu-Glu-Asp-Ile-Ala-Ile (SEQ ID NO: 5)
1 5 10 15
Gly-Leu-Xaa-Ala-Xaa-Ile-Pro-Ile-Pro-Xaa-Ile-Pro-Pro-Xaa-Phe- Phe (SEQ ID NO: 6)
1 5
Ser-Leu-Asp-Ile-Asp-Gly-Tyr (SEQ ID NO: 7)
1 5 10
Ser-Val-His-Asp-Ile-Ile-Leu-Val-Asp-Ala-Pro-Ala-Phe (SEQ ID NO: 8)
1 5
Ala-Ala-Asp-Leu-Ala- (SEQ ID NO: 9)
Gly-Ser-Asn-Ile (SEQ ID NO: 10)
1 5
Tyr-Pro-Gly-Leu-Thr-Pro-Tyr (SEQ ID NO: 11)
1 5 10
Asn-Val-Tyr-Thr-Trp-Ser-Ser-Asn-Gln-Met-Tyr-Met-Ile-Lys (SEQ ID NO: 12)
or a partial amino acid sequence, preferably an N-terminal amino acid sequence with a homology thereto of at least 70%, preferably at least 80%, more preferably at least 90%.
A preferred embodiment of the RGase according to the invention is characterized by the following partial amino acid sequence:
1 5 10 15
Ala-P e-Gly-Ile-Thr-Thr-Ser-Ser-Ser-Ala-Tyr-Val-Ile-Asp-T r-
20 25
Asp-Ala-Pro-Asn-Gln-Leu-Lys-Xaa-Thr-Val-Ser-Arg (SEQ ID NO: 13)
SUBSTITUTE SHEET A preferred embodiment of the RGase according to the invention is characterized by the following partial amino acid sequences
1 5 10
Asn-Val-Asn-Leu-Phe-Ile-Thr-Asp-Gly-Ala-Arg (SEQ ID NO : 14)
1 5
Ala-Pro-Asp-Gly-Pro-Ala- (SEQ ID NO: 15)
A preferred embodiment of the RGase according to the invention is characterized by being obtainable by means of Aspergillus aculeatus, CBS 101.43. A preferred embodiment of the RGase according to the invention is characterized by being obtainable by means of A. japonicus ATCC 20236.
A preferred embodiment of the RGase according to the invention is characterized by being obtainable by means of Irpex lacteus ATCC 20157.
Also, the invention comprises a recombinant DNA sequence, which comprises a DNA sequence coding for a polypeptide having RGase activity, or a DNA sequence having substantial sequence homology to such RGase coding sequence, preferably a homology of at least 70%, more preferably at least 80%, and most preferably at least 90%.
A preferred embodiment of the recombinant DNA sequence according to the invention comprises a DNA sequence selected from a) the A. aculeatus, A. japonicus or Irpex lacteus RGase DNA insert in any appropriate plasmid b) a DNA sequence which hybridizes to the coding region for the mature RGase DNA comprised by the DNA insert of a) and which comprises a structural gene for a polypeptide with RGase activity, and optionally a promoter, a coding region for a signal or leader peptide and/or transcriptional terminator c) a derivative of a DNA sequence defined in a) or b), or d) a DNA sequence which codes for a mature RGase or a signal peptide or a leader peptide thereof and which is degenerate within the meaning of the genetic code with respect to a DNA sequence of a) or b).
SUBSTITUTE SHEET A preferred embodiment of the recombinant DNA sequence according to the invention is characterized by the fact that the RGase activity originates from the RGase producible by means of Aspergillus aculeatus CBS 101.43 with the partial amino acid sequence according to the invention. A preferred embodiment of the recombinant DNA sequence according to the invention is characterized by the fact that the RGase activity originates from the RGase producible by means of Aspergillus japonicus ATCC 20236 with the partial amino acid sequence according to the invention.
A preferred embodiment of the recombinant DNA sequence according to the invention is characterized by the fact that the RGase acticity originates from the RGase producible by means of Irpex lacteus ATCC 20157 with the partial amino acid sequence according to the invention.
Also, the invention comprises a vector, which comprises the recombinant DNA sequence according to the invention. A preferred embodiment of the vector according to the invention is characterized by the fact that the promoter is the Aspergillus oryzae takaamylase promoter.
Also, the invention comprises a transformed host, which is characterized by the fact that it contains the vector according to the invention. A preferred embodiment of the transformed host according to the invention is characterized by the fact that the transformed host is an Aspergillus strain.
A preferred embodiment of the transformed host according to the invention is characterized by the fact that the transformed host is a strain belonging to the species Aspergillus aculeatus, Aspergillus niger, Aspergillus oryzae or Aspergillus awamori.
A preferred embodiment of the transformed host according to the invention is characterized by the fact that the transformed host is a microorganism, which in its non-transformed condition does not produce RGase or only produces RGase in insignificant amounts, preferably Bacillus sp., E. coli or S. cerevisiae. Also, the invention comprises a method for production of an RGase, which is characterized by the fact that a transformed host according to the invention is used for the production.
SUBSTITUTE SHEET Also, the invention comprises an RGase which is produced by means of the method according to the invention.
Also, the invention comprises an enzyme preparation comprising the
RGase according to the invention, which is characterized by the fact that it contains another plant cell wall degradation or modification agent, preferably a pectinase and/or cellulase and/or hemicellulase usable for degradation or modification of plant cell walls enriched with the RGase, preferably with an enrichment factor of at least
1.1 or deprived of an RGase, preferably with a deprivation factor of maximum 0.9.
A preferred embodiment of the enzyme preparation according to the invention is characterized by the fact that the other plant cell wall degradation or modification agent is producible by means of a microorganism belonging to the genus Aspergillus, preferably Aspergillus niger, Aspergillus aculeatus, Aspergillus awamori or Aspergillus oryzae.
Also, the invention comprises a use of the RGase according to the invention as an agent for degradation or modification of plant cell walls and/or plant cell wall components.
Also, the invention comprises a use of the enzyme preparation according to the invention as an agent for degradation or modification of plant cell walls and/or plant cell wall components. In the following it will be explained in detail how the recombinant DNA sequence according to the invention can be produced.
The strain Aspergillus aculeatus CBS 101.43 as a gene donor was fermented in a pilot plant scale in the following way.
An agar substrate with the following composition was prepared in a Fernbach flask:
SUBSTITUTE SHEET
pH was adjusted to between 5.30 and 5.35. Then 40 g of Agar Difco was added, and the mixture was autoclaved for 20 minutes at 120°C (the substrate is named E-agar).
The strain CBS 101.43 was cultivated on an E-agar slant (37°C). The spores from the slant were suspended in sterilized skim-milk, and the suspension was lyophilized in vials. The contents of one lyophilized vial was transferred to the Fernbach flask. The flask was then incubated for 13 days at 30°C.
A substrate with the following composition was prepared in a 500 litre seed fermenter:
Tap water was added to a total volume of around 240 litres. pH was adjusted to around 5.5 before addition of CaCO . The substrate was sterilized in the seed fermenter for 1 hour at 121°C. Final volume before inoculation was around 300 litres.
The Fernbach flask spore suspension was transferred to the seed fermenter. Seed fermentation conditions were:
SUBSTITUTE SHEET Fermenter type: Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.3.
Agitation: 300 rpm (two turbine impellers)
Aeration: 300 normal litre air per minute Temperature: 30 to 31 °C
Time: around 28 hours
Around 28 hours after inoculation 150 litres was transferred from the seed fermenter to the main fermenter.
A substrate with the following composition was prepared in a 2500 litre main fermenter:
Toasted soy meal 90 kg
KH2P04 20 kg
Pluronic antifoam agent 150 ml
Tap water was added to a total volume of around 900 litres. The toasted soy meal was suspended in water. pH was adjusted to 8.0 with NaOH, and the temperature was raised to 50°C. Thereafter around 925 Anson units of Alcalase® 0.6 L was added to the suspension. The mixture was held for 4 hours at 50°C and pH = 8.0 (Na2C03 addition) with no aeration and 100 rpm agitation. Thereafter the remaining substrate components were added and pH was adjusted to around 6.0 with phosphoric acid. The substrate was sterilized in the main fermenter for V/z hours at 123°C. Final volume before inoculation was around 1080 litres. Then 150 litres of seed culture was added. Fermentation conditions were:
Fermenter type: Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.7.
Agitation: 250 rpm (two turbine impellers)
Aeration: 1200 normal litre air per minute
Temperature: 30°C
Time: around 151 hours
From 24 fermentation hours to around 116 fermentation hours pectin solution was added aseptically to the main fermenter at a constant rate of around
SUBSTITUTE SHEET 8 litres per hour. The pectin solution with the following composition was prepared in a 500 litre dosing tank:
Pectin genu*) 22 kg
Phosphoric acid, cone. 6 kg Pluronic® antifoam agent 50 ml
*) Genu pectin (citrus type NF from the Copenhagen pectin factory Ltd.)
Tap water was added to a total volume of around 325 litres. The substrate was sterilized in the dosing tank for 1 hour at 121°C. Final volume before start of dosage was around 360 litres. When this portion ran out, another similar portion was made. Total volume of pectin solution for one fermentation was around 725 litres. After around 151 fermentation hours the fermentation process was stopped. The around 1850 litres of culture broth were cooled to around 5°C and the enzymes were recovered according to the following method. The culture broth was drum filtered on a vacuum drum filter (Dorr Oliver), which was precoated with Hyflo Super-Cell diatomaceous earth (filter aid). The filtrate was concentrated by evaporation to around 15% of the volume of the culture broth. The concentrate was filtered on a Seitz filter sheet (type supra 100) with 0.25% Hyflo Super-Cell as a filter aid (in the following table referred to as filtration I). The filtrate was precipitated with 561 g of (NH4)2S04/l at a pH of 5.5, and 4% Hyflo Super-Cell diatomaceous earth is added as a filter aid. The precipitate and the filter aid are separated by filtration on a frame filter. The filter cake is dissolved in water, and insoluble parts are separated by filtration on a frame filter. The filtrate is check filtered on a Seitz filter sheet (type supra 100) with 0.25% Hyflo Super-Cell as a filter aid (in the following table referred to as filtration II). The filtrate is diafiltered on an ultrafiltration apparatus. After diafiltration the liquid is concentrated to a dry matter content of 12.7% (in the following table referred to as dry matter content in concentrate).
The RGase was isolated from the above indicated Aspergillus aculeatus enzyme preparation broth in the manner described in Table 1 (Figs. 1 - 4).
SUBSTITUTE SHEET Table 1 ASPERGILLUS ACULEATUS: RHAMNOGALACTURONASE PURIFICATION
Aspergillus aculeatus enzyme broth
I 1 : ULTRAFILTRATION - DIALYSIS
Filtron Minisette, filter area 3500 cm2, membrane NMWL 10,000 20mM TRIS, pH 5.0; 5 x volume
I
2: IEC: WATER ACCELL QMA-PLUS, Fig. 1 (column: 5.0 x 23.0 cm, flow 60 ml/min) eluent = 20mM TRIS, pH 5.0, increasing NaCI-gradient 0.0M-linear-0.0125M-linear-0.25M-linear-0.5M
I
3: ULTRAFILTRATION - DIALYSIS Filtron Minisette, filter area 3500 cm2, membrane NMWL 10,000
20mM TRIS, pH 4.2; 5 x volume
I
4: CROSSLINKED ALGINATE, Fig. 2 (column, 4.9 x 17.5 cm, flow 10 ml/min) eluent 1 = 20mM TRIS, pH 4.2; eluent 2 = 20mM TRIS, pH 6.0
I
5: SAMPLE PREPARATION crosslinked alginate pool, pH adjustment to 6.0
I 6: IEC: PROTEIN PAC DEAE-8HR, Fig. 3
(column: 2.0 x 10.0 cm, flow 4.5 ml/min) eluent: 20mM TRIS, pH 6.0; increasing NaCI-gradient:
0.0M-step-0.038M-linear-0.1M-step-0.25M
I 7: ULTRAFILTRATION - DIALYSIS
Filtron minisette, filter area 1400 cm2, membrane NMWL 10,000 10mM Na-phosphate buffer, pH 7.6; 5 x volume
I
8: HAC: HYDROXYLAPATITE BIOGEL HT, Fig. 4 (column: 4.9 x 11.0 cm, flow 25 ml/min) eluent: Na-phosphate buffer, pH 7.6, increasing gradient in molarity: 10mM-linear-200mM-step-500mM
I
RHAMNOGALACTURONASE
SUBSTITUTE SHEET
IS S ad 1:
Buffer exchange in order to prepare for step 2, removal of small particles and about 50% of the colour, dilution to max. 15 mg protein/ml (otherwise the sample will not bind to the column in step 2).
ad 2: IEC is ion exchange chromatography. The rhamnogalacturonase fraction was pooled from 0.04 - 0.08 M NaCl.
ad 3:
Concentration and buffer exchange in order to prepare for step 4.
ad 4:
Affinity chromatography - the non retained fraction was pooled. The preparation of the crosslinked alginate was done according to Rombouts F.M., C.C.J.M. Geraeds, J. Visser, W. Pilnik, "Purification of various pectic enzymes on crosslinked polyuronides", in: Gribnau, T.C.J., J. Visser, R.J.F. Nivard (Editors), Affinity Chromatography and Related Techniques, Elsevier Scientific Publishing Company, Amsterdam, 255 - 260, 1982.
ad 5: pH adaption in order to prepare for step 6.
ad 6: HAC is hydroxylapatite chromatography. The rhamnogalacturonase fraction was pooled from 130 mM - 160 mM NaH2P04.
ad 7:
Concentration and buffer exchange in order to prepare for step 8.
SUBSTITUTE SHEET ad 8:
IEC is ion exchange chromatography. The rhamnogalacturonase fraction was pooled from 55 mM - 65 mM NaCl.
Now a part of the amino acid sequence is determined: The N-terminus of the RGase is blocked, and thus, direct amino acid sequencing is impossible. Following enzymatic digestion of the RGase with trypsin the following internal amino acid sequences have been obtained. Xaa designates undetermined amino acid residues that most likely carry carbohydrate.
Tryp-19: 1 5 10 15
Gly-Ala-Val-Gln-Gly-Phe-Gly-Tyr-Val-Tyr-His-Ala-Glu-Gly-Thr
Tyr-Gly-Ala-Arg (SEQ ID NO: 1)
Tryp-23:
1 5 10 15 Ser-Xaa-Asn-Ile-Leu-Ser-Tyr-Gly-Ala-Val-Ala-Asp-Xaa-Ser-Thr-
20 25
Asp-Val-Gly-Pro-Ala-Ile-Thr-Ser-Ala-Xaa-Ala-Ala-Arg-Lys (SEQ ID NO: 2 )
Ser-Arg-Asn-Ile (SEQ ID NO: 3 )
1 5 10
Ser- Ala-Tyr- Gly-Ser-Gly-Tyr-Xaa-Leu-Lys (SEQ ID NO: 4)
1 5
Thr-Leu-Glu-Asp-Ile-Ala-Ile (SEQ ID NO : 5)
1 5 10 15
Gly-Leu-Xaa-Ala-Xaa-Ile-Pro-Ile-Pro-Xaa-Ile-Pro-Pro-Xaa-Phe-
Phe (SEQ ID NO : 6)
SUBSTITUTE SHEET 1 5
Ser- Leu-Asp-Ile-Asp-Gly-Tyr (SEQ ID NO: 7)
1 5 10
Ser-Val-His-Asp-Ile-Ile-Leu-Val-Asp-Ala-Pro-Ala-Phe (SEQ ID NO: 8)
1 5
Ala-Ala-Asp-Leu-Ala- (SEQ ID NO: 9 )
Gly-Ser-Asn-Ile (SEQ ID NO: 10)
1 5
Tyr-Pro-Gly-Leu-Thr-Pro-Tyr (SEQ ID NO: 11)
1 5 10
Asn-Val-Tyr-Thr-Trp-Ser-Ser-Asn-Gln-Met-Tyr-Met-Ile-Lys (SEQ ID NO: 12)
The Aspergillus aculeatus RGase was further characterized, as follows.
Figs. 5 and 6 show the pH activity and pH stability, respectively.
The pH-optimum is around pH 5.0. The stability is good between pH 3 and 6.5 (> 80% residual activity), when treated for 1 hour at room temperature. The activity decreases slightly in the more acidic range; at pH 2.5 still around 70% of activity is found.
Figs. 7 and 8 shows the temperature activity dependency and the temperature stability dependency, respectively.
The temperature optimum is around 40°C, and the temperature activity range is relatively broad. For the fruit juice and wine industry the activity in the low temperature range is very remarkable: Around 50% activity at 10°C, and around 40% activity at 5°C.
In the temperature range of 5 - 50°C this RGase is not remarkably influenced after a treatment of 1 hour at pH 4.5 (> 80% of the initial activity), but rapidly inactivated at temperatures above 55°C. Molecular weight: 61 ,000 Dalton
Isoelectric point: pH 4.6
The RGase activity unit which is the same for A. aculeatus RGase, A. japonicus RGase and irpex lacteus RGase, is defined as follows. 5 1 unit of RGase is the amount of enzyme which at pH 5, 30°C and in 1 minute releases 1 μmole of molecules from Saponified Modified Hairy Regions (MHR- S) from apples as substrate.
This MHR-S substrate was made according to the method described in
Schols et al. in Carbohydrate Research 206 (1990), pages 105-115, o "Rhamnogalacturonase: a novel enzyme, that degrades the hairy regions of pectins".
The release of molecules is calculated from the change in distribution of molecular weights determined with High Performance Gel Permeation
Chromatography (HPGPC). Using commercial Gel Permeation Chromatography software, the Number Average Molecular Weight (Mn) was calculated before and after treatment with RGase. In relation to the substrate concentration, the number of glycosidic linkages cleaved were calculated and expressed in activity units according to the above mentioned unit definition.
The strain Aspergillus japonicus ATCC 20236 as a gene donor was fermented in a pilot plant scale in the following way. An agar substrate with the following composition was prepared in a
Fernbach flask:
HEET pH was adjusted to between 5.30 and 5.35. Then 40 g of Difco agar was added, and the mixture was autoclaved for 20 minutes at 120°C (the substrate is named E-agar).
The strain ATCC 20236 was cultivated on an E-agar slant (30°C). The spores from the slant were suspended in sterilized skim milk, and the suspension was lyophilized in vials. The contents of one lyophilized vial was transferred to the
Fernbach flask. The flask was then incubated for 27 days at 30°C.
A substrate with the following composition was prepared in a 500 litre seed fermenter: CaC03 1.2 kg
Glucose 7.2 kg
Rofec (corn steep liquor dry matter) 3.6 kg
Soy bean oil 1.2 kg
Tap water was added to a total volume of around 240 litres. pH was adjusted to around 5.5 before addition of CaC03. The substrate was sterilized in the seed fermenter for 1 hour at 121°C. The final volume before inoculation was around 300 litres.
The Fernbach flask spore suspension was transferred to the seed fermenter. Seed fermentation conditions were:
Fermenter type: Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.3.
Agitation: 300 rpm (two turbine impellers)
Aeration: 300 normal litre air per minute Temperature: 30 to 31 °C
Time: around 28 hours
Around 28 hours after inoculation 150 litres was transferred from the seed fermenter to the main fermenter.
A substrate with the following composition was prepared in a 2500 litre main fermenter:
Tap water was added to a total volume of around 900 litres. The toasted soy meal was suspended in water. pH was adjusted to 8.0 with NaOH, and the temperature was raised to 50°C. Thereafter around 925 Anson units of Alcalase® 0.6 L was added to the suspension. The mixture was held for 4 hours at 50°C and pH = 8.0 (Na2C03 addition) with no aeration and 100 rpm agitation. Thereafter the remaining substrate components were added and pH was adjusted to around 6.0 with phosphoric acid. The substrate was sterilized in the main fermenter for 1 . hours at 123°C. The final volume before inoculation was around 1100 litres. Then 150 litres of seed culture was added. Fermentation conditions were:
Fermenter type: Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.7.
Agitation: 250 rpm (two turbine impellers)
Aeration: 1200 normal litres of air per minute
Temperature: 30°C
Time: around 151 hours
From 24 fermentation hours to around 130 fermentation hours pectin solution was added aseptically to the main fermenter at a constant rate of around
8 litres per hour. The pectin solution with the following composition was prepared in a 500 litre dosing tank:
Pectin genu*) 22 kg Phosphoric acid, cone. 6 kg
Pluronic antifoam agent 50 ml
*) Genu pectin was of the citrus type NF from the Copenhagen pectin factory Ltd. Tap water was added to a total volume of around 325 litres. The substrate was sterilized in the dosing tank for 1 hour at 121°C. The final volume before start of dosage was around 360 litres. When this portion ran out, another similar portion was made. 5 After around 151 fermentation hours the fermentation process was stopped. The resulting culture broth with a volume of approximately 1850 litres was cooled to around 5°C, and the enzymes were recovered according to the following method.
The culture broth was drum filtered on a vacuum drum filter (Dorr Oliver), o which was precoated with Hyflo Super-Cell diatomaceous earth (filter aid) . The filtrate was concentrated by evaporation to around 15% of the volume of the culture broth. The concentrate was filtered on a Seitz filter sheet (type supra 100) with 0.25% Hyflo Super-Cell as a filter aid. The filtrate was precipitated with (NH4)2S04 at a pH of 5.5, and 4% Hyflo Super-Cell diatomaceous earth is added as a filter aid. The precipitate and the filter aid are separated by filtration on a frame filter. The filter cake is dissolved in water, and insoluble parts are separated by filtration on a frame filter. The filtrate is check filtered on a Seitz filter sheet (type supra 100) with 0.25% Hyflo Super-Cell as a filter aid. The filtrate is diafiltered on an ultrafiltration apparatus. After diafiltration the liquid is concentrated. The RGase was isolated from the above indicated Aspergillus japonicus enzyme preparation in the manner described in Table 2 (Figs. 9 - 11).
Table 2
ASPERGILLUS JAPONICUS: RHAMNOGALACTURONASE PURIFICATION
Aspergillus japonicus enzyme broth
1 : ULTRAFILTRATION - DIALYSIS
Filtron Minisette, filter area 3500 cm2, membrane NMWL 10,000
20mM TRIS, pH 6,0; 5 x volume
2: IEC: WATER ACCELL QMA-PLUS, Fig. 9
(column: 5.0 x 26.5 cm, flow 60 ml/min) eluent = 20mM TRIS, pH 6.0, increasing NaCI-gradient:
0.0M-step-0.05M-linear-0.225M-linear-0.35M-step-0.5M
3: SAMPLE PREPARATION
Rhamnogalacturonase pool, addition of solid (NH4)2S04 to 2M concentration, pH-adjustment to pH 5.0
4: HIC: PHENYL TOYOPEARL 650 (M), Fig. 10
(column: 2.6 x 23.0 cm, flow 16 ml/min) eluent: water, decreasing (NH4)2S04gradient: 2M-concave decrease (=linear decrease of conductivity)-0.5M-step-0.0M
5: ULTRAFILTRATION - DIALYSIS Filtron Minisette, filter area 1400 cm2, membrane NMWL 10,000 10mM TRIS-buffer, pH 5.0; 5 x volume
6: IEC: PROTEIN PAC DEAE-8HR, Fig. 11 (column: 2.0 x 10.0 cm, flow 4.5 ml/min) eluent: 10mM TRIS, pH 5.0; increasing NaCI-gradient:
0.0M - linear - 0.15M
RHAMNOGALACTURONASE ad 1:
Buffer exchange in order to prepare for step 2, removal of small particles and about 50% of the colour, dilution to maximum 15 mg protein/ml (otherwise the sample will not bind to the column in step 2).
ad 2:
IEC is ion exchange chromatography. The rhamnogalacturonase fraction was pooled from 0.05 to 0.06 M NaCl.
ad 3:
Buffer adaption in order to prepare for step 4.
ad 4:
HIC is hydrophobic interaction chromatography. The rhamnogalacturonase fraction was pooled from 1.18 to 1.41 M (NH4)2S04.
ad 5:
Buffer exchange in order to prepare for step 6.
ad 6:
IEC is ion exchange chromatography. The rhamnogalacturonase fraction was pooled from 0.014 to 0.024 M NaCl.
Now a part of the amino acid sequence is determined, i.e. the N-terminal amino acid sequence of the rhamnogalacturonase from Aspergillus japonicus ATCC 20236.
1 5 10 15
Ala-P e-Gly-Ile-T r-Thr-S er-S er-S er-Ala-Tyr-Val -I le-Asp-Thr-
20 25
Asp-Ala-Pro-Asn-Gln-Leu-Lys-Xaa-T r-Val-Ser-Arg (SEQ ID NO : 13 ) The sequence has no homology to other proteins in the databases. In addition, there is no homology to the peptide sequences from the rhamnogalacturonase from Aspergillus aculeatus.
The Aspergillus japonicus RGase was further characterized, as follows. Figs. 12 and 13 show the pH activity and pH stability, respectively.
The pH-optimum is around pH 6.5 - 7.0. Especially remarkable is the activity in the neutral and alkaline range: bewteen pH 5.5 and 12 the activity is > 80% of the maximum activity.
The stability is good between pH 5.5 and 12, when treated for 1 hour at room temperature, whereas at lower pH the stability decreases significantly.
Figs. 14 and 15 show the temperature activity dependency and the temperature stability dependency, respectively.
The temperature optimum is around 40°C, and the temperature activity range is relatively broad: between 20 and 60°C the activity is > 80% of the maximum activity.
For the fruit juice and wine industry the activity in the low temperature range is very remarkable: > 60% activity at 5-10°C. In the temperature range of 5 - 40°C this RGase is not remarkably influenced after a treatment of 1 hour at pH 4.5 (> 80% of the initial activity); but above 40°C it is remarkably influenced. Molecular weight: 53,000 Dalton
Isolelectric point: pH 5.3
The strain Irpex lacteus ATCC 20157 as a gene donor was fermented in a pilot plant scale in the following way.
An agar substrate with the following composition was prepared in a Fernbach flask:
pH was adjusted to between 5.30 and 5.35. Then 40 g of Difco agar was added, and the mixture was autoclaved for 20 minutes at 120°C (the substrate is named E-agar).
The strain ATCC 20157 was cultivated on an E-agar slant (37°C). The spores from the slant were suspended in sterilized skim milk, and the suspension was lyophilized in vials. The contents of one lyophilized vial was transferred to the Fernbach flask. The flask was then incubated for 18 days at 37°C.
A substrate with the following composition was prepared in a 500 litre seed fermenter:
Tap water was added to a total volume of around 240 litres. pH was adjusted to around 5.5 before addition of CaC0 . The substrate was sterilized in the seed fermenter for 1 hour at 121°C. The final volume before inoculation was around 300 litres.
The Fernbach flask spore suspension was transferred to the seed fermenter. The seed fermentation conditions were: Fermenter type: Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.3.
Agitation: 300 rpm (two turbine impellers)
Aeration: 300 normal litre air per minute Temperature: 37°C
Time: around 59 hours
Around 59 hours after inoculation 150 litres was transferred from the seed fermenter to the main fermenter.
A substrate with the following composition was prepared in a 2500 litre main fermenter:
Toasted soy meal 120 kg
Maltose 30 kg
Cellulose powder 50 kg
(Arbocel CB-200) Pluronic antifoam agent 200 ml
Tap water was added to a total volume of around 1200 litres. The toasted soy meal was suspended in water. The pH was adjusted to 6.2 before the substrate was sterilized in the main fermenter for 11/2 hours at 123°C. The final volume before inoculation was around 1550 litres. Then 150 litres of seed culture was added.
Fermentation conditions were:
Fermenter type: Conventional aerated and agitated fermenter with a height/diameter ratio of around 2.7.
Agitation: 250 rpm (two turbine impellers) Aeration: 1200 normal litre air per minute
Temperature: 37°C
Time: around 120 hours
SUBSTTTUTE SHEET From 24 fermentation hours to around 130 fermentation hours water was added asepticaily to the main fermenter at a constant rate of around 4 litres per hour.
The RGase was isolated from the above indicated Iφex lacteus enzyme preparation broth in the manner described in Table 3 (Figs. 16 - 18).
Table 3
IRPEX LACTEUS: RHAMNOGALACTURONASE PURIFICATION
Irpex lacteus enzyme broth
1 : ULTRAFILTRATION - DIALYSIS
Filtron Minisette, filter area 3500 cm2, membrane NMWL 10,000 distilled water; 5 x volume
2: SAMPLE PREPARATION addition of solid (NH4)2S04 to 2M concentration, pH adjustment to pH 5.0
3: HIC: PHENYL TOYOPEARL 650 (M), Fig. 16
(column: 5.0 x 26.5 cm, flow 60 ml/min) eluent: water, decreasing (NH4)2S04-gradient M-concave decrease (=linear decrease of conductivity)-0.5M-step-0.0M
4: ULTRAFILTRATION - DIALYSIS
Filtron Minisette, filter area 3500 cm2, membrane NMWL 10,000
20mM TRIS-buffer, pH 8.0; 5 x volume
5: IEC: WATER ACCELL QMA-PLUS, Fig. 17 (column: 26.0 x 23.0 cm, flow 16 ml/min) eluent = 20mM TRIS, pH 8.0, increasing NaCI-gradient: 0.0M-step-0.05M-step-0.1 M-step-0.25M-step-0.5M
6: ULTRAFILTRATION - DIALYSIS Filtron Minisette, filter area 700 cm2, membrane NMWL 10,000 20mM NaCI/HCI, pH 3.6; 5 x volume
7: IEC: FRACTOGEL EMD SO3-650 (M), Fig. 18 (column: 2.6 x 8.4 cm, flow 15 ml/min) eluent: 20mM NaCI/HCI, pH 3.6; increasing NaCI-gradient:
0.02M-linear-0.32M-linear-0.52M
RHAMNOGALACTURONASE
SUBSTTTUTE SHEET ad 1:
Liquid exchange in order to prepare for step 2, removal of small particles and about 50% of the colour, dilution to max. 15 mg protein/ml (otherwise the sample will not bind to the column in step 2).
ad 2:
Liquid adaption in order to prepare for step 3.
ad 3:
HIC is hydrophobic interaction chromatography. The rhamnogalacturonase fraction was pooled from 1.07 m to 1.16 M (NH4)2S04.
ad 4:
Buffer exchange in order to prepare for step 5.
ad 5:
IEC is ion exchange chromatography. The rhamnogalacturonase fraction that did not bind to the column was pooled and used for step 6.
ad 6:
Buffer exchange in order to prepare for step 7.
ad 7:
IEC is ion exchange chromatography. The rhamnogalacturonase fraction was pooled from 0.18 M to 0.22 M NaCl.
Now a part of the amino acid sequences is determined:
1 5 10
Asn-Val-Asn-Leu-Phe-Ile-Thr-Asp-Gly-Ala-Arg (SEQ ID NO: 14)
1 5
Ala-Pro-Asp-Gly-Pro-Ala- (SEQ ID NO: 15) The Irpex lacteus RGase was further characterized, as follows.
Figs. 19 and 20 show the pH activity and pH stability, respectively.
The pH-optimum is around pH 5.5.
The stability is good between pH 3 and 11 (residual activity > 80%), when treated for 1 hour at room temperature. Remarkable is the activity in the neutral and alkaline pH-range: At pH 7 still more than 50% of the activity is found, and at pH 8
- 12 still around 30 - 35% activity is found. Furthermore, the excellent pH-stability has to be mentioned, a residual activity > 80% being found at pH 3 - 12.
Figs. 21 and 22 show the temperature activity dependency and the temperature stability dependency, respectively.
The temperature optimum is around 40°C, and the temperature activity range is relatively broad: at 10 - 17°C the activity is > 80% of the maximum activity, and even at 80°C more than half of the activity is still present.
For the fruit juice and wine industry the activity in the low temperature range is remarkable: Around 80% activity at 10°C, and around 70% activity at 5°C.
In the temperature range of 5 to 50°C this RGase is not remarkably influenced after a treatment of 1 hour at pH 4.5 (> 80% of the initial activity). Around 70% of the activity is still found at 60°C, whereas the stability decreases rapidly at temperatures of 70°C and above. Molecular weight: 45,000 Dalton
Isoelectric point: pH 7.2
The below table shows some characteristics of the different RGases isolated from the three identified strains. 10
On the basis of the above indicated amino acid sequences sequence probing processes were carried out for the corresponding cDNA. After isolation of the mRNA, the cDNA was synthetized.
Recombinant DNA molecules according to the invention are constructed 15 and identified in the following manner.
Construction of a A. aculeatus cDNA library in E. coli
Total RNA is extracted from homogenized A. aculeatus mycelium, collected at the time for maximum activity of the RGase, using methods as described by Boel et al. (EMBO J., 3: 1097-1102, 1984) and Chirgwin et al. (Biochemistry (Wash), 18: 20 5294-5299, 1979). Poly(A)-containing RNA is obtained by two cycles of affinity chromatography on oligo(dT)-cellulose as described by Aviv and Leder (PNAS, USA 69:1408-1412, 1972). cDNA is synthesized with the use of a cDNA synthesis kit from Invitrogen according to the manufacturer's description. Identification of A. aculeatus RGase specific cDNA recombinants by use of synthetic oliαodeoxyribonucleotides
A mixture of synthetic oligodeoxyribonucleotides corresponding to a part of the determined amino acid sequence is synthesized on an Applied Biosystems. Inc. DNA synthesizer and purified by polyacylamide gel electrophoresis. Approximately 150.000 E. coli recombinants from the A. aculeatus cDNA library is transferred to Whatman 540 paper filters. The colonies are lysed and immobilized as described by Gergen et al. (Nucleic Acids Res. 7, 2115-2135, 1979). The filters are hybridized with the ■^■^P-labelled RGase specific oligo mixture as described by Boel et al. (EMBO J., 3, 1097-1102, 1984). Hybridization and washing of the filters are done at a temperature 10°C below the calculated Tm, followed by autoradiography for 24 hours with an intensifier screen. Following autoradiography, the filters are washed at increasing temperatures followed by autoradiography for 24 hours with an intensifier screen. Miniprep plasmid DNA is isolated from hybridizing colonies by standard procedures (Birnboim and Doly Nucleic Acids Res. 7, 1513-1523, 1979), and the DNA sequence of the cDNA insert is established by the Sanger dideoxy procedure. The RGase cDNA fragment is exised from the vector by cleavage with Hindlll/Xbal (or other appropriate enzymes) and is purified by agarose gel electrophoresis electroeluted and made ready for ligation reactions. The cDNA fragment is ligated to Hindlll/Xbal digested ρHD414 to generate pHD RGase in which the cDNA is under transcriptional control of the TAKA promotor from Aspergillus oryzae and the AMG terminator from Aspergillus niger.
Identification of A. aculeatus RGase specific cDNA recombinants using immunological screening procedures. The cDNA library was split in 50 pools each containing approximately 3000 different cDNA clones. DNA was isolated from the pools and transformed into an appropriate yeast strain. Approximately 20.000 yeast clones (10 plates) were obtained from each of the original pools, in order to ensure that all clones were represented in the yeast library. The yeast clones were replica plated onto minimal agar plates containing galactose. Nitrocellulose filters were placed on top of the yeast colonies followed by incubation of the plates for 2 days at 30°C. The nitrocellulose filters were peeled off and incubated with a monosDecific antibodv raised against the RGase, using standard immunological procedures. Positive clones were purified twice and rescreened using the same antibody preparations. DNA was isolated from the positive yeast clones, and transformed into E. coll MC1061 in order to get higher quantities of DNA. DNA was isolated and analyzed by use of restriction enzymes.
The cDNA was exised from the yeast/E coli vector using Hindlll/Xbal, purified on gel and inserted into the Aspergillus expression vector pHD414 as described in this specification.
Construction of an Aspergillus expression vector The vector pHD414 (Fig.23) is a derivative of the plasmid p775 (described in EP 238023). In contrast to this plasmid, pHD414 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 Narl (positioned in the pUC vector) and Xbal Oust 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 Stul (positioned in the 5'end of the promoter) and Pvull (in the pUC vector), fractionated on gel and religated, resulting in pHD414. Fig. 23 is a map of plasmid pHD414, wherein "AMG Terminator" indicates the A. niger glucoamylase terminator, and 'TAKA Promoter" indicates the A. oryzae TAKA amyiase promoter.
Transformation of Asoeraillus orvzae or Aspergillus niαer (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, A. niger or argB mutants hereof and incubated with shaking at 37°C for about 2 days. The mycelium is harvested by filtration through miracloth and washed with 200 ml of 0.6 M MgS04. The mycelium is suspended in 15 ml of 1.2 M MgS04. 10 mM NaH2P04, pH = 5.8. The suspension is cooled on ice and 1 ml of buffer containing 120 mg of Novozvm® 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°C until a large number of protoplasts is visible in a sample inspected under the microscope. 5 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-HCI, pH = 7.0. Centrifugation is performed for 15 minutes at 100 g and the protoplasts are collected from the top of the MgS0 cushion. 2 volumes of STC (1.2 M sorbitol, 10 mM Tris- HCI, pH = 7.5. 10 mM CaCI2) are added to the protoplast suspension and the o 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 μl of protoplast suspension is mixed with 5-25 μg of the appropriate DNA in 10μl of STC. Protoplasts from the argB strains are mixed with pSal43 DNA (an A. nidulans argB gene carrying plasmid) and protoplasts from the argB+ strains 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 CaCI2 and 10 mM Tris-HCI, 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 from the argB strains transformed with pSal43 are spread on minimal plates (Cove Biochem.Biophys.Acta 113 (1966) 51-56) with glucose and urea as carbon and nitrogen sources, respectively, and containing 1.2 M sorbitol for osmotic stabilization. Protoplasts from the argB-strains transformed with p3SR2 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 CsCI to inhibit background growth. After incubation for 4-7 days at 37°C spores are picked, suspended in sterile water 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. Production of RGase in high yield with this transformed host: Expression of recombinant A. aculeatus RGase in an A. oryzae strain pHD RGase is transformed into A. oryzae IFO 4177 by cotransformation with p3SR2 containing the amdS gene from A. nidulans as described with a mixture of equal amounts of pHD RGase and p3SR2 (approximately 5 μg of each). 5 Transformants which can use acetamide as sole nitrogen source are reisolated twice. After growth on YPD (Sherman et al. 1981) for three days culture supernatants are analysed by SDS-PAGE. The gels are stained with coomassie brilliant blue R. The best transformants are selected for further studies and grown in a 2 liter Kieler fermentor on 4% soy bean meal and supplied with glucose during growth. The culture is heavily agitated during fermentation. The recombinant product is isolated from the culture broth by removal of the cells by centrifugation, ultrafiltration of the supernatant and freeze drying.
Expression of RGase in an A. nicer strain pHD RGase is transformed into A. niger argB by cotransformation with pSaI43 containing the argB gene form A. nidulans as described earlier. Protoplast are incubated with equal amounts, approximately 5 μg of each plasmid.
Transformants are selected on minimal plates (Cove Biochem.Biophys.Acta 113
(1966), 55-56) by relief of argenine requirement.
After two reisolations of conidiospores the transformants are cultured for seven days in YPD (Sherman et al., 1981) at 30°C. The culture supernatants are analyzed by SDS-PAGE. Most of the transformants produced RGase in their supernatants.
Production of RGase using a transformed host other than Aspergillus species without significant amounts of accompanying similar enzymes.
Expression of RGase in S. cerevisiae
The RGase gene is isolated from pHD RGase and introduced into the yeast expression vector pYHD5 in which the cDNA is under transcriptional control of the Gal 1-10 promoter and the α-factor terminator. A URA3 mutant yeast strain is transformed with the yeast expression plasmid by the Li/salt procedure. Transformants are selected on minimal aαar olates without uracil. The transformants are replica plated to minimal agar plate without uracil, but supplemented with galactose (in order to induce the promoter) and tested for expression of RGase by use of antibodies and by measurement of the enzyme activity.
Expression of RGase in E. coli The RGase cDNA is excised from pHD RGase using Hindlll/Xbal. The fragment is treated with Klenow DNA polymerase and dNTP in order to make blunt ended DNA molecules and purified on gel. The fragment is cloned into the vector pHD282 in the Pvull site (Dalboege et al., Gene, 79, 325-332, 1989). and in a subsequent mutationstep using standard site directed mutagenesis techniques, fused directly in frame to the OmpA signal peptide in pHD282.
The OmpA-RGase chimeric gene is transferred to the expression vector pHD 234 as a Clal/BamHI fragment and transferred into E. coli MC1061 (Casadaban and Cohen, J. Mal.Biol., 138, 179-207, 1980) to generate recombinant clones. E. coli MC1061 containing the expression plasmid is grown in 1.5 liter MBR reactor equipped with temperature, pH, air-flow rate and agitation controllers. The medium contained 40 mg tryptone/ml (Difco) and 20 mg yeast extract/ml. Production of RGase is induced by raising the temperature from 28°C to above 37°C at an A525 = 50.
The bacteria samples are analyzed by SDS-PAGE and activity measurements.
The RGase according to the invention can be used as a plant cell wall degrading enzyme, thus including the applications shown on page 35 of GB 2115820A.
If the RGase according to the invention is used together with Pectinex Ultra SP and/or an acetyl esterase, a synergistic effect can be demonstrated. EXAMPLE 1
Pectin extraction
Pectins have gelation and stabilisation properties, which make them useful for the food industry. They are commercially extracted from waste materials of the food industry, e.g. citrus peels, apple pomace or sugar-beet pulp.
Most often the extraction with acids (sulphuric acid or nitric acid) is used for the production of pectins. At a pH around 2 and at an elevated temperature the pectins are extracted from plant material and precipitated with alcohol after precipitation. This acid extraction has several disadvantages: water pollution, corrosion, filtering problems due to desintegration of the plant cell walls, partial break down of the wanted pectin polymers (the degree of polymerisation is one of the most important parameters of a commercial pectin). Thus, it is obvious, that an extraction of pectins with enzymes, which do not decompose native pectin polymers would be of great advantage.
Industrial apple pomace for the pectin production was used to compare the amount of pectin extractable either by chemicals or RGases.
Chemical extraction of pectin (prior art)
To 1 part of pomace 19 parts of distilled water was added and the mixture was heated to the boiling point in order to bring the soluble part of the pomace into solution. The pH value was adjusted to 1.9 by means of 2N H2S04. The mixture is held at this pH for 2.5 hours at 90°C and afterwards cooled to room temperature. The mixture is filtered and the pomace residues washed with 10 parts of distilled water. To 1 part of the filtrate 6 parts of methanol is added. After 30 minutes standing the mixture is filtered and pressed. The alcohol insoluble substance (AIS) is washed with 4 parts of methanol and filtered and pressed again. The obtained AIS is dried at 60°C for one hours. From this AIS the amount of starch is determined with the test kit from Boehringer Mannheim (order no. 207748). The amount of obtained pectin is calculated by determination of the amount of AIS in % obtained from the dry matter substance from the pomace and subtracting the amount of starch in the AIS.
Enzymatic extraction of pectin To 1 part of pomace 19 parts of 0.1 m sodium acetate buffer of pH 5.0
(with 0.02% NaN3) is added. At 30°C the mixture is treated for 20 hours with solutions of the purified RGases according to the invention originating from A. aculeatus and A. japonicus. Afterwards the mixture is filtered and the pomace residues washed with 10 parts of distilled water. The AIS is obtained in the way described above.
Results
With the chemical extraction 17.5% pectin was obtained whereas with the enzymatic extraction between 9 and 11% were obtained, depending upon the type and amount of RGase used. These results prove, that the RGase is one of the key enzymes for enzymatic extraction of pectins from plant material. Also, it appears from the above that 50 to 60% of the pectin extractable by chemial means and with all the accompanying disadvantages can be extracted enzymatically in an environmental sound manner, especially when this enzyme will be combined with other pectin liberating activities, e.g. /S-1 ,4-galactanase. This ability of extracting pectins from the plant cell wall proves that RGase is important for the production of cloudy juices, nectars and purees (stabilization of the cloud and the desired consistency of a product).
EXAMPLE 2
Citrofiber DF50 (from Citrosuco Paulisto S/A, Matao, Brazil) is a commercially available dietary fiber product, derived from orange juice pulp. It is a by-product from citrus juice processing containing the juice vesicle membranes and segment walls from oranges. This product consists of cellulolytic and non-cellulolutic polysaccharides such as pectins and hemicellufoses.
A liquefaction of this citrofiber in order to change the soluble/insoluble solids ratio of this fiber will result in a better application value and offers new possibilities for formulating this fibers in other products: e.g. juices, soft drinks, and liquid health products.
Fig. 24 shows that RGase is one of the key activities for the liquefaction of this citrofiber. RGase alone can increase the soluble part from around 15% to 25- 30% in respect to the RGase used. The functionality of Pectinex® Ultra SP-L, a multi-enzyme complex for liquefaction containing RGase (Aspergillus aculeatus) in certain amounts, could even be improved by boosting the RGase activity. By doubling of the amount of RGase in Pectinex® Ultra SP-L an increase of 5 - 10% of the soluble solids (in respect to treatment time) was obtained. Besides the higher degree of liquefaction a shortening of the processing time is possible. This again proves the importance of RGase for liquefaction of plant cell walls.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: NOVO NORDISK A/S
(ii) TITLE OF INVENTION: RHAMNOGALACTURONASE, CORRESPONDING DNA SEQUENCE, RHAMNOGALACTURONASE CONTAINING ENZYME PREPARATION AND USE OF THE ENZYME PREPARATION
(iii) NUMBER OF SEQUENCES: 15
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: NOVO NORDISK A/S, Patent Dept.
(B) STREET: Novo Alle
(C) CITY: Bagsvaerd
(E) COUNTRY: Denmark
(F) ZIP: DK-2880
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: BACH, Niels et al.
(B) REGISTRATION NUMBER: (EPO) GA 24307
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: +4544448888
(B) TELEFAX: +4544493256
(C) TELEX: 37304
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus aculeatus
(B) STRAIN: CBS 101.43 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Gly Al a Val Gin Gly Phe Gly Tyr Val Tyr His Al a Glu Gly Thr Tyr 1 5 10 15
Gly Ala Arg
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus aculeatus
(B) STRAIN: CBS 101.43
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Ser Xaa Asn He Leu Ser Tyr Gly Ala Val Ala Asp Xaa Ser Thr Asp 1 5 10 15
Val Gly Pro Ala He Thr Ser Ala Xaa Ala Ala Arg Lys 20 25
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (i i) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus aculeatus
(B) STRAIN: CBS 101.43 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Ser Arg Asn He 1
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus aculeatus
(B) STRAIN: CBS 101.43
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Ser Ala Tyr Gly Ser Gly Tyr Xaa Leu Lys 1 5 10
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus aculeatus
(B) STRAIN: CBS 101.43
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Thr Leu Glu Asp He Ala He
1 5 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus aculeatus
(B) STRAIN: CBS 101.43
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Gly Leu Xaa Ala Xaa He Pro He Pro Xaa He Pro Pro Xaa Phe Phe 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: l inear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus aculeatus
(B) STRAIN: CBS 101.43
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ser Leu Asp He Asp Gly Tyr 1 5
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus aculeatus
(B) STRAIN: CBS 101.43
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Ser Val His Asp He He Leu Val Asp Ala Pro Ala Phe 1 5 10
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus aculeatus
(B) STRAIN: CBS 101.43
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Ala Ala Asp Leu Ala 1 5
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus aculeatus
(B) STRAIN: CBS 101.43
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Gly Ser Asn He
1
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(i ) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus aculeatus
(B) STRAIN: CBS 101.43
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Tyr Pro Gly Leu Thr Pro Tyr
1 5
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Aspergillus aculeatus
(B) STRAIN: CBS 101.43
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Asn Val Tyr Thr Trp Ser Ser Asn Gin Met Tyr Met He Lys 1 5 10
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) PROTEIN FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: A. japonicus
(B) STRAIN: ATCC 20236
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Ala Phe Gly He Thr Thr Ser Ser Ser Ala Tyr Val He Asp Thr Asp 1 5 10 15
Ala Pro Asn Gin Leu Lys Xaa Thr Val Ser Arg 20 25
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
TTT TE SHEET (vi) ORIGINAL SOURCE:
(A) ORGANISM: Irpex lacteus
(B) STRAIN: ATCC 20157
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Asn Val Asn Leu Phe He Thr Asp Gly Ala Arg 1 5 10
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Irpex lacteus
(B) STRAIN: ATCC 20157
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Ala Pro Asp Gly Pro Ala 1 5

Claims

1. Rhamnogalacturonase (RGase) with the following partial amino acid sequences:
1 5 10 15 Gly-Ala-Val-Gln-Gly-Phe-Gly-Tyr-Val-Tyr-His-Ala-Glu-Gly-Thr
Tyr-Gly-Ala-Arg (SEQ ID NO : 1)
1 5 10 15
Ser-Xaa-Asn-Ile-Leu-Ser-Tyr-Gly-Ala-Val-Ala-Asp-Xaa-Ser-Thr-
20 25 Asp-Val-Gly-Pro-Ala-Ile-Thr-Ser-Ala-Xaa-Ala-Ala-Arg-Lys (SEQ ID NO: 2 )
Ser-Arg-Asn-Ile (SEQ ID NO: 3 )
1 5 10 Ser-Ala-Tyr-Gly-Ser-Gly-Tyr-Xaa-Leu-Lys (SEQ ID NO: 4 )
l 5
Thr-Leu-Glu-Asp-Ile-Ala-Ile (SEQ ID NO: 5)
1 5 10 15
Gly-Leu-Xaa-Ala-Xaa-Ile-Pro-Ile-Pro-Xaa-Ile-Pro-Pro-Xaa-Phe-
Phe (SEQ ID NO: 6)
1 5
Ser-Leu-Asp-Ile-Asp-Gly-Tyr (SEQ ID NO : 7 )
1 5 10
Ser-Val-His-Asp-Ile-Ile-Leu-Val-Asp-Ala-Pro-Ala-Phe (SEQ ID NO : 8 )
1 5
Ala-Ala-Asp-Leu-Ala- (SEQ ID NO : 9 )
SUBSTITUTE SHEET Gly-Ser-Asn-Ile (SEQ ID NO: 10)
1 5
Tyr-Pro-Gly-Leu-Thr-Pro-Tyr (SEQ ID NO: 11)
1 5 10
Asn-Val-Tyr-Thr-Trp-Ser-Ser-Asn-Gln-Met-Tyr-Met-Ile-Lys (SEQ ID NO: 12)
1 5 10 15
Ala-Phe-Gly-Ile-Thr-Thr-Ser-Ser-Ser-Ala-Tyr-Val-Ile-Asp-Thr- 20 25
Asp-Ala-Pro-Asn-Gln-Leu-Lys-Xaa-Thr-Val-Ser-Arg (SEQ ID NO: 13)
1 5 10
Asn-Val-Asn-Leu-Phe-Ile-Thr-Asp-Gly-Ala-Arg (SEQ ID NO: 14)
1 5 Ala-Pro-Asp-Gly-Pro-Ala- (SEQ ID NO: 15)
or a partial amino acid sequence, preferably an N-terminal amino acid sequence with a homology thereto of at least 70%, preferably at least 80%, more preferably at least 90%.
2. RGase according to Claim 1, with the following partial amino acid sequences:
1 5 10 15
Gly-Ala-Val-Gln-Gly-Phe-Gly-Tyr-Val-Tyr-His-Ala-Glu-Gly-Thr
Tyr-Gly-Ala-Arg (SEQ ID NO: 1)
1 5 10 15 Ser-Xaa-Asn-Ile-Leu-Ser-Tyr-Gly-Ala-Val-Ala-Asp-Xaa-Ser-Thr-
20 25
Asp-Val-Gly-Pro-Ala-Ile-Thr-Ser-Ala-Xaa-Ala-Ala-Arg-Lys ( SEQ ID
NO: 2) Ser-Arg-Asn-Ile (SEQ ID NO: 3 )
1 5 10
Ser-Ala-Tyr-Gly-Ser-Gly-Tyr-Xaa-Leu-Lys (SEQ ID NO: 4)
1 5
Thr-Leu-Glu-Asp-Ile-Ala-Ile (SEQ ID NO: 5)
1 5 10 15
Gly-Leu-Xaa-Ala-Xaa-Ile-Pro-Ile-Pro-Xaa-Ile-Pro-Pro-Xaa-Phe-
Phe (SEQ ID NO: 6)
1 5
Ser-Leu-Asp-Ile-Asp-Gly-Tyr (SEQ ID NO: 7)
1 5 10
Ser-Val-His-Asp-Ile-Ile-Leu-Val-Asp-Ala-Pro-Ala-Phe (SEQ ID NO: 8)
1 5
Ala-Ala-Asp-Leu-Ala- (SEQ ID NO: 9)
1
Gly-Ser-Asn-Ile (SEQ ID NO: 10)
1 5 Tyr-Pro-Gly-Leu-Thr-Pro-Tyr (SEQ ID NO: 11)
1 5 10
Asn-Val-Tyr-Thr-Trp-Ser-Ser-Asn-Gln-Met-Tyr-Met-Ile-Lys (SEQ ID NO: 12 )
or a partial amino acid sequence, preferably an N-terminal amino acid sequence with a homology thereto of at least 70%, preferably at least 80%, more preferably at least 90%.
3. RGase according to Claim 1, with the following partial amino acid sequence:
1 5 10 15
Ala-Phe-Gly-Ile-Thr-Thr-Ser-Ser-Ser-Ala-Tyr-Val-Ile-Asp-Thr- 20 25
Asp-Ala-Pro-Asn-Gln-Leu-Lys-Xaa-Thr-Val-Ser-Arg (SEQ ID NO: 13)
4. RGase according to Claim 1, with the following partial amino acid sequences
1 5 10 Asn-Val-Asn-Leu-Phe-Ile-Thr-Asp-Gly-Ala-Arg (SEQ ID NO: 14)
1 5
Ala-Pro-Asp-Gly-Pro-Ala- (SEQ ID NO: 15)
5. RGase according to Claims 1 and 2, obtainable by means of Aspergillus aculeatus, CBS 101.43.
6. RGase according to Claims 1 and 3, obtainable by means of A. japonicus
ATCC 20236.
7. RGase according to Claims 1 and 4, obtainable by means of Iφex lacteus ATCC 20157.
8. A recombinant DNA sequence comprising a DNA sequence coding for a polypeptide having RGase activity, or a DNA sequence having substantial sequence homology to such RGase coding sequence, preferably a homology of at least 70%, more preferably at least 80%, and most preferably at least 90%.
9. A recombinant DNA sequence according to Claim 8, comprising a DNA sequence selected from a) the A. aculeatus, A. japonicus or Irpex lacteus RGase DNA insert in any appropriate plasmid 5 b) a DNA sequence which hybridizes to the coding region for the mature
RGase DNA comprised by the DNA insert of a) and which comprises a structural gene for a polypeptide with RGase activity, and optionally a promoter, a coding region for a signal or leader peptide and/or transcriptional terminator 10 c) a derivative of a DNA sequence defined in a) or b), or d) a DNA sequence which codes for a mature RGase or a signal peptide or a leader peptide thereof and which is degenerate within the meaning of the genetic code with respect to a DNA sequence of a) or b).
10. A recombinant DNA sequence according to Claims 8 - 9, wherein the 15 RGase activity originates from the RGase producible by means of Aspergillus aculeatus CBS 101.43 with the partial amino acid sequence according to Claims 1 and 2.
11. A recombinant DNA sequence according to Claims 8 - 9, wherein the RGase activity originates from the RGase producible by means of Aspergillus
20 japonicus ATCC 20236 with the partial amino acid sequence according to Claims 1 and 3.
12. A recombinant DNA sequence according to Claims 8 - 9, wherein the RGase acticity originates from the RGase producible by means of Irpex lacteus ATCC 20157 with the partial amino acid sequence according to the Claims 1 and 4.
25 13. Vector comprising the recombinant DNA sequence according to Claims
8-12.
SUBSTITUTE SHEET ISA/3E
14. Vector according to Claim 13, wherein the promoter is the Aspergillus oryzae takaamylase promoter.
15. Transformed host containing the vector according to Claim 13 or 14.
16. Transformed host according to Claim 15, wherein the transformed host is 5 an Aspergillus strain.
17. Transformed host according to Claim 16, wherein the transformed host is a strain belonging to the species Aspergillus aculeatus, Aspergillus niger, Aspergillus oryzae or Aspergillus awamori.
18. Transformed host according to Claim 16, wherein the transformed host is 10 a microorganism, which in its non-transformed condition does not produce RGase or only produces RGase in insignificant amounts, preferably Bacillus sp., E. coli or S. cerevisiae.
19. Method for production of a RGase by use of a transformed host according to Claims 15 - 18.
15 20. RGase produced by the method according to Claim 19.
21. Enzyme preparation comprising RGase according to Claim 1 - 7 or 20, characterized by the fact that it contains another plant cell wall degradation or modification agent, preferably a pectinase and/or cellulase and/or hemicellulase usable for degradation or modification of plant cell walls enriched with the RGase, 0 preferably with an enrichment factor of at least 1.1 or deprived of an RGase, preferably with a deprivation factor of maximum 0.9.
22. Enzyme preparation according to Claim 21 , wherein the other plant cell wall degradation or modification agent is producible by means of a microorganism
SUBSf ITUTE SHEET belonging to the genus Aspergillus, preferably Aspergillus niger, Aspergillus aculeatus, Aspergillus awamori or Aspergillus oryzae.
23. Use of the RGase according to Claims 1 - 7 or 20 as an agent for degradation or modification of plant cell walls and/or plant cell wall components.
24. Use of the enzyme preparation according to Claims 21 - 22, as an agent for degradation or modification of plant cell walls and/or plant cell wall components.
SUBSTITUTE SHEET I
EP92909677A 1991-05-02 1992-05-01 Rhamnogalacturonase, corresponding dna sequence, rhamnogalacturonase containing enzyme preparation and use of the enzyme preparation Withdrawn EP0583313A1 (en)

Priority Applications (2)

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EP92909677A EP0583313A1 (en) 1991-05-02 1992-05-01 Rhamnogalacturonase, corresponding dna sequence, rhamnogalacturonase containing enzyme preparation and use of the enzyme preparation
EP00118463A EP1067180A1 (en) 1991-05-02 1992-05-01 Aspergillus japonicus and Irpex lacteus rhamnogalacturonase and its use

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EP91610039 1991-05-02
EP91610039 1991-05-02
EP92909677A EP0583313A1 (en) 1991-05-02 1992-05-01 Rhamnogalacturonase, corresponding dna sequence, rhamnogalacturonase containing enzyme preparation and use of the enzyme preparation

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