WO2003106502A1

WO2003106502A1 - Methods for producing dextrins using enzymes

Info

Publication number: WO2003106502A1
Application number: PCT/DK2003/000400
Authority: WO
Inventors: Yasuhito Takeda; Isao Hanashiro; Michiko Ihara; Shinobu Takagi
Original assignee: Novozymes A/S
Priority date: 2002-06-17
Filing date: 2003-06-17
Publication date: 2003-12-24
Also published as: AU2003232182A1

Abstract

A dextrin having an average degree of polymerization (DP) of at least about 4,000; which is essentially free of cyclic structures, or which is at least 90% degradable by treatment with a glucoamulase (EC 3.2.1.3).

Description

Methods for producing dextrins using enzymes

FIELD OF THE INVENTION

The present invention relates to high-molecular weight non-cyclic dextrins, and methods o1 enzymatic production of such dextrins.

BACKGROUND OF THE INVENTION

Branching enzyme (EC 2.4.1.18) hereinafter denoted BE, catalyzes transglycosylation to forrr the alpha-1 ,6-glucosidic linkages (branch points) of glycogen, dextrin, and amylopectin ir microorganisms, plants and higher organisms. Glycogen and amylopectin are highly branchec starch materials used for energy storage in microorganisms, plants and higher organisms. No only does BE form a branch between different molecules (intermolecular transfer), it alsc catalyzes the transfer of a multi-branched glucan to another site on the same molecule (intramolecular transfer).

Highly branched starch materials have unique properties like high solubility, low viscosity anc less tendency to retrograde compared to unmodified starch, which make them interesting fo use i n a dhesive c ompositions i ncluding s urface s izing a nd c oating i n t he p aper i ndustry a . described in EP 0690170 (Avebe B.A.), food and drink additives and anti-starch retrogradatioi agents as described in US 4454161.

Branching enzymes from several different organisms have been isolated and disclosed, e.g. US 4454161 describes a BE from Bacillus megaterium; Boyer and Preiss, Biochemistry, vo! 16 (16), pp. 3693-3699, 1977, describe a BE from Escherichia coli; Zevenhuizen, Biocherr Biophys. Acta (81), pp. 608-611 , 1964, describes a BE from Arthrobacter globiformis; Walke and Builder, Eur. J. of Biochem., vol. 20 (1), pp. 14-21 , 1971, describe a BE fron Streptococcus mitis; Kiel et al., Gene, vol. 78 (1), pp. 9-18, 1989, describe a BE from tin cyanobacterium Synechococcus sp. PCC7942; Rumbak et al., Journal of Bacteriology , vo 173 (21), pp. 6732-6741 , 1991 , describe a BE from Butyrivibrio fibrisolvens; Kiel et al., DN/ Sequence, vol. 3 (4), pp. 221-232, 1992, describe a BE from Bacillus caldolyticus.

A common characteristic of the above mentioned references is that none of the reporte temperature optima are higher than 45°C. Two branching enzymes were described as havin a higher temperature optimum: Kiel et al., Molecular & General Genetics, vol. 230 (1-2), pf 136-144, 1991 (also in EP 0418945): a Bacillus stearothermophilus 1503-4R branchin enzyme having a temperature optimum of 53°C; and Takata et al., Applied and Environment; Microbiology, vol. 60 (9), pp. 3096-3104, 1994: a Bacillus stearothermophilus TRBE14 branching enzyme having a temperature optimum of around 50°C.

Because of the increased reaction rates obtained at higher temperatures it is industrially advantageous to use branching enzymes with a high temperature optimum. Thereby a higher capacity is obtainable with the same amount of enzyme, and a better production economy is achieved. Furthermore it is beneficial to run processes at high temperatures due to prevention of infections. Thermophilic branching enzymes from Rhodothermus sp., having a temperature optimum above 60°C were disclosed in WO 00/58445 (Novozymes A/S) which is incorporated herein in its entirety by reference.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide high-molecular weight essentially non-cyclic dextrins, and methods for producing such dextrins. The present inventors surprisingly found that dextrins of hitherto unseen high-molecular weight, and almost completely devoid of cyclic structures, could be produced by treatment of compositions comprising amylose or amylopectin with microbial branching enzymes (EC 2.4.1.18). The produced dextrins were thus highly susceptible to degradation by treatment with a glucoamylase (EC 3.2.1.3).

Accordingly in a first aspect the invention relates to a dextrin having an average degree of polymerization (DP) of at least about 4,000; which is essentially free of cyclic structures, or which is at least 90% degradable by treatment with a glucoamulase (EC 3.2.1.3).

In a second aspect the invention relates to a dextrin having an average degree of polymerization (DP) of at least about 4,000; which is essentially free of cyclic structures, or which is at least 90% degradable by treatment with a glucoamulase (EC 3.2.1.3), said dextrin being obtainable by treatment of a composition comprising amylose or amylopectin with a branching enzyme (EC 2.4.1.18).

A third aspect of the invention relates to a dextrin having an average degree of polymerization (DP) of at least about 4,000; which is essentially free of cyclic structures, or which is at least 90% degradable by treatment with a glucoamulase (EC 3.2.1.3), and which is produced by treatment of a composition comprising amylose or amylopectin with a branching enzyme (EC 2.4.1.18).

In the present context the term "essentially free of cyclic structures" means that the dextrin is at least about 90% free of cyclic structures. Since cyclic structures in dextrins are not degradable by treatment with glucoamylase enzyme, the dextrins of the three first aspects are substantially degradable by treatment with a glucoamylase (EC 3.2.1.3). Preferably, the dextrins of the invention are at least 92% degradable by treatment with a g lucoamylase, or 94%, or even 96%, an more preferably they are at least 98% degradable by treatment with a glucoamylase (EC 3.2.1.3). A non-limiting example of a treatment of a dextrin with a glucoamylase is shown in the examples below.

A fourth aspect of the invention relates to a method of producing a dextrin as defined in any of the preceeding aspects, comprising a step of treating a composition comprising amylose or amylopectin with a branching enzyme (EC 2.4.1.18), whereby the dextrin is produced.

In a final aspect the invention relates to the use of a dextrin, as defined in the three first aspects, or produced by a method as defined in the preceeding aspect, as a medical device or part of a medical device, in the formulation of a pharmaceutical product, in adhesive compositions including surface sizing and coating in the paper industry, as food and drink additives, or as anti-starch retrogradation agents.

DEFINITIONS

For purposes of the present invention, alignments of sequences and calculation of homology scores may be done using a full Smith-Waterman alignment, useful for both protein and DNA alignments. The default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively. The penalty for the first residue in a gap is -12 for proteins and -16 for DNA, while the penalty for additional residues in a gap is -2 for proteins and -4 for DNA. Alignment may be made with the FASTA package version v20u6 (W. R. Pearson and D. J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448, and W. R. Pearson (1990) "Rapid and Sensitive Sequence Comparison with FASTP and FASTA", Methods in Enzymology, 183:63-98).

Multiple alignments of protein sequences may be made using " ClustalW" (Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680). Multiple alignment of DNA sequences may be done using the protein alignment as a template, replacing the amino acids with the corresponding codon from the DNA sequence. DETAILED DESCRIPTION OF THE INVENTION

The first three aspects of the invention relate to a dextrin having an average degree of polymerization (DP) of at least about 4,000; which is essentially free of cyclic structures, or which is at least 90% degradable by treatment with a glucoamulase (EC 3.2.1.3); a dextrin having an average degree of polymerization (DP) of at least about 4,000; which is essentially free of cyclic structures, or which is at least 90% degradable by treatment with a glucoamulase (EC 3.2.1.3), said dextrin being obtainable by treatment of a composition comprising amylose or amylopectin with a branching enzyme (EC 2.4.1.18); or a dextrin having an average degree of polymerization (DP) of at least about 4,000; which is essentially free of cyclic structures, or which is at least 90% degradable by treatment with a glucoamulase (EC 3.2.1.3), and which is produced by treatment of a composition comprising amylose or amylopectin with a branching enzyme (EC 2.4.1.18).

The DP of a dextrin molecule can be determined as described in the non-limiting examples of the present application {vide infra). In a preferred embodiment of the first three aspects, the dextrin has a DP of at least about 5,000; or 6,000; or even 7,000; more preferably 8,000; or 9,000; even more preferably 10,000; or 11,000; and most preferably 13,000; or 15,000.

A preferred embodiment relates to a dextrin of the first three aspects, which is at least 95% degradable by treatment with a glucoamulase, more preferably 97%, and most preferably at least 99%) degradable by treatment with a glucoamulase.

The non-limiting examples herein show how the dextrins of the invention may be produced from potato amylose, purified amylose, synthetic amylose, and mixtures of those. Accordingly, a preferred embodiment relates to a dextrin of the first three aspects, wherein the composition comprising amylose or amylopectin comprises potato amylose, or purified amylose, or a mixture of the two.

A preferred embodiment relates to a dextrin of the second or third aspects, wherein the branching enzyme is of microbial origin; preferably the branching enzyme is thermophilic, and preferably the branching enzyme has a temperature optimum of at least 60°C; more preferably the branching enzyme is derived from a Rhodothermus species, preferably from a R. obamensis or marinus; and most preferably the branching enzyme has an amino acid sequence which is a) at least 65% identical with the mature amino acid sequence of SEQ ID NO:2 shown in WO 00/58445; b) at least 65% identical w ith the mature branching enzyme amino acid sequence encoded by the nucleic acid sequence contained in plasmid pT7Blue contained in the E. coli DH12S, deposited as DSM 12607; or c) which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) the nucleotides of the nucleic acid sequence of SEQ ID NO:1 shown in WO 00/58445, corresponding to the mature enzyme, (ii) the cDNA sequence of SEQ ID NO:1 shown in WO 00/58445, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii).

In a preferred embodiment, the branching enzyme is derived from a m icroorganism of any genus. For purposes of the present invention, the term "derived from" as used herein in connection with a given source shall mean that the BE encoded by a nucleic acid sequence is produced by the source or by a cell in which the nucleic acid sequence from the source has been inserted. In a preferred embodiment, the BE is secreted extracellularly.

A preferred BE of the present invention may be a bacterial BE. For example, the BE may be derived from a Gram-positive bacterium such as a Bacillus sp., e.g. a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis; or a Streptomyces sp., e.g. a Streptomyces lividans or Streptomyces murinus; or a Gram-negative bacterium, e.g. an E. coli or a Pseudomonas sp.

A preferred BE of the present invention may be a fungal BE, and more preferably derived from a yeast such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia; or more preferably derived from a filamentous fungal genus such as Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma.

In a preferred embodiment, the branching enzyme is derived from Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis.

In another preferred embodiment, the branching enzyme is derived from Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. For example, taxonomic equivalents of Rhodothermus obamensis are defined by Sako et al., International Journal of Systematic Bacteriology , Vol. 46 (4 ) pp. 1099-1104 (1996).

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Japan Collection of Microorganisms (JCM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

Furthermore, such branching enzymes may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The nucleic acid sequence may then be derived by similarly screening a genomic or cDNA library of another microorganism. Once a nucleic acid sequence encoding a polypeptide has been detected with the probe(s), the sequence may be isolated or cloned by utilizing techniques which are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

In another preferred embodiment of the second and third aspects, the branching enzyme is a variant, a shuffled, a synthetic, or a hybrid enzyme derived from one or more branching enzymes of microbial origin; preferably the branching enzyme is thermophilic, preferably the branching enzyme has a temperature optimum of at least 60°C.

An aspect of the invention relates to a method of producing a dextrin as defined in any of the preceeding claims, comprising a step of treating a composition comprising amylose or amylopectin with a branching enzyme (EC 2.4.1.18), whereby the dextrin is produced.

In a preferred embodiment of the fourth aspect, the branching enzyme is of microbial origin; preferably the branching enzyme is thermophilic, and preferably the branching enzyme has a temperature optimum of at least 60°C; even more preferably the branching enzyme is derived from a Rhodothermus species, preferably from a R. obamensis or R. marinus; and most preferably the branching enzyme has an amino acid sequence which is a) at least 65% identical with the mature amino acid sequence of SEQ ID NO:2 shown in WO 00/58445; b) at least 65% identical with the mature branching enzyme amino acid sequence encoded by the nucleic acid sequence contained in plasmid pT7Blue contained in the E. coli DH12S, deposited as DSM 12607; or c) which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) the nucleotides of the nucleic acid sequence of SEQ ID NO:1 shown in WO 00/58445, corresponding to the mature enzyme, (ii) the cDNA sequence of SEQ ID NO:1 shown in WO 00/58445, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii).

In a preferred embodiment, the branching enzyme of the present invention comprises the amino acid sequence of SEQ ID NO:2 shown in WO 00/58445, or an allelic variant thereof; or a fragment thereof that has branching enzyme activity. In a more preferred embodiment, the branching enzyme comprises the amino acid sequence of SEQ ID NO:2 shown in WO 00/58445. In another preferred embodiment, the branching enzyme consists of the amino acid sequence of SEQ ID NO:2 shown in WO 00/58445, or an allelic variant thereof; or a fragment thereof that has branching enzyme activity. In another preferred embodiment, the branching enzyme consists of the amino acid sequence of SEQ ID NO:2 shown in WO 00/58445.

A fragment of SEQ I D NO:2 shown in WO 00/58445 is a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of this amino acid sequence.

An allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

In another embodiment, the branching enzyme is encoded by nucleic acid sequences which hybridize under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with a nucleic acid probe which hybridizes under the same conditions with (i) the nucleotides of SEQ ID NO:1 shown in WO 00/58445, (ii) the cDNA sequence contained in the nucleotides of SEQ ID NO:1 shown in WO 00/58445, (iii) a subsequence of (i) or (ii), or (iv) a complementary strand of (i), (ii), or (iii) (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York). The subsequence of SEQ ID NO:1 may be at least 100 nucleotides or preferably at least 200 nucleotides. Moreover, the subsequence may encode a polypeptide fragment which has branching enzyme activity. The polypeptides may also be allelic variants or fragments of the polypeptides that have branching enzyme activity.

The nucleic acid sequence of SEQ ID NO:1 shown in WO 00/58445 or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO:2 shown in WO 00/58445, or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having branching enzyme activity from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, and more preferably at least 35 nucleotides in length. Longer probes can also be used. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed by the present invention.

Thus, a genomic DNA or cDNA library prepared from such other organisms may be screened for DNA which hybridizes with the probes described above and which encodes a polypeptide having branching enzyme activity. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA which is homologous with SEQ ID NO:1 shown in WO 00/58445, or a subsequence thereof, the carrier material is used in a Southern blot. For purposes of the present invention, hybridization indicates that the nucleic acid sequence hybridizes to a labeled nucleic acid probe corresponding to the nucleic acid sequence of SEQ ID NO:1 shown in WO 00/58445, its complementary strand, or a subsequence thereof, under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions are detected using X-ray film.

In a preferred embodiment, the nucleic acid probe is a nucleic acid sequence which encodes the polypeptide of SEQ ID NO:2 shown in WO 00/58445, or a subsequence thereof. In another preferred embodiment, the nucleic acid probe is SEQ ID NO:1 shown in WO 00/58445. In another preferred embodiment, the nucleic acid probe is the nucleic acid sequence contained in plasmid pT7Blue, which is contained in Escherichia coli DSM 12607, wherein the nucleic acid sequence encodes a polypeptide having branching enzyme activity.

For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures.

For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2 x SSC, 0.2% SDS preferably at least at 45°C (very low stringency), more preferably at least at 50°C (low stringency), more preferably at least at 55°C (medium stringency), more preferably at least at 60°C (medium-high stringency), even more preferably at least at 65°C (high stringency), and most preferably at least at 70°C (very high stringency).

For short probes which are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at 5°C to 10°C below the calculated T_m using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCI, 0.09 M Tris- HCI pH 7.6, 6 mM EDTA, 0.5% NP-40, 1X Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6X SSC plus 0.1% SDS for 15 m inutes and twice each for 15 minutes using 6X SSC at 5°C to 10°C below the calculated T_m.

In a fourth embodiment, the present invention relates to branching enzyme variants comprising a substitution, deletion, and/or insertion of one or more amino acids. The amino acid sequences of the variant polypeptides may differ from the amino acid sequence of SEQ ID NO:2 shown in WO 00/58445 by an insertion or deletion of one or more amino acid residues and/or the substitution of one or more amino acid residues by different amino acid residues. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions which do not generally alter the specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, LeuΛ al, Ala/Glu, and Asp/Gly as well as these in reverse.

In another preferred embodiment of the fourth aspect, the branching enzyme is a variant, a shuffled, a synthetic, or a hybrid enzyme derived from one or more branching enzymes of microbial origin; preferably the branching enzyme is thermophilic, preferably the branching enzyme has a temperature optimum of at least 60°C.

A final preferred embodiment of the fourth aspect, the dextrin is subsequently purified or isolated in one or more steps.

EXAMPLES

Microbial branching enzyme (BE)

The microbial branching enzyme used in these examples was isolated from the thermophilic bacterium Rhodothermus obamensis as described in WO 00/58445 (EMBL accession No: AB060080). Branching enzyme activity was measured by iodine staining assay as described in Appl Micobiol Biotechnol (2001 ) 57:653-659. It was determined by the decrease in the color of glucan-iodine complex at OD 660 nm using amylose type III from Sigma Chemical. One unit is defined as the amount of enzyme which decreases the absorbance 660 nm by 1 % per minute.

Materials Potato amylose (type II) was purchased from Sigma Chemical and purified as described elsewhere (Y Takeda, H P Guan and J Preiss, Carbohydr. Res., 240 (1993) 253-263). Synthetic amylose AS-110 and AS-1000 were obtained from Nakano Vinegar Co., Ltd. and amylose A0847 (mw 15,000) was purchased from Tokyo Kasei Kogyo Co. Ltd. 2-Aminopyridine of special grade for fluorescent labeling and sodium cyanoborohydride were purchased from Wako Pure Chemical Industries Ltd. and Aldrich Chemical Co. Inc. Isoamylase and Rhizopus glycoamylase were the products of Hayashibara Biochemical Laboratories and Toyobo respectively.

Preparation of amylose solution (pH=7.5)

A certain amount of amylose (e.g. 20 mg) was gelatinized with 2N NaOH (333 μl) for 30 minutes then diluted with water of 100 μl and MOPS buffer pH=7.5 (120 μl) to be the final concentration 30mM MOPS. The pH is adjusted to 7.5 with 2N HCI and total volume is adjusted to make the amylose concentration e.g. 10 mg/ml

Fluorescent labeling of dextrin Fluorescent labeling of the reducing residue of amylose or dextrin was carried out as described in Carbohydrate Research 306 (1998) 421-426.

2-Aminopyridine (2-AP) solution was prepared by dissolving 1 g of 2-AP in 760 μl of 12 N HCI. Aqueous s odium cyanoborohydride ( NaCNBH₃) was p repared j ust b efore u se by d issolving 52.9 mg of NaCNBH₃ in 100 μl of water. The same volume of 2-AP solution was added to the dextrin-solution containing dextrin up to 20 mg/ml in a teflon-lined screw capped tube and incubation at 60 °C for 1 hour in darkness. Then half the volume of aqueous NaCNBH₃ was added to the mixture followed by further incubated for 24 hours. After the incubation, the labeled dextrin was precipitated by 3 volumes of ethanol for over one hour at -20°C. The precipitate was recovered by centrifugation at 2000 rpm at 4°C for 10 minutes, then washed with 75% ethanol containing 50 mM NaCl at least three times to remove excess amount of reagents.

Gel Permeation Column (GPC) chromatographv GPC was p erformed w ith two d ifferent H PLC systems. S ystem A a nd B were d esigned for analyses of large and small molecules, respectively. System A consists of a HPLC pump (PU- 1580, Jasco), an analytical column (see below), a fluorescence detector (FP-2020 Plus, Jasco) and a refractive index detector (RI-2031 Plus, Jasco). The column (HR10/30, Pharmacia) was packed with Toyopearl HW-75S, HW-50S and HW-40S (Tosoh) at the volume ratio of 2:3:1 and kept at 37°C. System B consists of a HPLC pump (PU-1580, Jasco), a Shodex OHpak SB-803 HQ and two Shodex OHpak SB-802.5 HQ analytical columns (8 x 300 mm each, Showa Denko) connected in series, a fluorescence detector (FP-920, Jasco) and a differential refractometer (ERC-7512, Erma). The column temperature was kept at 50°C. Excitation and emission wave-lengths were 315 and 400 nm, respectively.

Average Degree of Polymerization (DP)

Fluorescence response corresponds to a molar distribution of molecules and refractive index corresponds to a weight distribution of molecules. The eluent was 50% dimethylsulfoxide (DMSO)-50 mM NaCl and flow rate for system A and B was set to 0.2 and 0.25 ml/min, respectively. A specimen labeled with 2-AP was dissolved in water or 50% DMSO and passed through a membrane filter with pore size of 0.22 mm prior to injection to the columns. The number-average degree of polymerization (DPn) of the specimen was calculated from the response of refractive index (Rl) and fluorescence (F) based on the Rl/F values of standard amyloses e.g. AS-1000 (DPn: 4400) or AS-10 (DPn: 52) using the following equation: DP sample = [(RI/F) sample/ (Rl/F) standard] x DP standard.

High-performance anion-exchange chromatographv with pulsed amperometric detection

(HPAEC-PAD)

HPAEC-PAD was performed with a Dionex system equipped with a pulsed amperometric detector (PAD-II, Dionex). Two CarboPac PA-1 columns (Dionex) were connected in tandem.

The gradient elution was carried out at flow rate of 1.0 ml/min using eluent A (150 mM NaOH) and eluent B (150 mM NaOH containing 500 mM sodium acetate) according to the time program shown in table 1 :

Table t

Example 1 : Production of large dextrin from potato amylose by BE

Potato amylose solution (pH7.5) was treated with BE at the concentration of 111 U/mg amylose at 60°C for 6 hours. To terminate the reaction, 1/10 volume of 1-butanol was added to the reaction mixture and boiled for 5 minutes followed by the incubation at 4°C for 3 hours to precipitate un-reacted amylose. The precipitate was removed by centrifugation at 3000 rpm for 10 minutes at 4°C then the dextrin products were recovered from the supernatant by precipitation with 3 volumes of ethanol and incubation on ice. The recovered dextrin was dissolved in water at the concentration of -1.8 mg/ml then subjected for the fluorescent labeling as described in Carbohydrate Research 306 (1998) 421-426. As the control, potato amylose without BE treatment was also fluorescent labeled. Fluorescent labeled dextrin or amylose was dissolved in 50% DMSO and after filtration it was subjected to GPC chromatography. DP of each dextrin was calculated using AS-1000 (DP 4400) as the standard.

The amyloses used as substrate showed a monomodal distribution with a peak at DP 676 for potato amylose and DP 95 for amylose A0847. Both dextrin products from the BE treatment showed two distinct populations, as judged from their fluorescence profile. For the product from potato amylose, the DP at the peaks were estimated to be 6860 and 151 , and the molar ratio of the two populations appeared to be 1:1. For the product from amylose A0847, the DP at the peaks were estimated to be 4690 and 134 and the smaller product was the more abundant by mole. The results showed that a dextrin having a much higher DP, 10-50 times higher, than the substrate was being produced by the branching enzyme reaction. The results also showed that the size of the dextrin products from the BE reaction could vary depending on the size of amylose substrate.

Example 2: Production of large dextrin from the mixed size of amylose

Equal volume of the solutions of potato amylose and amylose A0847 at 4 mg/ml each, both pH 7.5, were mixed and treated with BE (55.5 U/mg amylose) at 60°C for 6 hours. The dextrin product was labeled with 2-AP and subjected to GPC with system A as described in Example 1.

Production of large dextrin by the BE treatment was confirmed by GPC. Again, two distinct populations in the dextrin product were observed in a fluorescent profile, and the DP at the peaks was estimated at 11,400 and 125. The DP for the larger dextrin product (11,400) was much higher than those of the products obtained from either one of the two amyloses alone (Example 1 ). The results showed that the large dextrin over DP 11 ,000 can be produced by the BE using a mixture of amyloses with different size as substrates.

Example 3: Time course of dextrin production by BE from potato amylose The potato amylose solution was treated with BE (92.4 U/mg amylose) at 60°C for 0.5, 2 or 18 hours. After terminating the reaction by boiling for 2 minutes, 3 volumes of ethanol was added and the mixtures were kept at -20°C for over one hour. The precipitated dextrin-products were collected by centrifugation at 2000 rpm for 10 minutes at 4°C, and dissolved in 111 μl of 90% dimethylsulfoxide (DMSO) by heating followed by addition of 89 μl of water with extra heating.

For fluorescence labeling, 200 μl of 2-aminopyridine (1 g per 760 μl of 12N HCI) was added to the 200 μl of dextrin solution and incubated at 60°C for one hour in dark, then 200 μl of sodium cyanoborohydride solution (52.92 mg per 100 μl of water) was mixed a nd incubated for 24 hours under the same condition. After the reaction, the labeled dextrin was precipitated with 3 volumes of 99% ethanol at -20°C for over one hour and recovered by centrifugation at 2000 rpm for 10 minutes at 4°C. The dextrin was washed with 2.4 ml of 75% ethanol - 50 mM NaCl three times by mixing and centrifugation under the same condition. Then again it was dissolved in 111 μl of 90%) DMSO by heating followed by addition of 89 μl of water with extra heating and precipitated by 600 μl of 99% ethanol containing 1 μl of 1 M NaCl at -20°C for over one hour. To remove excess amount of reagent completely, the dextrin was washed 2 times with 2.4 ml of 75% ethanol - 50 mM NaCl. Finally, the washed dextrin was dissolved again in 111 μl of 90% DMSO and 89 μl of water and 1 μl of 1 M NaCl was added to the solution. Three volumes of 99% ethanol was added and kept at -20°C until analysis.

The labeled dextrins were subjected to GPC with system A. The size of dextrin product from potato amylose (DP 1190) increased from DP 2800, DP 12,800 and DP 17,200 after 0.5, 2 and 18 hours of treatment with the BE. Based on the total area of Rl response, 95% by weight of the initial amount of amylose substrate was retained in the dextrin product after 18 hours of BE treatment. On the other hand, based on the fluorescent response, number of glucan molecules decreased to 8% of the initial amount. The results suggested that the most of the glucosyl residues that was originally present in amylose substrate was integrated into the large dextrin product and very limited number of molecules was enlarged to be the large dextrin product. The smaller dextrin product was considered to be removed by the ethanol precipitation employed in this example after BE treatment.

Example 4: BE treatment of fluorescent labeled potato amylose The purified potato amylose (DP1190) was fluorescent labeled with 2-AP as described. The labeled amylose was dissolved with 2N NaOH, water and 2N HCI, and finally the pH was adjusted with MOPS buffer (final concentration 30mM) to pH7.5. The concentration of labeled amylose in the solution was 10 mg/ml. It was treated with BE (92.4 U/mg amylose) at 60°C for 18 hours.

After the reaction was terminated by boiling for 2 minutes, the reaction mixture was divided into two portions of equal volume, and a portion was kept for GPC analysis. To another portion, 3 volumes of 99% ethanol were added and the mixture was stored at -20°C for over one hour. The each of BE product with or without ethanol precipitation was analyzed by GPC with system A. The size of ethanol-precipitated dextrin was DP14,000. The reaction mixture without ethanol precipitation contained small molecules of DP -10 other than the dextrin of DP14,000; and the molar ratio of the large and small dextrins was 1:11. This result supported the previous observation in Example 3.

Example 5: Analysis of the small dextrin after BE treatment The ethanol-soluble fraction of the BE treated potato amylose (18 hours) in the Example 3 was divided into some portions of equal volume and then air-dried using a centrifugal vacuum- evaporator.

A portion of the ethanol-soluble fraction was labeled and subjected to GPC with system B. Another portion was treated with isoamylase (0.1 U/mg dextrin) at pH 3.5, 50°C for 2.5 hours. After terminating the reaction by heating, the reaction mixture was dried with a CVE. The hydrolyzate was labeled and subjected to GPC with system B. The dextrin in the ethanol- soluble fraction showed a size distribution from DP 5 to about 20. The most dominant dextrin was DP 9. Isoamylase treatment of the fraction did not cause any significant changes in elution profiles by both Rl and fluorescence detectors, indicating that the small dextrin consisted of linear molecules.

Example 6: Chain-length distribution of dextrin produced by BE

Potato amylose was treated with the BE for 18 hours as described in Example 3. The dextrin product was recovered by precipitation with one volume of ethanol. The precipitate was dissolved in 1N NaOH then diluted with water and pH was adjusted to 3.5 with acetate buffer and 2N HCI. Final concentration of the dextrin was 8 mg/ml in 10mM acetate buffer.

To release alpha-1,6 b ranches, the dextrin solution was treated with i soamylase (0.1 U/mg dextrin) at 50°C for 2.5 hours. After the reaction was terminated by heating for 2 minutes, the reaction mixture was a ir-dried with a CVE. The d ried d extrin was s ubjected to a nalyses by HPAEC-PAD and GPC with system B.

For HPAEC-PAD analysis, the dried dextrin was dissolved in 1 N NaOH, diluted with water and then analyzed by HPAEC-PAD after filtration with a 0.22-mm filter. HPAEC-PAD revealed that the chain-length distribution of the dextrin product was from DP 3 to DP about 20. The majority of the unit chains were distributed from DP 4 to DP 13, which corresponded to -90% (by weight) of total dextrin.

The chain-length distribution obtained by GPC was consistent with the result by HPAEC-PAD. The fluorescence responses for the chains of DP 4 to 13 comprised more than 90% of total peak area, indicating that the unit chains having these lengths are dominant on a molar basis as well as on a weight basis.

Based on these results, average chain-length (CL) was estimated at -9. Therefore the number of unit chains per molecule of the large dextrin product was estimated to be -1900 by dividing its DP (17,200, see Example 3) by the average CL (9). Also, using the DP and CL, branch- linkage content in the large dextrin product was calculated to be approx. 11.

Example 7: Large dextrin produced by BE and its amyloglucosidase treatment Potato amylose (DPn 650) and synthetic amylose AS-110 (DPn 521) were treated by the BE (55.6 U/mg amylose) at 60°C for 6 hours. Un-reacted amylose was precipitated by addition of 1/10 volume of 1-butanol followed by incubation for 3 hours at 4°C. After centrifugation, the supernatant was mixed with 1 volume of ethanol and left overnight at -20°C. The precipitate was collected by centrifugation and subjected to fluorescent labeling. After washing to remove excess reagents with 75% ethanol, a portion of the precipitate was dissolved in 50% DMSO and subjected to GPC with system B. Another portion was dissolved in 50 mM acetate buffer pH 4.5 then treated with glucoamylase (0.2 U/mg dextrin) at 40 °C for 16 hours. After terminating the reaction by heating, the reaction mixture was filtrated with a membrane with pore size of 0.22 mm then subjected to GPC with system B.

The estimated size of the dextrin product from potato amylose and synthetic amylose was DP 8,550 and DP 21 ,700, respectively. After glucoamylase treatment, Rl peak for the large dextrin disappeared and only a trace Rl-response was observed around 55-65 min. No difference in the trace Rl-response after glucoamylase treatment was observed between the amylose used as substrate and the large dextrin product by the BE, indicating that the action of the BE on amylose did not produce dextrins that are resistant to glucoamylase. The results mean that the large dextrin product does not contain cyclic structures since such cyclic molecules are resistant to the hydrolytic action of glucoamylase.

Example 8: Dextrin from potato amylose by potato branching enzyme

Potato branching enzyme was purified from potato tuber as following: 1 kg of diced potato was homogenized with 1 liter of buffer K (50mM Tris-acetate, 10mM EDTA, 30% glycerol, 2.5mM dithiothreitol, 1mM phenylmethanelsulfonyl fluoride, pH7.5) using electric mixer. After cloth filtration, the soluble part was collected by centrifugation at 9000 rpm at 4°C for 15 minutes. Ammonium sulfate was added to the supernatant at 70% saturation, and precipitated protein was harvested by centrifugation at 9000 rpm at 4°C for 15 minutes. The precipitation was dissolved in 200ml of the buffer K and dialyzed with 15 times volume of the same buffer at 4°C for 4 hours, 8 hours and 3.5 hours by refreshing the buffer. Undissolved particle was removed by ultracentrifuge (33,500 rpm, 4°C for 1 hours) and grass-filtration at 4°C. The solution was subjected to DEAE Sepharose FF (Pharmacia) equilibrated with the buffer K and eluted with the gradient of 0-0.5 M KCI to be fractionated. The fractions with BE activity were collected and subjected to amylose bound Sepharose 6B column with the buffer K at 4°C then bound BE was eluted with the same buffer at 25°C. As the result, BE was purified with over 90% purity on SDS-gel electrophoresis and free from α-amylase activity. BE activity was determined by branching linkage assay, where 1 unit is defined as the enzyme amount necessary to form one μmol of branching linkage per minute (Carbohydr.Res.240 (1993) 253-263).

The purified potato amylose was fluorescense labeled as described in Example 3. The labeled amylose was precipitated with 1/10 volume of 1-butanol and 20 mM NaCl with the incubation at 4°C over one hour. The obtained labeled amylose was washed with 10% 1-butanol containing 6 mM NaCl by centrifuge (2500 rpm, 4°C, 5 minutes) three times. It was once again dissolved in water and precipitation and washing with 1-butanol was repeated two times. The labeled amylose was dissolved in water and MOPS buffer to be pH 7.5 then treated with potato BE (10 mU/mg amylose, BL unit) at 30°C for 6 hours. After terminating the reaction by boiling for 2 minutes, the reaction mixture was filtrated and subjected to GCP with system B. For comparison, the substrate without BE treatment was also analyzed with the same system.

As the results the used amylose showed the peak at DP 900 with the fluorescence response, while the dextrin after potato BE treatment had the distribution with the peaks at DP 1190 and DP 30. The result suggests that, unlike bacterial BE, potato BE does not produce large molecule dextrin of its size 10-15 times of the substrate.

Deposit of Biological Material

An E. coli clone containing the BE gene from Rhodothermus obamensis inserted into plasmid pT7Blue (see Example 1 ) has been deposited under the terms of the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, and given the following accession number:

Deposit Accession Number Date of Deposit

NN049443 DSM 12607 1998-Dec-23

The deposit was made by Novo Nordisk A/S and was later assigned to Novozymes A/S. The strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

1. A dextrin having an average degree of polymerization (DP) of at least about 4,000; which is essentially free of cyclic structures, or which is at least 90% degradable by treatment with a glucoamulase (EC 3.2.1.3).

2. A dextrin having an average degree of polymerization (DP) of at least about 4,000; which is essentially free of cyclic structures, or which is at least 90%) degradable by treatment with a glucoamulase (EC 3.2.1.3), said dextrin being obtainable by treatment of a composition comprising amylose or amylopectin with a branching enzyme (EC 2.4.1.18).

3. A dextrin having an average degree of polymerization (DP) of at least about 4,000; which is essentially free of cyclic structures, or which is at least 90% degradable by treatment with a glucoamulase (EC 3.2.1.3), and which is produced by treatment of a composition comprising amylose or amylopectin with a branching enzyme (EC 2.4.1.18).

4. The dextrin of any preceeding claim, which has a DP of at least about 5,000; or 6,000; or even 7,000; more preferably 8,000; or 9,000; even more preferably 10,000; or 11 ,000; and most preferably 13,000; or 15,000.

5. The dextrin of any preceeding claim, which is at least 95% degradable by treatment with a glucoamulase, more preferably 97%, and most preferably at least 99% degradable by treatment with a glucoamulase.

6. The dextrin of any of claims 2 - 5, wherein the composition comprising amylose or amylopectin comprises potato amylose, or purified amylose, or a mixture of the two.

7. The dextrin of any of claims 2 - 6, wherein the branching enzyme is of microbial origin.

8. The dextrin of claim 7, wherein the branching enzyme is thermophilic, preferably the branching enzyme has a temperature optimum of at least 60°C.

9. The dextrin of claim 7 or 8, wherein the branching enzyme is derived from a Rhodothermus species, preferably from a R. obamensis or R. marinus.

10. The dextrin of any of claims 7 - 9, wherein the branching enzyme has an amino acid sequence which is a) at least 65%) identical with the mature amino acid sequence of SEQ ID NO:2 shown in WO 00/58445; b) at least 65% identical with the mature branching enzyme amino acid sequence encoded by the nucleic acid sequence contained in plasmid pT7Blue contained in the £. coli DH12S, deposited as DSM 12607; or c) which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) the nucleotides of the nucleic acid sequence of SEQ ID NO:1 shown in WO 00/58445, corresponding to the mature enzyme, (ii) the cDNA sequence of SEQ ID NO:1 shown in WO 00/58445, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii).

11. The dextrin of any of claims 2 - 6, wherein the branching enzyme is a variant, a shuffled, a synthetic, or a hybrid enzyme derived from one or more branching enzymes of microbial origin.

12. The dextrin of claim 11 , wherein the branching enzyme is thermophilic, preferably the branching enzyme has a temperature optimum of at least 60°C.

13. A method of producing a dextrin as defined in any of the preceeding claims, comprising a step of treating a composition comprising amylose or amylopectin with a branching enzyme

(EC 2.4.1.18), whereby the dextrin is produced.

14. The method of claim 13, wherein the branching enzyme is of microbial origin.

15. The method of claim 14, wherein the branching enzyme is thermophilic, preferably the branching enzyme has a temperature optimum of at least 60°C.

16. The method of claim 14 or 15, wherein the branching enzyme is derived from a Rhodothermus species, preferably from a R. obamensis or R. marinus.

17. The method of any of claims 13 - 16, wherein the branching enzyme has an amino acid sequence which is a) at least 65% identical with the mature amino acid sequence of SEQ ID NO:2 shown in WO 00/58445; b) at least 65% identical with the mature branching enzyme amino acid sequence encoded by the nucleic acid sequence contained in plasmid pT7Blue contained in the E. coli DH12S, deposited as DSM 12607; or c) which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) the nucleotides of the nucleic acid sequence of SEQ ID NO:1 shown in WO 00/58445, corresponding to the mature enzyme, (ii) the cDNA sequence of SEQ ID NO:1 shown in WO 00/58445, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii).

18. The method of claim 13, wherein the branching enzyme is a variant, a shuffled, a synthetic, or a hybrid enzyme derived from one or more branching enzymes of microbial origin.

19. The method of claim 18, wherein the branching enzyme is thermophilic, preferably the branching enzyme has a temperature optimum of at least 60°C.

20. The method of claims 13 - 19, wherein the dextrin is subsequently purified or isolated in one or more steps.

21. Use of a dextrin, as defined in any of claims 1 - 12, or produced by a method as defined in any of claims 13 - 20, as a medical device or part of a medical device, in the formulation of a pharmaceutical product, in adhesive compositions including surface sizing and coating in the paper industry, as food and drink additives, or as anti-starch retrogradation agents.