Classifications

• • 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
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
• • 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
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

• the present invention relates to an enzyme.
• the present invention also relates to a nucleotide sequence coding for the enzyme.
• Boos et al (I) observed the formation of acetyl-maltose and acetyl-oligomaltosides after accumulation of maltose or maltooligosides in E. coli. They also observed the formation of acetyl-maltose and acetyl-oligomaltosides in vitro when maltose or maltotriose, acetyl-coenzyme A and a cytosolic E. coli extract were mixed together Boos et al (2).
• E. coli linkage map In addition, they cloned a 3.4 kb DNA fragment containing the gene in a high copy plasmid. Over-expressed maltose transacetylase was then purified to homogeneity from cell free extracts of an E. coli strain harbouring the above mentioned plasmid. The enzyme was shown to be a homodimer with two identical subunits of 20 kDa. The km (mM) and Vmax ( ⁇ mol/min x mg enzyme) values of this enzyme for the substrates glucose, maltose and acetyl-coenzyme A were
• an enzyme having ⁇ (l ,4) glucan acetyl-transferase activity wherein the enzyme comprises the amino acid sequence shown as SEQ ID No. 1, or a variant, homologue or fragment thereof.
• a recombinant enzyme having ⁇ (l ,4) glucan acetyl-transferase activity, wherein the enzyme comprises the amino acid sequence shown as SEQ ID No. 1, or a variant, homologue or fragment thereof.
• a recombinant enzyme having ⁇ (l,4) glucan acetyl-transferase activity wherein the enzyme has the amino acid sequence shown as SEQ ID No. 1.
• a recombinant enzyme having (l ,4) glucan acetyl-transferase activity, wherein the recombinant enzyme is immunologically reactive with an antibody raised against a purified recombinant enzyme according to the above-mentioned aspect of the present invention.
• nucleotide sequence coding for the enzyme of the present invention or a sequence that is complementary thereto.
• nucleotide sequence comprising the sequence shown as SEQ ID No. 2, or a variant, homologue or fragment thereof or a sequence that is complementary thereto.
• nucleotide sequence or the enzyme of the present invention there is provided a construct comprising or expressing the nucleotide sequence or the enzyme of the present invention.
• a vector comprising or expressing the construct or the nucleotide sequence or the enzyme according to the present invention.
• a plasmid comprising or expressing the vector, the construct or the nucleotide sequence or the enzyme according to the present invention.
• a transgenic organism comprising or expressing the plasmid, the vector, the construct or the nucleotide sequence or enzyme according to the present invention.
• a modified carbohydrate (preferably starch) prepared by a method comprising or expressing or using the present invention.
• the enzyme of the present invention may be obtainable from any one of a bacterium, a fungus, an alga, a yeast, or a plant.
• the enzyme is obtainable from E. coli.
• the ⁇ (l,4) glucan acetyl-transferase of the present invention is sometimes referred to as Mac.
• the gene coding for the ⁇ (l,4) glucan acetyl-transferase of the present invention is also sometimes referred to as the mac gene. 3
• nucleotide sequence having the sequence shown as SEQ ID No. 2.
• nucleotide sequence or the enzyme of the present invention there is provided a construct comprising or expressing the nucleotide sequence or the enzyme of the present invention.
• a vector comprising or expressing the construct or the nucleotide sequence or the enzyme according to the present invention.
• a plasmid comprising or expressing the vector, the construct or the nucleotide sequence or the enzyme according to the present invention.
• a transgenic organism comprising or expressing the plasmid, the vector, the construct or the nucleotide sequence or enzyme according to the present invention.
• a modified carbohydrate (preferably starch) prepared by a method comprising or expressing or using the present invention.
• the enzyme of the present invention may be obtainable from any one of a bacterium, a ungus, an alga, a yeast, or a plant.
• the enzyme is obtainable from E.coli.
• the (l ,4) glucan acetyl-transferase of the present invention is sometimes referred to as Mac.
• the gene coding for the ⁇ (l,4) glucan acetyl-transferase of the present invention is also sometimes referred to as the mac gene. 4
• the enzyme comprises the amino acid sequence shown as SEQ ID No 1 , or a variant, homologue or fragment thereof.
• the enzyme has the amino acid sequence shown as SEQ ID No 1.
• the enzyme is encoded by a nucleotide sequence comprising the nucleotide sequence shown as SEQ ID No 2, or a variant, homologue or fragment thereof or a sequence that is complementary thereto.
• the enzyme is encoded by the nucleotide sequence shown as SEQ ID No 2.
• the organism is a plant.
• the nucleotide sequence is a DNA sequence.
• the enzyme or nucleotide sequence(s) coding for same may be used in vitro or in vivo in combination with one or more other enzymes or nucleotide sequence(s) coding for same, which enzymes or nucleotide sequence(s) coding for same are preferably prepared by use of recombinant DNA techniques.
• an in vivo enzymatic modification process can be followed by an in vitro enzymatic modification process.
• the enzymes used need not necessarily be the same enzymes.
• variant in relation to the enzyme include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence providing the resultant amino acid sequence has ⁇ (l,4) glucan acetyl-transferase activity, preferably having at least the same activity of the enzyme shown as SEQ ID No. 1.
• homologue covers homology with respect to structure and/or function 5 providing the resultant enzyme has ⁇ (l ,4) glucan acetyl-transferase activity.
• sequence homology preferably there is at least 75 % . more preferably at least 85% , more preferably at least 90% homology to the sequence shown as SEQ ID No. 1. More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown as SEQ ID No. 1.
• variant in relation to the nucleotide sequence coding for the enzyme include any substitution of, variation of, modification of. replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for an enzyme having (l ,4) glucan acetyl-transferase activity, preferably having at least the same activity of the enzyme shown as SEQ ID No. 1.
• homologue covers homology with respect to structure and/or function providing the resultant nucleotide sequence codes for an enzyme having ⁇ (l,4) glucan acetyl-transferase activity.
• sequence homology preferably there is at least 75 % , more preferably at least 85% , more preferably at least 90% homology to the sequence shown as SEQ ID No. 2. More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown as SEQ ID No. 2.
• nucleotide sequence is not a native nucleotide sequence.
• native nucleotide sequence means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment. 6
• the enzyme of the present invention may be used in conjunction with other enzymes.
• the enzyme is not a native enzyme.
• native enzyme means an entire enzyme that is in its native environment and when it has been expressed by its native nucleotide sequence.
• construct which is synonymous with terms such as “conjugate”, “cassette” and “hybrid” - includes the nucleotide sequence directly or indirectly attached or fused to a promoter.
• An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shl- intron or the ADH intron, intermediate the promoter and the nucleotide sequence.
• the terms do not cover the natural combination of the gene coding for the enzyme ordinarily associated with the wild type gene promoter and when they are both in their natural environment.
• One highly preferred embodiment of the present invention therefore relates to the nucleotide sequence of the present invention operatively linked to a heterologous promoter.
• the construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a plant, such as potato, into which it has been transferred.
• a marker which allows for the selection of the genetic construct in, for example, a plant, such as potato, into which it has been transferred.
• markers exist which may be used, such as for example those encoding mannose-6-phosphate isomerase (especially for plants) or those markers that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.
• expression vector means a construct capable of in vivo or in vitro 7 expression.
• transformation vector means a construct capable of being transferred from one species to another - such as from an E.coli plasmid to an ⁇ grobacterium to a plant.
• tissue includes tissue and organ, which tissue and organ can be isolated tissue and isolated organ, as well as tissue and organ when within an organism.
• organism in relation to the present invention includes any organism that could comprise the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, and/or wherein the nucleotide sequence according to the present invention can be e; ⁇ pressed when present in the organism.
• the organism is a plant.
• transgenic organism in relation to the present invention includes any organism that comprises the nucleotide sequence coding for the enzyme according to the present invention and/or the products obtained therefrom, and/or wherein the nucleotide sequence according to the present invention can be expressed within the organism.
• the nucleotide sequence is incorporated in the genome of the organism.
• the transgenic organism is a plant.
• the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the nucleotide sequence coding for the enzyme according to the present invention, constructs according to the present invention, vectors according to the present invention, piasmids according to the present invention, cells according to the present invention, tissues according to the present invention, or the products thereof.
• the transgenic organism can 8 also comprise the nucleotide sequence coding for the enzyme of the present invention under the control of a heterologous promoter.
• the transgenic organism does not comprise the combination of a promoter and the nucleotide sequence coding for the enzyme according to the present invention, wherein both the promoter and the nucleotide sequence are native to that organism and are in their natural environment.
• promoter is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Mond theory of gene expression.
• the promoter could additionally include one or more features to ensure or to increase expression in a suitable host.
• the features can be conserved regions such as a Pribnow Box or a TATA box.
• the promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention.
• suitable other sequences include the 5 ⁇ -intron or an ADH intron.
• Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements.
• TMV 5' signal sequence see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97).
• the nucleotide sequence according to the present invention is under the control of a promoter that allows expression of the nucleotide sequence.
• the promoter may be a cell or tissue specific promoter. If, for example, the organism is a plant then the promoter can be one that affects expression of the nucleotide sequence in any one or more of seed, stem, tuber, sprout, root and leaf tissues.
• the basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
• the vector system may comprise one vector, but it can comprise two vectors. In the case of two vectors, the vector system is normally referred to as a binary vector system.
• Binary vector systems are described in further detail in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.
• the nucleotide sequence or construct of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appear to be essential for insertion of modified T-DNA into the plant genome.
• the vector system of the present invention is preferably one which contains the sequences necessary to infect me plant (e.g. the vir region) and at least one border part of a T- DNA sequence, the border part being located on the same vector as the genetic construct.
• the vector system is preferably an Agrobacterium tumefaciens Ti- plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, as these piasmids are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these piasmids or derivatives thereof.
• the nucleotide sequence or construct of the present invention may be first constructed in a microorganism in which the vector can replicate and which is easy to manipulate before insertion into the plant.
• An example of a useful microorganism is E. coli, but other microorganisms having the above properties may be used.
• a vector of a vector system as defined above has been constructed in E. coli, it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens.
• the Ti-plasmid harbouring the nucleotide sequence or construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the nucleotide sequence or construct of the invention, which DNA is subsequently transferred into the plant cell to be modified.
• cloning vectors which contain a replication system in E. coli and a marker which allows a selection of the transformed cells.
• the vectors contain for example pBR 322, pUC series. M13 mp series, pACYC 184 etc.
• the nucleotide or construct of the present invention can be introduced into a suitable restriction position in the vector.
• the contained plasmid is used for the transformation in E.coli.
• the E. coli cells are cultivated in a suitable nutrient medium and then harvested and lysed.
• the plasmid is then recovered.
• sequence analysis there is generally used sequence analysis, restriction analysis, electrophoresis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each sequence can be cloned in the same or different plasmid.
• T-DNA for the transformation of plant cells has been intensively studied and is described in EP-A-120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B.B. , Alblasserdam, 1985, Chapter V; Fraley. et al. , Crit. Rev. Plant Sci. , 4: 1-46; and An et al. , EMBO J. (1985) 4:277-284.
• a plant to be infected is wounded, e.g. by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive.
• the wound is then inoculated with the Agrobacterium.
• the inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop into mature plants.
• tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc.
• Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
• the present invention relates to an enzyme having ⁇ (l,4) glucan acetyl- transferase activity and a nucleotide coding for same.
• the present invention also provides a modified carbohydrate (preferably starch) obtainable from use of the same.
• NCIMB National Cancer Institute
• DH5 ⁇ -pM AC3 (which contains a 3.2 kb EcoP ⁇ -Pstl fragment from E. coli comprising the mac gene).
• the deposit number is NCIMB 40789.
• This deposit concerns the plasmid pMAC3.
• NCIMB National Collections of Industrial and Marine Bacteria Limited
• NF1830-pMAC5 (which contains the E.coli mac gene).
• the deposit number is NCIMB 40790.
• This deposit concerns the plasmid pMAC5.
• a highly preferred aspect of the present invention therefore relates to an enzyme having ⁇ (l,4) glucan acetyl-transferase activity, wherein the enzyme comprises the amino acid sequence shown as SEQ ID No. 1, or a variant, homologue or fragment thereof; and wherein the enzyme is expressed by a nucleotide sequence obtainable from either deposit number NCIMB 40789 or deposit number NCIMB 40790.
• nucleotide sequence comprising the sequence shown as SEQ ID No. 2, or a variant, homologue or fragment thereof or a sequence that is complementary thereto, and wherein the nucleotide sequence is obtainable from either deposit number NCIMB 14
• the present invention also provides a modified carbohydrate (preferably starch) obtainable from use of this same plasmid.
• Figure 1 which shows the nucleotide sequence corresponding to SEQ ID No. 2:
• Figure 2 which shows the amino acid sequence corresponding to SEQ ID No. 1 ;
• Figure 3 which shows a nucleotide sequence comprising the sequence corresponding to SEQ ID No. 2;
• Figure 4 which is a plasmid map of pMACl
• FIG. 5 which is a plasmid map of pMAC2
• FIG. 6 which is a plasmid map of pMAC3
• Figure 7 which is a plasmid map of pMAC5 ;
• FIG. 8 which is a plasmid map of pMAC8
• Figure 9 which is a plasmid map of pMAC9.
• Figure 10 which is a plasmid map of pMAClO.
• the 704 bp PCR product was digested with EcoRl and HindlU and inserted in pUH ⁇ 21-2 digested with the same restriction enzymes.
• the PCR product was digested with BamHI and PstI and inserted m pBETP5 digested with the same enzymes.
• the SBE TV-mac fusion was control sequenced with primer # C028
• the 35S terminator-m ⁇ c fusion was sequenced with primer # B456 og # C027.
• Plasmid name pMACIO Plasmid size: 9.37 kb
• the mac gene was isolated from the 4.3 kb EcoRI fragment from ⁇ phage 8C4 (151) from the Kohara collection (4).
• the fragment was inserted into the EcoRI site of plasmid pBluescript II SK (+) in both orientations yielding piasmids pMACl and pMAC2 ( Figures 4 and 5).
• these piasmids gave rise to highly elevated maltose acetyltransferase levels indicating that the 4.3 kb EcoRI fragment contains the mac gene.
• the nucleotide sequence of the 3.2 kb EcoRI-PstI insert in pMAC3 was then determined by automated sequencing on an A.L.F. sequencer.
• the 3137 bp DNA sequence revealed a 372 bp region of the 3' end of the E. coli acrB gene and three open reading frames potentially encoding proteins of 124, 126, and 183 amino acids ( Figure 3).
• the 183 codon orf which encodes a protein of a predicted molecular weight of 20073 ( Figure 2) is the mac gene, since the E. coli maltose acetyl-transferase has an estimated subunit molecular weight of 20.000 (3).
• the mac gene was inserted after an isopropylthiogalactosidase (IPTG) inducible phage T7-promoter Al in pUHE21-2 to give pMAC5 (Figure 7).
• IPTG isopropylthiogalactosidase
• pMAC5 Figure 7
• Cultures of E coli strain NF1830 (MC1000. recAl . F * lacIqlZ: :tm5, a gift from Niels Fiil, University of Copenhagen) harbouring pMAC5 was found to have highly elevated levels of maltose acetyltransferase, when expression of the mac gene is induced by addition of IPTG to the growth medium.
• a 1 L LB culture of NF1830-pMAC5 supplemented with ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml) was grown at 37 °C with vigorous shaking until the A600 reached 0.7.
• IPTG was added to a final concentration of 2mM and growth was continued for four hours.
• the cells were harvested by centrifugation (10 min. at 4 000 x g) and washed by resuspension in 200 ml 0.9% NaCl. The cell pellet was then resuspended in 250 ml 20 mM potassium phosphate pH 7.5 containing 0.4 mM PMSF, 0.4 mg/ml pepstatin and 1.6 mM EDTA.
• the suspension was sonicated 5 x 1 min. using a Vibra Cell VC 600 with a 19 mm High Gain Horn and extender (all from Sonics and Materials Inc. , USA).
• the homogenate was clarified by centrifugation for 60 min. at 90 000 x g at 4°C and subsequent filtration through a 0.22 ⁇ m filter. 19
• the resulting crude extract was applied to a Q-Sepharose 26/10 column (Pharmacia Biotech) equilibrated with 20 mM potassium phosphate pH 7.5 (hereinafter called "buffer A") at a flow rate of 2 ml/min.
• buffer A 20 mM potassium phosphate pH 7.5
• the column was washed with 300 ml of buffer A and the bound protein was eluted by applying a 0 to 0.3 M NaCl linear gradient in buffer A (300 ml).
• the fractions containing enzyme activity were pooled and applied to a 8 ml Affi-Gel Blue (Biorad) column (16 mm x 26 cm) equilibrated with buffer A at a flow rate of 1 ml/min.
• the column was washed with 50 ml of the same buffer containing 0.4 M NaCl.
• the enzyme was then eluted with the same buffer containing 2 M NaCl.
• the active pool was dialysed overnight against buffer A and subsequently concentrated to approximately 3 ml in a Centriprep-30 (Amicon). This fraction was applied to a 6 ml Acetyl-coA-Minileak column equilibrated with buffer A at a flow rate of 0.3 ml/min.
• This affinity resin was made by coupling 200 mg of Acetyl-coA to 5 g (dry weight) of Minileak High (Kem-En-Tek, Denmark) in 10 ml of 1 M NaCO 3 pH 11 for 20h at room temperature.
• the column was washed with 20 ml of buffer A. It was then turned upside down and the pure enzyme was eluted in less than 20 ml with buffer A containing 0.5 M NaCl.
• the purification of the maltose acetyltransferase to homogeneity was achieved after three chromatographic steps. From 11 culture we were able to get 5.8 mg pure Mac. The yield was 29% and the enzyme was purified 80-fold. The purity of the enzyme was assessed both by SDS-PAGE and mass spectrometry. The latter revealed a molecular mass of 19,982 Da.
• the concentration of pure Mac solutions was estimated spectrophotometrically at 280 nm using an extinction coefficient of 0.66 as determined from the amino acid composition of Mac according to (5).
• the acetyl-transferase activity of Mac was assayed spectrophotometrically according to a modified Alpers' assay (6).
• a Perkin Elmer Lambda 18 spectrophotometer was used.
• the assay mixture of a total volume of 1 ml contained a 50 mM potassium phosphate, 2 mM EDTA buffer at pH 7.5, 100 ⁇ l of maltose 1M, 100 ⁇ l of Acetyl-coA 0.4 mM, 10 ⁇ l 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) 40 mM dissolved in methanol and 10 ⁇ l enzyme.
• the reaction was started by the addition of enzyme or maltose and was monitored at 412 nm at 25°C.
• One activity unit was defined as the amount of enzyme that produced an increase in absorbance of 1 per minute at 25°C.
• An extinction coefficient of 13 600 M ' 1 x cm "1 was used for DTNB in order to calculate the consumption of acetyl coenzyme A.
• N-terminal sequencing of pure Mac was performed using an Applied Biosystems 476A protein sequencer. One nanomole of protein was desalted by RP-HPLC on a C2 column (4.6/30) prior to loading onto the sequencer. The N-terminal sequence of Mac was determined up to residue was determined up to residue 48 and was in complete concordance with the nucleotide sequence of the mac gene ( Figure 1). Furthermore, the N-terminal methionine residue was not present on the mature protein ( Figure 2).
• Rabbits were immunised subcutaneously at 2-week intervals during 6 weeks and at 4- week intervals thereafter with 90 ⁇ g of pure protein emulsified (1 : 1 , vol/vol) with Freund ' s adjuvant. Antisera were tested against Mac in immunoblots and were found highly specific. 21
• the isoelectric point of Mac was determined by isoelectric focusing on a PhastGel IEF 4-6.5 (Pharmacia) and was found to be 5.7.
• the pH profile of Mac was investigated between pH 5 and 8.5 at a 100 mM maltose concentration in 50 mM buffers containing 100 mM NaCl. Under these conditions, the pH optimum was 7.7.
• Mac The pH stability of Mac was examined at 25 °C between pH 3.0 and 10.0. Mac was instantaneously inactivated at pH 3.0 but was stable between pH 4.0 and 10.0 for at least six hours.
• thermostability of Mac was investigated at pH 7.5 between 40 and 70°C. After incubation for four hours at 40°C and 50°C, the remaining activity of Mac was 100% and 75%, respectively. Its half-life was 70 min, and 22 min at 60°C and 70°C, respectively.
• the substrate preference of Mac towards the carbohydrate acetyl-acceptor substrate was investigated by measuring the initial rate of the acetylation of various carbohydrates (used at 50 and 100 mM concentrations) following the procedure described in "Determination of enzyme concentration and activity". The results are presented in Tables 1 , 2 and 3. Among the monosaccharides tested, glucose was the best substrate and among the disaccharides tested, maltose and isomaltose were the best substrates. 22
• the SBE-Mac fusion was inserted in the E. coli expression vector pAL781 (Invitrogene, San Diego. USA) in order to over-express the fusion enzyme in E. coli and analyse the activity.
• pAL781 Invitrogene, San Diego. USA
• a comparison of the highly over-expressed SBE-Mac fusion and the purified wild type Mac enzyme on SDS gels showed that the fusion migrated slightly slower due to the 27 amino acid extension.
• the fusion retained the ability to use maltose as a substrate for acetylation.
• the fusion enzyme appears to be intact and is fully active in E. coli. Therefore, it may be assumed, that the SBE-Mac fusion enzyme will be active in potatoes.
• the E coli mac gene was amplified with primers:
• the PCR product was digested with BamHI and PstI and inserted in pBETP5 (see PCT patent application No. WO 94/24292, the contents of which are incorporated herein by reference) digested with the same enzymes yielding pMAC8.
• the mac gene is inserted in an expression cassette that provides tuber specific expression from a patatin promoter and transcription termination at a CaMV 35S terminator.
• the Mac enzyme is fused to 102 amino acids of the N-terminus of the potato starch branching enzyme including a 75 amino acid transit peptide that directs the mac gene product to the potato tuber amyloplasts.
• a segment containing the nodium - i.e. a segment taken from 2 mm above and 5 mm below the nodium - was cut from in vitro grown potato plants or mannose selected shoots (for mannose selection see our earlier patent applications WO 93/05163 and/or WO 94/20627).
• the leaf was removed from the nodium segment, and the segment was placed vertically on agar plates with MS medium (Sigma) supplemented with 60 g sucrose/1 and 2 mg 6-benzyl-aminopurine/l.
• the nodium segments were grown for 7 days at 20°C with a 16 hour light period and an 8 hour dark period. Subsequently, the plates were wrapped in alu-foil and placed in the dark at 20°C.
• the minitubers were harvested after about 28 days and applied for western analysis in order to detect Mac expression.
• Potato minitubers transformed with the pMAC9 or pMACIO constructs were examined by Western analysis for expression of the E. coli mac gene with antibodies raised towards the E. coli maltose acetyltransferase.
• the analysis clearly demonstrated that 3 out of 5 MAC9 minitubers and 5 out of 7 MAC 10 minitubers gave a distinct expression of the E. coli maltose acetyltransferase.
• These results indicate that the 75 amino acid SBE transit peptide, that was originally fused to the 209 amino acid SBE-Mac fusion, has been removed from the SBE-fusion. Furthermore, this implies that the transit peptide was correctly processed by the signal peptidase in the amyloplast membrane, and that the SBE-Mac fusion has been directed to the amyloplast.
• Potato tubers of comparable sizes were chosen and cut into pieces and homogenised in extraction buffer and Dowex (1 % , w/vol) using a mortar and pestle or an electric blender.
• 5 ml extraction buffer 50 mM potassium phosphate pH 7.5, 2 mM EDTA, 0.5 mM PMSF
• the mixture was allowed to stand on ice for 30 min and the insoluble material was removed by centrifugation. Protein concentration was measured using the BCA reagent (Pierce).
• Mac activity was measured in duplicates or triplicates as follows: 0, 50, 100 or 200 ⁇ l potato extract, 10 ⁇ l of 1 mM acetyl-coenzyme A, 25 ⁇ l of 1 M glucose and assay buffer (50 mM potassium phosphate, 2 mM EDTA, pH 7.5) were mixed per microtiter plate well to give a total volume of 250 ⁇ l. The reaction was started by the addition of acetyl-coenzyme A. After 10 min. reaction at room temperature, 25 ⁇ l of freshly made 4 mM DTNB was added and A 405 was measured immediately. Two wells were prepared for each single assay, one with glucose and one without. Activity was calculated by subtracting the absorbance of the well without glucose (background absorbance) from that of the well with glucose. 28
• GATCATCATT GATACAACGT GTAATCACTA AGGCCGCGTA AGCGGCCTTT riTATGCATA 360
• CTCCAGCCAG CCTTTTTCCA CAATCAGATA TACTTTCCCT ACACTGTGTT AATAAGGATA 2940 TGCTGGTGAG AACACGACAT CTGGTCGGCC TTATTTCGGG AGTACTGATT CTTTCAGTAT 3000
• t ne incicaiions made oeiow rent ⁇ tne microorganism referred to in its. description on sace 22 ⁇ ' ⁇ ne 1 ? - 7 1
• NCIMB National Collections of Industrial and Marine Bacteria Limi e
• Tne incicaiions l isted seiow wi l l oe suomittea to tne international Bureau later tsptc ⁇ /ytntgt ⁇ rancr ⁇ r ⁇ af ⁇ t ⁇ na ⁇ cs ⁇ on ⁇ ⁇ .g. Aeeatton .Sumbtr ofOtpanO

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• 1996
  • 1996-03-13 GB GBGB9605274.1A patent/GB9605274D0/en active Pending
• 1997
  • 1997-03-07 CN CN97194352A patent/CN1259997A/zh active Pending
  • 1997-03-07 JP JP9532251A patent/JP2000506023A/ja active Pending
  • 1997-03-07 CA CA002248540A patent/CA2248540A1/en not_active Abandoned
  • 1997-03-07 PL PL97328829A patent/PL328829A1/xx unknown
  • 1997-03-07 EP EP97908181A patent/EP0906413A2/en not_active Withdrawn
  • 1997-03-07 BR BR9708029-2A patent/BR9708029A/pt unknown
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  • 1997-03-07 AU AU20243/97A patent/AU720991B2/en not_active Ceased

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