Classifications

• • 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

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• 1992
  • 1992-05-01 EP EP92909677A patent/EP0583313A1/de not_active Withdrawn
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