DE19846635A1 - New nucleic acid encoding a branching enzyme, useful for in vitro synthesis of branched glucans and to prepare transgenic plants producing modified starch - Google Patents

Abstract

A nucleic acid (I) isolated from Neisseria encodes a branching enzyme (II). (I) has a fully defined 2475 bp sequence (given in the specification and encodes; (i) a protein with a fully defined 762 amino acid sequence (given in the specification); or (ii) a protein encoded by the insert in plasmid DSM 12425. Independent claims are also included for the following: (1) a vector containing (I); (2) host cells transformed with (I) or the vector in (1); (3) production of (II) by in vitro transcription and translation of (I); (4) antibodies (Ab) against (II); (5) transgenic plant cell containing (I), linked to regulatory sequences functional in plants; (6) transgenic plants containing the cells of (5) and their harvestable parts; (7) production of the plants of (h); (8) native regulatory region (RR) that controls transcription of (I) in bacteria; and in vitro production of alpha -1,6-branched alpha -1,4-glucans (III) using sucrose as substrate and a combination of an amylosucrase and a branching enzyme (BE).

Description

The present invention relates to in-vitro methods for producing α-1,6-branched α- 1,4-glucans based on sucrose and an enzyme combination of one Amylosucrase and a branching enzyme and nucleic acid molecules coding Branching enzyme from bacteria of the genus Neisseria. The invention further relates to the glucans obtainable by the process described.

α-1,6 branched α-1,4-glucans are of extraordinary interest in various ways, since they are used, for example, for the manufacture of products in the pharmaceutical sector and cosmetic industry. For example, they can be used as binders for tablets Carriers for active pharmaceutical ingredients, as packaging material, as carriers for Powder additives, as a UV-absorbing additive in sunscreen creams and as Carrier material of aromas or fragrances can be used.

α-1,6-branched α-1,4-glucans occur mainly as amylopectin in the plant kingdom a component of strength before. These branched glucans occur in the animal kingdom and in bacteria mainly in the form of glycogen.

The polysaccharide starch is made up of chemically uniform building blocks, the Glucose molecules, built up, however, represents a complex mixture of different Molecular forms represent the differences in the polymerization and the Have degrees of branching and thus their physicochemical properties differ greatly from each other. One differentiates between amylose strength, an im essential unbranched polymer from α-1,4-glycosidically linked glucose units, and the amylopectin starch, a branched polymer in which the branches through the Additional α-1,6-glycosidic linkages occur. To Textbook information (Voet and Voet, Biochemistry, John Wiley & Sons, 1990) occur the α-1,6- Branches average every 24 to 30 glucose residues. This corresponds to one Degree of branching of approx. 3% -4%. The information on the degree of branching is variable and depending on the origin (e.g. plant species, plant variety, etc.) of the respective starch. In The amount of amylose typical of plants used in industrial starch production varies in total starch content between 10 and 25%. For the production of α-1,6-branched α-1,4-  Glucans of different degrees of branching have already been found described various approaches involving the use of (transgenic) plants. For example, heterologous expression of a bacterial glycogen synthase leads to Potato plants to slightly lower the amylose content, to increase the Degree of branching and a change in the branching pattern of amylopectin in Comparison to wild-type plants (Shewmaker et al., Plant. Physiol. 104 (1994), 1159-1166).

It was also observed that heterologous expression of the branching enzyme from E. coli (glgB) in amylose-free potato mutants (amf) (Jacobsen et al., Euphytica 44 (1989), 43-48) led to amylopectin molecules with 25% more branching points (Kortstee et al., Plant J. 10 (1996), 83-90) as the control (amf). Isolation of those generated in transgenic plants Different glucans need different degrees of branching to remove z. B. the Amylose component of additional cleaning steps, which are very complex and therefore time and are expensive. Furthermore, a targeted adjustment of the degree of branching with such Approaches not possible. Furthermore, such in vivo methods have fluctuating Test conditions (environmental factors, location) a high variability in terms of Product quality.

Glycogen has a higher degree of branching than amylopectin. This too Polysaccharide contains α-1,6-branched α-1,4-glucans. Glycogen is different from starch also in the average side chain length and in the degree of polymerization. Contains glycogen according to textbook information (Voet and Voet, Biochemistry, John Wiley & Sons, 1990) an α-1,6 branch point on average every 8 to 12 glucose residues. This matches with a degree of branching of approximately 8% to 12%. For the molecular weight of glycogen various data ranging from 1 million to over 1,000 million (D.J. Manners in: Advances in Carbohydrate Chemistry, Ed. M. L. Wolfrom, Academic Press, New York (1957), 261-298; Geddes et al., Carbohydr. Res. 261 (1994), 79-89). This information too are strongly dependent on the respective original organism, its nutritional status and on the type of isolation of the glycogen. Glycogen is usually made from mussels (e.g. Mytillus edulis), obtained from mammalian liver or muscles (e.g. rabbits, rats) (Bell et al., Biochem. J. 28 (1934), 882; Bueding and Orrell, J. Biol. Chem. 236 (1961), 2854). This does Manufacturing on an industrial scale is time and cost intensive.

The naturally occurring α-1,6-branched α-1,4-glucans, starch and Glycogen, behave very depending on their proportion of 1,6-glycosidic branches differently. This applies u. a. in terms of their solubility, transparency, enzymatic  Hydrolysis, rheology, gelling and retrogradation properties. Such Fluctuations in properties, however, are not for many industrial applications tolerable.

An alternative to the production of α-1,6-branched α-1,4-glucans from plants or animal organisms are in vitro approaches. These stand out from the in-vivo Processes generally characterized by better controllability and higher reproducibility, because the reaction conditions in vitro, as opposed to the conditions in a living Organism, can be set defined. This usually enables production of consistent products of great uniformity and purity and thus of great Quality that is of great importance for further industrial use. The workup of products of consistent quality leads to cost reductions because the Process parameters that are required for the processing, not for everyone Processing approach need to be re-optimized. Another benefit of certain in vitro The process is that the products are free of organisms by the in vivo process be used. For certain applications in the food industry and the This is urgently required in the pharmaceutical industry.

The in vitro methods can generally be divided into two different groups.

In one group of methods, various substrates, such as. B. amylose, Amylopectin and glycogen, exposed to the activity of a branching enzyme.

For example, Borovsky et al. (Eur. J. Biochem. 59 (1975), 615-625) show that the use of the branching enzyme from potatoes in connection with the substrate amylose to products leads, which are similar to amylopectin, but differ structurally from this.

Boyer and Preiss (Biochemistry 16 (1977), 3693-3699) also showed that purified Branching enzyme (α-1,4-glucan: α-1,4-glucan 6-glycosyltransferase) from E. coli was used can be used to increase the degree of branching of amylose or amylopectin.

However, if glycogen from E. coli or rabbit liver is used with the branching enzyme from E. incubated coli, only a slight increase in the degree of branching is achieved (Boyer and Preiss, a. a. O.).

Rumbak et al. (J. Bacteriol. 173 (1991), 6732-6741) were able to determine the degree of branching of Amylose, amylopectin and glycogen can be increased by adding these substrates with the Branching enzyme from Butyrivibrio fibrisolvens.

A similar approach was developed by Okada et al. (U.S. Patent No. 4,454,161) to the To improve the properties of starch-containing foods. Thereby substances  such as amylose, amylopectin, starch or dextrin together with one Branching enzyme incubated. This had beneficial effects on the durability of Food containing the appropriately modified substances. Also describes patent application EP-A1 0 690 170 the reaction of gelled starch in aqueous solution Use of a branching enzyme. This leads to strengths with advantageous properties in papermaking.

The in vitro methods described above, however, have the disadvantage that already due to the fluctuating degree of branching of the starting materials (e.g. starch, amylopectin, etc.) it is not possible to manufacture uniform products. Furthermore, targeted control of the Degree of branching is not possible and the substrates used are also relatively expensive.

The other group of in vitro methods includes de novo synthesis of α-1,6-branched α- 1,4-glucans starting from various substrates (glucose-1-phosphate, ADP-glucose, UDP-glucose) using an enzyme combination consisting of a 1,4- Glucan chain-forming enzyme (phosphorylase, starch synthase, glycogen synthase) and one Branching enzyme.

Illingwort et al. (Proc. Nat. Acad. Sci. USA 47 (1961), 469-478) for an in vitro Method using a phosphorylase a from muscles (organism unknown) in Combination with a branching enzyme (organism unknown) shows that the de novo Synthesis of glycogen-like molecules using the substrate glucose-1-phosphate is possible. Boyer and Preiss (op. Cit.) Combined the enzymatic activity of one Phosphorylase from rabbit muscles or a glycogen synthase from E. coli with one Branching enzyme from E. coli using the substrate glucose-1-phosphate or UDP-glucose and thus produced branched α-glucans. Borovsky et al. (Eur. J. Biochem. 59 (1975), 615-625) examined the de novo synthesis of α-1,6-branched α-1,4-glucans from glucose-1-phosphate using a branching enzyme Potato in combination with a phosphorylase (1,4-α-D-glucan: orthophosphate α-glycosyltransferase [EC 2.4.1.1]) from maize. Doi (Biochimica et Biophysica Acta 184 (1969), 477-485) showed that the enzyme combination of a starch synthase (ADP-D-glucose: α- 1,4-glucan α-4-glucosyltransferase) from spinach and a branching enzyme from potato leads to amylopectin-like products using the substrate ADP-glucose. Parodi et al. (Arch. Biochem. Biophys. 132 (1969), 11-117) used for the de novo synthesis  branched glucans from UDP-glucose combined a glycogen synthase from rat liver with a rat liver branching enzyme. They received a polymer that was native Glycogen is similar and that of the polymers based on glucose-1-phosphate differs.

This second group of in vitro methods also has the disadvantage that the substrates, e.g. B. Glucose-1-phosphate, UDP-glucose and ADP-glucose, are very expensive. Also appears a targeted control of the degree of branching is not possible here.

Büttcher et al. (J. Bacteriol. 179 (1997), 3324-3330) describe an in vitro method for Preparation of water-insoluble α-1,4-glucans using an amylosucrase and Sucrose as a substrate. However, only linear α-1,4-glucans are used here Branches synthesized.

The object of the present invention is therefore to provide a method represent the cost-effective production of α-1,6-branched α-1,4-glucans for enables industrial purposes, as well as to provide nucleic acid molecules that are in such Enzymes that can be used in methods, in particular branching enzymes, are encoded.

This object is achieved by the embodiments described in the claims.

The invention thus relates to an in vitro method for producing α-1,6-branched α-1,4- Glucans using the substrate sucrose and an enzyme combination of one Amylosucrase and a branching enzyme. An "in vitro method" is used in Within the scope of the present invention an implementation, i. H. understood a reaction that takes place outside of a living organism. "In vitro" means in particular that the The method according to the invention takes place in a reaction vessel. Particularly preferably "in vitro "that the reaction takes place in the absence of living cells.

An advantage of a method according to the invention is that it is possible to increase the degree of branching control and that by this control the properties of the synthesized glucan to the each planned use of the glucan can be adapted. So there is possibly in the field of use as encapsulation material in the pharmaceutical Area the possibility of a specific setting of the degree of branching Achieve optimization of drug release rate.  

In connection with the present invention, an amylosucrase (sucrose: 1,4-α-D-glucan 4-α-glucosyltransferase, EC 2.4.1.4.) Is understood to mean an enzyme which converts sucrose to water-insoluble α-1,4 -Glucans and fructose catalyzed. The following reaction scheme is proposed for this enzyme:

Sucrose (α-1,4-D-glucan) _n → D-fructose + (α-1,4-D-glucan) _{n + 1}

It is a transglycosylation reaction. The products of this reaction are water-insoluble α-1,4-glucans and fructose. The transglucosylation can be in the absence or take place in the presence of acceptor molecules. Such acceptor molecules can e.g. B. Polysaccharides such as e.g. B. maltooligosaccharides, dextrin or glycogen. If such If the acceptor molecule is a linear, oligomeric α-1,4-glucan, the resulting product is the Transglucosylation reaction by the amylosucrase a polymeric linear α-1,4-glucan. If the transglucosylation reaction by means of the amylosucrase in the absence of any Acceptor molecules is carried out, a glucan with a terminal fructose molecule receive. In the context of the present invention, all are by means of an amylosucrase, in Absence or presence of acceptor molecules, products available as α-1,4- Called glucans.

The following reaction scheme is proposed for the reaction mechanism of a transglucosylation using an amylosucrase in the absence of an acceptor molecule:

G-F + n (GF) → G _n -G-F + nF,

where GF is sucrose, G is glucose, F is fructose and G _n -GF is an α-1,4-glucan. The following reaction scheme is proposed for the reaction mechanism of transglucosylation using amylosucrase in the presence of an acceptor molecule:

mG-F + G _n → G _nm + mF,

where Q is a polysaccharide acceptor molecule, G _{nm is} a polysaccharide consisting of acceptor plus synthesized α-1,4-glucan chain, GF is sucrose, F is fructose and G is glucose.

No cofactors are required for transglucosylation by an amylosucrase. In principle, all amylosucrases are required to carry out the process according to the invention suitable that catalyze the synthesis of linear α-1,4-glucans starting from sucrose.  

Amylosucrases have hitherto been known from some types of bacteria, in particular mainly from Neisseria species (MacKenzie et al., Can. J. Microbiol. 24 (1978), 357-362). An amylosucrase of prokaryotic origin is therefore preferably used. Amylosucrases are known, for example, from Neisseria perflava (Okada and Hehre, J. Biol. Chem. 249 (1974), 126-135; MacKenzie et al., Can. J. Microbiol. 23 (1977), 1303-1307) or Neisseria canis, Neisseria cinerea, Neisseria denitrificans, Neisseria sicca and Neisseria subflava (MacKenzie et al., Can. J. Microbiol. 24 (1978, 357-362). Furthermore, WO 95/31553 an amylosucrase from Neisseria polysaccharea. One is particularly preferred amylosucrase naturally secreted by a prokaryote.

In a preferred embodiment of the method according to the invention, a Amylosucrase from Neisseria polysaccharea is used.

The enzyme, which is expressed in Neisseria polysaccharea, is extremely stable and binds very tightly the polymerization products and is competitively inhibited by the reaction product fructose (MacKenzie et al., Can. J. Microbiol. 23 (1977) 1303-1307). In the Neisseria species Neisseria polysaccharea, the amylosucrase is secreted (Riou et al., Can. J. Microbiol. 32 (1986), 909- 911), whereas with other Neisseria species it remains in the cell. Most notably an amylosucrase is preferred with the in Seq ID No. 5 given amino acid sequence used.

In a further preferred embodiment, a purified amylosucrase is used. A purified amylosucrase is understood to mean an enzyme that is largely free of cell components of the cells in which the protein is synthesized. Preferably means the term "purified amylosucrase" means an amylosucrase that has a purity of at least 70%, preferably at least 85% and particularly preferably at least 90% having.

The use of a purified protein for the production of α-1,4-glucans offers various Benefits. Compared to processes that use partially purified protein extracts, the reaction medium of the process according to the invention contains no residues of Production strain (microorganism) that is used to purify the protein or to manufacture genetically.

Furthermore, the use of the purified protein offers advantages for use in the Food and pharmaceutical industries to see. By the defined and unnecessary of all  Ingredient-free composition of the reaction medium is also the product in its components more precisely. This leads to a much less extensive one Approval procedures for these biotechnologically produced products in the food and Pharmaceutical industry, especially because these products have no traces of a transgenic Microorganism should have.

In the context of the present invention, under a branching enzyme (α-1,4-glucan: α-1,4-glucan 6-glycosyltransferase, E.C. 2.4.1.18) understood a protein that a Transglycosylation reaction catalyzed in the α-1,4 linkages of an α-1,4-glucan donor hydrolyzed and the thereby released α-1,4-glucan chains onto an α-1,4-glucan acceptor chain transferred and thereby converted into α-1,6 links.

In principle, all branching enzymes are used to carry out the method according to the invention of any origin (bacterial, fungal, vegetable, animal) suitable (see e.g. Baba et al., Biochem. Biophys. Res. Commun. 181: 87-94 (1991); Kossmann et al., Mol. Gen. Genet. 203: 237-244 (1991); Nakamura and Yamanouchi, Plant Physiol. 99: 1265-1266 (1992); Baker et al., 1986, J. Biol. Chem. 261: 8738-8743; Kiel et al., Gene (1989), 9-17, etc.).

The isolation of appropriate genes is known to those skilled in the art using molecular biological Standard methods possible, such as u. a. in Sambrook et al. (Sambrook et al., Molecular cloning: A laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, NY, USA (1989)) described.

In a preferred embodiment of the invention, it is a Branching enzyme from a prokaryote, preferably from a bacterium of the genus Neisseria and particularly preferably from Neisseria denitrificans and very particularly preferably around a branching enzyme according to the invention as described below. In particular preferred is a branching enzyme with the in Seq ID No. 1 amino acid sequence shown.

In a further preferred embodiment, it is a cleaned one Branching enzyme. Under a purified branching enzyme, an enzyme is created understood that is largely free of cell components of the cells in which the protein is synthesized. Preferably, "purified branching enzyme" means that the enzyme a degree of purity of at least 70%, preferably of at least 85% and particularly preferably has at least 90%.  

Furthermore, recombinantly produced are preferred in the inventive method Proteins used. In the context of the present invention, these include proteins understood, which were produced in that a DNA coding for the respective protein is introduced into a host cell and expressed there. The protein can then subsequently obtained from the host cell and / or from the culture medium. The The host cell is preferably a bacterium or a protist (e.g. fungi, especially yeasts, Algae) such as B. defined in Schlegel "General microbiology" (Georg Thieme Verlag, 1985, 1-2). The proteins are particularly preferably secreted by the host cell. The production Such host cells for the production of a recombinant protein can according to the expert known methods.

An overview of various expression systems can be found e.g. B. in Methods in Enzymology 153: 385-516 (1987), Bitter et al. (Methods in Enzymology 153 (1987), 516-544), Sawers et al., Applied Microbiology and Biotechnology 46 (1996), 1-9, Billman-Jacobe, Current Opinion in Biotechnology 7 (1996), 500-504, Hockney, Trends in Biotechnology 12 (1994), 456- 463, and Griffiths et al., Methods in Molecular Biology 75 (1997), 427-440. Expression vectors are widely described in the literature. They contain alongside a selection marker gene and one which ensures replication in the selected host The origin of replication is usually a bacterial or viral promoter, as well as usually one Termination signal for transcription. Located between promoter and termination signal there is at least one restriction site or a polylinker that allows the insertion of a codie DNA sequence. As a promoter sequence, provided that it is in the selected Host organism is active, which is naturally the transcription of the corresponding gene controlling DNA sequence can be used. This sequence can also be used against others Promoter sequences are exchanged. Both promoters can be used which cause a constitutive expression of the gene, as well as inducible promoters which a allow targeted regulation of the expression of the downstream gene. Bacterial and viral Promoter sequences with these properties are described in detail in the literature. Regulatory sequences for expression in microorganisms (e.g. E. coli, S. cerevisiae) are adequately described in the literature. Promoters that have a particularly strong expression of allow downstream genes are z. B. the T7 promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89), lacuv5, trp, trp-lacUV5 (DeBoer et al., In Rodriguez and Chamberlin (Eds), Promotors, Structure and Function; Praeger, New York, (1982), 462-481;  DeBoer et al. Proc. Natl. Acad. Sci. USA (1983), 21-25), λp1, rac (Boros et al., Gene 42 (1986), 97-100). As a rule, the protein amounts reach from the middle to the end of the logarithmic phase of the growth cycle of the microorganisms culminated. For Synthesis of proteins are therefore preferably used inducible promoters. This often lead to higher yields of protein than constitutive promoters. The usage strong constitutive promoters leads to the constant transcription and translation of one often cloned genes to lose energy for other essential cell functions and this slows down cell growth (Bernard R. Glick / Jack J. Pasternak, Molecular Biotechnology (1995), Spektrum Akademischer Verlag GmbH, Heidelberg Berlin Oxford, S. 342.). In order to achieve an optimal amount of protein, a two-step process is often used applied. First, the host cells are raised to a relatively high under optimal conditions cell density cultivated. In the second step, the transcription is then depending on the type of used promoter induced. A through is particularly suitable in this context Lactose or IPTG (= isopropyl-β-D-thiogalactopyranoside) inducible tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1983). Termination signals for transcription are also described in the literature.

The transformation of the host cell with the DNA encoding a corresponding protein can generally be carried out according to standard methods, such as, for. B. described in Sambrook et al. (Molecular Cloning: A Laboratory Course Manual, ^2nd edition (1989), Cold Spring Harbor Press, New York). The host cell is cultivated in nutrient media which meet the needs of the host cell used, in particular taking into account pH, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements, etc. The enzyme produced by the host cells can be cleaned using conventional cleaning methods such as precipitation, ion exchange chromatography, affinity chromatography, gel filtration, HPLC reverse phase chromatography, etc.

By modifying the DNA expressed in the host cells, one can get into the host cell Produce polypeptide that, due to certain properties, is easier to remove from the culture medium can be isolated. So there is the possibility of the protein to be expressed as To express fusion protein with another polypeptide sequence, the specific Binding properties the isolation of the fusion protein via affinity chromatography enable (e.g. Hopp et al., Bio / Technology 6 (1988), 1204-1210; Sassenfeld, Trends Biotechnol. 8: 88-93 (1990).  

In a preferred embodiment of the method according to the invention Enzymes used that were produced recombinantly and from the host cell into the nutrient medium dium was secreted

the so that no disruption of cells and no further purification of the Protein is required because the secreted protein can be obtained from the supernatant. To remove residual components of the culture medium, use chemical engineering common methods, such as B. dialysis, reverse osmosis, chromatographic methods etc. be used. The same applies to the concentration of the in the culture medium secreted protein. The secretion of proteins by microorganisms is normal mediated by N-terminal signal peptides (signal sequence, leader peptide). Proteins with this Signal sequences can penetrate the cell membrane of the microorganism. A secretion from Proteins can be achieved by the DNA sequence encoding this signal peptide is added to the corresponding region coding for the enzyme.

An optionally naturally present signal peptide is preferred, for example that the amylosucrase from Neisseria polysaccharea.

The signal peptide of the α-CGTase from Klebstella oxytoca M5A1 is very particularly preferred (Fiedler et al., J. Mol. Biol. 256 (1996), 279-291) or a signal peptide as described by the Nucleotides 11529-11618 of the GenBank accessible under accession number X86014 Sequence is encoded.

Alternatively, the enzymes used in the method according to the invention can also be found under Use of an in vitro transcription and translation system which is used to express the Proteins leads to have been produced without the use of microorganisms.

In a further preferred embodiment, the amylosucrase and / or that Branching enzyme immobilized on a carrier material.

Immobilizing the enzymes offers the advantage that the enzymes act as catalysts for the Synthesis reaction easily recovered from the reaction mixture and several times can be used. Since the purification of enzymes is usually cost and is time consuming, immobilization and recycling enables a significant amount Cost cutting. Another benefit is the purity of the reaction products, which are none Contains protein residues.

A large number of carrier materials are available for the immobilization of proteins Available, the coupling to the carrier material via covalent or non-covalent bin can be done (for an overview see: Methods in Enzymology 135, 136, 137). Vastness  Spread as a carrier material have z. B. agarose, algae, cellulose, polyacrylamide, Silica or nylon.

In a further preferred embodiment of the method, a (partially purified) Enzyme raw extract of an amylosucrase and / or a branching enzyme is used. Under In this context, a crude extract is an amylosucrase and / or Branching enzyme preparation with an as opposed to a purified enzyme (see for example Examples 5 and 6) understood reduced degree of purity.

In a preferred embodiment, the Change in the degree of branching of the α-1,6-branched α-1,4-glucans by the Change in protein activity ratio from branching enzyme to amylosucrase reached. Under the protein activity ratio, the ratio of those used Protein activities (u) understood from amylosucrase to branching enzyme. The determination the protein activities can be carried out as described in Examples 7 and 8. In the The process according to the invention (see Example 9) can be carried out Protein activity ratio (units amylosucrase / units branching enzyme) in the range of 1/4000 to 2000/1 can be used.

In a preferred embodiment, the protein activity ratio is in a range from 1/1500 to 1500/1.

In a further preferred embodiment, the protein activity ratio is in one Range from 1/800 to 1300/1.

In a particularly preferred embodiment, the protein activity ratio reads in one Range from 1/400 to 1200/1.

About the change in the protein-activity ratio is the change in Degree of branching of the α-1,6-branched α-1,4-glucans obtained from 0.05% to 35% possible.

In a preferred embodiment, the change in the degree of branching is α-1,6-branched α-1,4-glucans obtained in the 6-position from 0.15% to 25% possible, in particular from 0.20% to 15% and very particularly preferably from 0.25% to 12%.

With the aid of the method according to the invention, it is in particular possible to produce products which have a higher degree of branching than glycogen.  

Under the degree of branching in the context of the present invention average proportion of branches in O-6 position compared to all different linked glucose units understood, which are determined by methylation analysis can (see example 10).

In a further preferred embodiment, the Change in the molecular weight of the product by changing the Protein activity ratio of branching enzyme to amylosucrase reached. Here it is especially possible that the protein activity ratio during the reaction leading to Synthesis of α-1,6-branched α-1,4-glucans leads, is changed.

In a further preferred embodiment of the method according to the invention provided that the process be carried out at different sucrose concentrations becomes. In principle, it is possible to carry out in a concentration range of preferably 1% to 80% sucrose (w / v), preferably in a range from 5% to 50% and particularly preferably in a range from 10% to 40%.

In the context of the present invention, the molecular weight is determined by Light scattering experiments (Light Scattering from Polymer solutions, editor: Huglin, M. B., Academic Press, London, 1972) according to Berry (J. Chem. Phys. 44 (1966), 4550ff).

With the aid of the method according to the invention, it is in particular possible to adjust the molecular weight of the α-1,6-branched α-1,4-glucans produced by means of the method in the range from 1000 to 3000 × 10 ⁶ . The α-1,6-branched α-1,4-glucans produced preferably have a molecular weight in the range from 100,000 to 1500 × 10 ⁶ , in particular in the range from 100,000 to 1000 × 10 ⁶ , preferably in the range from 262,000 to 1000 × 10 ⁶ and particularly preferably in the range from 262,000 to 499 × 10 ⁶ .

The invention further relates to α-1,6-branched α-1,4-glucans, which are described by the above Methods of the invention are available. These α-1,6-branched α-1,4-glucans have a degree of branching that is above the degree of branching that is reached, if only the activity of an amylosucrase is used and which is at most 25 mol%.

In a preferred embodiment of the invention, α-1,6-branched α-  1,4-glucans with a degree of branching of 0.05% to 20%, preferably in the range of 0.15% to 17%, in particular in the range from 0.2% to 15%, particularly preferably in the range from 0.25% to 13%, and very particularly preferably in the range from 0.3% to 12%. In a In another preferred embodiment, the degree of branching is in the range from 0.35% to 11%, and particularly in the range of 0.4% to 10.5%.

The α-1,6-branched α-1,4-glucans according to the invention can, for. B. in the field of textile, the paper, cosmetics, food and pharmaceutical industries. In general, the use of the glucans according to the invention can be carried out in two Structure areas of application:

1. Food industry

The glucans according to the invention can be similar to starch in the range of Food industry are used.

Starch is a classic additive for many foods in which they are used takes on the essential function of binding aqueous additives or a Increasing the viscosity or an increased gel formation. Important Characteristic features are the flow and sorption behavior, the swelling and ver paste temperature, viscosity and thickening performance, solubility of starch, the transparency and paste structure, the heat, shear and acid stability, the inclination for retrogradation, the ability to form films, the freeze / thaw stability, the Digestibility and the ability to form complexes with e.g. B. inorganic or organic ions.

2. Non-food industry

The glucans according to the invention can also be used in the non-food sector like the classic polysaccharide starch. That is, in this big For example, the glucans can be used as an auxiliary for different areas Manufacturing processes or used as an additive in technical products. In the The use of glucans as an auxiliary here is in particular paper and Cardboard industry to name. Starch is primarily used here for retardation (Retention of solids), the setting of filler and fine particles, as Firming substance and for drainage. It also has favorable properties  the strength in terms of rigidity, hardness, sound, grip, gloss, the Smoothness, the splitting strength and the surfaces exploited. Just like strength, can glucans according to the invention are used here.

2.1 Paper and cardboard industry

There are four areas of application within the paper making process, namely Differentiate between surface, line, mass and spray.

The requirements for the glucans in relation to the surface treatment are in the essentially a high degree of whiteness, an adapted viscosity, a high Viscosity stability, good film formation and low dust formation. In the Use in the stroke plays the solid content, an adjusted viscosity, a high Binding ability and high pigment affinity play an important role. As an addition to Mass is a quick, even, lossless distribution, high mechanical Stability and complete restraint in the paper flow are important. At the Use of the glucans in the spray area is also an adapted solids content, high Viscosity and a high binding capacity are important.

2.2 Adhesives industry

The glucans according to the invention can optionally also be used in the adhesive industry instead of replacing strength.

A large area of use of starch is in the adhesive industry, where the Possible uses are divided into four sections: use as a pure one Starch glue, the use in starch glues prepared with special chemicals, the use of starch as an additive to synthetic resins and polymer dispersions and the use of starches as extenders for synthetic adhesives. 90% of Starch-based adhesives are used in the fields of corrugated cardboard manufacture and manufacture of paper bags, pouches and bags, manufacture of composite materials for paper and aluminum, production of cardboard and rewetting glue for letters envelopes, stamps, etc. used.

2.3 Textile and textile care products industry

A large field of application for glucans instead of starch is as an auxiliary and additive the production of textiles and textile care products. Within the  The textile industry can be divided into the following four areas of application: The application as a sizing agent, d. H. as an auxiliary for smoothing and strengthening the Velcro behavior for Protection against the tensile forces attacking during weaving and to increase the Abrasion resistance when weaving, especially as a means of textile upgrading quality-impairing pretreatments, such as bleaching, dyeing, etc., as Thickeners in the manufacture of color pastes to prevent Dye diffusion and as an additive to chaining agents for sewing threads.

2.4 Building materials industry

The fourth area of application is the use as an additive in building materials. An example is the production of plasterboard, in which the glucans mixed in the gypsum pulp with gelatinize the water, diffuses to the surface of the plasterboard and there the Can bind cardboard to the plate. Mixing is another area of application Plaster and mineral fibers. In ready-mixed concrete, glucans according to the invention can may be used to delay the setting.

2.5 Soil stabilization

Another market for glucans would be in the manufacture of agents for Soil stabilization, which is used for the temporary protection of artificial earth movements Soil particles are used against water. Combination products from the According to current knowledge, glucans and polymer emulsions could be used in their erosion and incrustation-reducing effect can be equated with the previously used products, are priced but significantly below these.

2.6 Use in crop protection and fertilizers

Another area of application is in the possible use of glucans in Plant protection products for changing the specific properties of the preparations. So could use the glucans to improve the wetting of crop protection and Fertilizers, for the dosed release of the active ingredients, for the conversion of liquid, volatile and / or malodorous agents in microcrystalline, stable, moldable Substances to mix incompatible compounds and to extend the Duration of action can be used by reducing the decomposition.  

2.7 Pharmaceuticals, medicine and cosmetics industry

Another area of application is in the field of pharmaceuticals, medicine and Cosmetics industry. In the pharmaceutical industry, the invention could Glucan instead of starch as a binder for tablets or for binder dilution in Capsules are used. Furthermore, they could serve as tablet disintegrants because they absorb liquid after swallowing and swell as soon as possible should that the active ingredient is released. Medical lubricants and wound powders are based for quality reasons on strength. In the field of cosmetics, strengths become for example as a carrier of powder additives such as fragrances and salicylic acid used. A relatively large area of application for starch is toothpaste. In In this area, the glucans according to the invention could replace the starch.

2.8 Addition to coals and briquettes

One area of use is also the addition of the glucans according to the invention instead of starch Coal and briquette. Coal can be quantitatively high-quality with an addition of starch be agglomerated or briquetted, whereby an early disintegration of the briquettes ver is prevented. The added starch is between 4 and 6% for charcoal, for calorified Coal between 0.1 and 0.5%. Starches are also gaining ground as binders Significance, since their addition to coal and briquette means that harmful substances are emitted can be significantly reduced.

2.9 Ore and coal sludge processing

The glucans can also be used instead of starch in ore and coal sludge processing Flocculants are used.

2.10 Foundry auxiliary

Another area of application is as an additive to foundry additives. At different Casting processes require cores that are made from binder-mixed sands become. Bentonite is used predominantly as a binder today modified starches, mostly source starches, is offset.

The purpose of the starch addition is to increase the flow resistance and to improve it the binding strength. In addition, the source strengths can further produk technical requirements such as dispersible in cold water, rehydratable, good  miscible in sand and have high water retention capacity. Also the glucans according to the invention could replace starch here.

2.11 Use in the rubber industry

In the rubber industry, the glucans according to the invention could be used instead of starch Improvement of the technical and optical quality can be used. Reasons are thereby improving the surface gloss, improving the grip and Look, for that the glucans are sticky before cold vulcanization rubberized surfaces strewn by rubber materials, as well as improving the Printability of the rubber.

2.12 Manufacture of leather substitutes

Another sales opportunity for glucan could be in the manufacture of Leather substitutes exist.

2.13 Glucans in synthetic polymers

The following areas of application are emerging in the plastics sector: integration into the processing process (the glucans are only filler, there is no direct one) Binding between synthetic polymer and glucan) or alternatively the integration in the production of polymers (glucan and polymer form a firm bond).

The use of glucans as a pure filler is like compared to the other substances Talc is probably not competitive. It looks different if the specific Properties of Glucane come into play and thereby the property profile of End products is significantly changed. An example of this is the use of Starch products in the processing of thermoplastics, such as polyethylene. Here, the Starch and the synthetic polymer by co-expression in a ratio of 1: 1 to one "Master batch" combined from using granulated polyethylene using Various products can be manufactured using conventional process technologies. Through the integration of starch in polyethylene films can increase the permeability of hollow bodies to a improved water vapor permeability, an improved antistatic behavior, an improved Antiblock behavior and improved printability with aqueous inks can be achieved. Here the starch could be replaced by the glucans according to the invention.  

Another option is to use glucans instead of starch in buttocks polyurethane foam. With the adaptation of the glucans as well as through the process engineering It is possible to optimize the reaction between synthetic polymers and the To specifically control the hydroxyl groups of the glucan. The result is polyurethane films that pass through the use of glucans could get the following property profiles: a reduction the coefficient of thermal expansion, reducing the shrinkage behavior, improving the Pressure / tension behavior, increase in water vapor permeability without changing the Water absorption, reduction of flammability and tear density, no dripping flammable parts, freedom from halogen and reduced aging. Disadvantages currently still there is reduced compressive strength and reduced impact resistance. The product development is not limited to foils. Even fixed Plastic products such as pots, plates and bowls are with a glucan content of over 50% producible. Furthermore, glucan / polymer mixtures should be judged favorably because they have a much higher biodegradability.

Extraordinary importance could continue due to their extreme Have water-binding capacity graft polymers in which instead of starch according to the invention Glucans can be used. Starch graft polymers are products with a backbone Strength and a side rail grafted on according to the principle of the radical chain mechanism of a synthetic monomer. The starch graft polymers available today stand out with a better binding and retention capacity of up to 1000 g water per g starch high viscosity. The areas of application for these superabsorbents have changed in recent years Years have expanded significantly and are in the hygiene sector with products such as diapers and Documents as well as in the agricultural sector, e.g. B. seed pilling.

Furthermore, the present invention relates to nucleic acid molecules encoding a branching enzyme (EC 2.4.1.18) from bacteria of the genus Neisseria selected from the group consisting of

• a) Nucleic acid molecules that encode a protein that the in Seq ID No. 2 shown Includes amino acid sequence;
• b) Nucleic acid molecules that the in Seq ID No. 1 nucleotide sequence of the encoding region;
• c) nucleic acid molecules encoding a protein comprising the amino acid sequence which is encoded by the insertion of plasmid DSM 12425;  
• d) Nucleic acid molecules that are a branching enzyme from Neisseria denitrificans encoding region of the insertion of the plasmid DSM 12425;
• e) nucleic acid molecules, their complementary strand with a nucleic acid molecule hybridized according to (a), (b), (c) and / or (d) and which is a branching enzyme from a Encode bacterium of the genus Neisseria; and
• f) nucleic acid molecules, the nucleotide sequence of the sequence of a Nucleic acid molecule according to (e) due to the degeneration of the genetic code deviates.

The in Seq ID No. 1 nucleic acid sequence shown is a genomic sequence that a coding region for a branching enzyme from Neisseria denitrificans. A plasmid containing this DNA sequence was stored as DSM 12425. With the help of this sequence or of this molecule, it is now possible for the person skilled in the art to generate homologous sequences from other Neisseria Isolate species or strains. This can be done, for example, with the help of conventional Methods such as screening cDNA or genomic libraries with suitable ones Hybridization tests are carried out. The isolation of homologous sequences can also be done as in Example 1 described. In this way it is possible, for example, Identify and isolate nucleic acid molecules that correspond to those listed under Seq ID No. 1 hybridize specified sequence and which encode a branching enzyme.

In principle, the nucleic acid molecules according to the invention can be a branching enzyme encode any bacterium of the genus Neisseria, preferably encode them Branching enzyme from Neisseria denitrificans.

In the context of the present invention, the term “hybridization” means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrock et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY). "Hybridization" particularly preferably means hybridization under the following conditions:

Hybridization buffer:
2xSSC; 10x Denhardt's solution (Fikoll 400 + PEG + BSA; ratio 1: 1: 1); 0.1% SDS; 5mM EDTA; 50 mM Na ₂ HPO ₄ ; 250 µg / ml herring sperm DNA; 50 µg / ml tRNA; or
25 M sodium phosphate buffer pH 7.2; 1mM EDTA; 7% SDS
Hybridization temperature: T = 65 to 68 ° C
Wash buffer: 0.2xSSC; 0.1% SDS
Washing temperature: T = 65 to 68 ° C.

Nucleic acid molecules which hybridize with the nucleic acid molecules according to the invention, can in principle originate from any bacterium of the genus Neisseria that contains an ent expressing speaking protein, preferably they come from Neisseria denitrificans. Nucleic acid molecules that hybridize with the molecules of the invention can e.g. B. can be isolated from genomic or from cDNA libraries. Identification and Isolation of such nucleic acid molecules can be done using the inventions nucleic acid molecules according to the invention or parts of these molecules or the reverse Complements of these molecules are made, e.g. B. by means of hybridization according to standard methods (See, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) or by amplification using PCR.

As a hybridization sample, e.g. B. nucleic acid molecules can be used that exactly or essentially the one under Seq ID No. 1 specified nucleotide sequence or parts thereof Have sequence. The fragments used as a hybridization sample may differ also act on synthetic fragments that are made with the help of common synthetic techniques were prepared and their sequence essentially with that of an inventive Nucleic acid molecule matches. Has one identified and isolated genes that are related to the inventions? Hybridize nucleic acid sequences according to the invention, a determination of the sequence and the properties of the proteins encoded by this sequence are analyzed in order to determine whether it is a branching enzyme. Are particularly suitable for this Homology comparisons at the level of the nucleic acid or amino acid sequence as well as the Determination of the enzymatic activity.

The molecules hybridizing with the nucleic acid molecules according to the invention include in particular fragments, derivatives and allelic variants of the nucleic acid described above remolecules, which are preferably a branching enzyme from bacteria of the genus Neisseria Encode Neisseria denitrificans. The term derivative in this context means that the sequences of these molecules differ from the sequences described above Differentiate nucleic acid molecules at one or more positions and a high degree have homology to these sequences. Homology means a sequence identity over the entire length of at least 60%, in particular an identity of at least 70%,  preferably over 80%, particularly preferably over 90% and in particular at least 95%. The deviations from the nucleic acid molecules described above can z. B. by deletion, addition, substitution, insertion or recombination.

Homology also means that functional and / or structural equivalence between the relevant nucleic acid molecules or the proteins encoded by them. Both Nucleic acid molecules homologous to the molecules described above and derivatives of these molecules, there are usually variations of these molecules that Represent modifications that perform the same biological function. It can be both are naturally occurring variations, for example sequences from others Neisseria species or strains or to mutations, these mutations being natural May have occurred or were introduced by targeted mutagenesis. Furthermore, the variations are synthetically produced sequences. With the allelic Variants can be naturally occurring variants as well variants produced synthetically or by recombinant DNA techniques.

Those encoded by the different variants of the nucleic acid molecules according to the invention Proteins have certain common characteristics. For this, e.g. B. biological Activity, molecular weight, immunological reactivity, conformation etc. include, as well physical properties such as B. the running behavior in gel electrophoresis, chromato graphic behavior, sedimentation coefficient, solubility, spectroscopic Properties, stability; pH optimum, temperature optimum etc.

The molecular weight of the branching enzyme derived from the amino acid sequence Neisseria denitrificans is 86.3 kDa. The derived molecular weight of an invented Protein according to the invention is therefore preferably in the range from 70 kDa to 100 kDa in the range from 77 kDa to 95 kDa and particularly preferably around 86 kDa.

The nucleic acid molecules according to the invention can be any nucleic acid molecules in particular DNA or RNA molecules, for example cDNA, genomic DNA, mRNA etc. They can be naturally occurring molecules, or by genetic engineering or chemical ones Molecules produced by synthetic methods. They can be single-stranded molecules that either which contain the coding or the non-coding strand, or double-stranded Molecules.

The present invention further relates to nucleic acid molecules of at least 15,  preferably more than 50 and particularly preferably more than 200 nucleotides in length hybridize specifically with at least one nucleic acid molecule according to the invention. To hybridize specifically here means that these molecules with nucleic acid molecules hybridize, which encode a protein according to the invention, but not with Nucleic acid molecules that encode other proteins. Hybridization means preference wise hybridization under stringent conditions (see above). In particular, the invention relates those nucleic acid molecules, which with transcripts of the invention Hybridize nucleic acid molecules and thereby prevent their translation. Such Nucleic acid molecules that are specific to the nucleic acid molecules of the invention can hybridize, for example components of antisense constructs or ribozymes or can be used as primers for amplification by means of PCR.

The invention further relates to vectors, in particular plasmids, cosmids, viruses, Bacteriophages and other vectors common in genetic engineering that he described above contain nucleic acid molecules according to the invention.

In a preferred embodiment, those contained in the vectors Nucleic acid molecules linked in sense orientation to regulatory elements that make up the Ensure expression in prokaryotic or eukaryotic cells. The term "Expression" can mean transcription as well as transcription and translation.

The expression of the nucleic acid molecules according to the invention in prokaryotic cells, for example in Escherichia coli, enables, for example, a more precise characterization of the enzymatic activities of the encoded proteins. In addition, it is possible to introduce various types of mutations into the nucleic acid molecules according to the invention by means of common molecular biological techniques (see, for example, Sambrook et al., Loc. Cit.), Which leads to the synthesis of proteins with possibly changed biological properties. It is possible to generate deletion mutants in which progressive deletions from the 5 'or from the 3' end of the coding DNA sequence produce nucleic acid molecules which lead to the synthesis of correspondingly shortened proteins. It is also possible to introduce point mutations at positions which have an influence, for example, on the enzyme activity or the regulation of the enzyme. In this way, e.g. B. mutants are produced which have a changed K _m value or are no longer subject to the regulatory mechanisms normally present in the cell via allosteric regulation or covalent modifications. Furthermore, mutants can be produced which have an altered substrate or product specificity. Furthermore, mutants can be produced which have a changed activity-temperature profile. Genetic engineering manipulation in prokaryotic cells can be carried out according to methods known to the person skilled in the art (cf. Sambrook et al., Loc. Cit.).

Regulatory sequences for expression in prokaryotic organisms, e.g. B. E. coli, and in Eukaryotic organisms have been adequately described in the literature, especially those for expression in yeast, such as. B. Saccharomyces cerevisiae. An overview of different Systems for expression for proteins in various host organisms can be found e.g. B. in Methods in Enzymology 153: 383-516 (1987) and Bitter et al. (Methods in Enzymology 153 (1987), 516-544).

Preferably, the one according to the invention inserted in a vector according to the invention is Nucleic acid molecule modified in such a way that the encoded protein after expression in a suitable host organism can be isolated more easily from the culture medium. So there is for example the possibility of using the coded branching system as a fusion protein to express another polypeptide sequence, the specific binding properties of which Allow isolation of the fusion protein via affinity chromatography (see e.g. Chong et al., Gene 192 (1997), 271-281; Hopp et al., 1988, Bio / Technology 6: 1204-1210; Sassenfeld, Trends in biotechnology. 8: 88-93 (1990).

It is further preferred that that contained in a vector according to the invention Nucleic acid molecule comprises nucleotide sequences which are responsible for the secretion of the branching enzyme allow the culture medium. Preferably a sequence is used which is the signal peptide encoded the α-CGTase from Klebstella oxytoca M5A1 (Fiedler et al., J. Mol. Biol. 256 (1996), 279-291; Genebank Acc. No. X86014, CDS 11529-11618). By the secretion of the enzyme in the culture medium simplifies the extraction and purification. It will be an information of the cells avoided and the enzyme can be obtained from the culture medium, with the Removal of residual components of the culture medium using common methods, such as. e.g. B. dialysis, Osmosis, chromatographic methods etc. can be used.

Furthermore, the vectors according to the invention can comprise further functional units which stabilize the vector in a host organism, such as. B. a bacterial  Origin of replication or the 2µ DNA for stabilization in S. cerevisiae.

In a further embodiment, the invention relates to host cells, in particular prokaryotic or eukaryotic cells containing a nucleic acid described above Molecule or a vector were transformed, as well as cells derived from such host cells descend and contain the described nucleic acid molecules or vectors. The Host cells can be bacterial (e.g. E. coli) or fungal cells (e.g. yeast, especially S. cerevisiae), as well as plant or animal cells. The term "transformed" means thereby that the cells according to the invention with a nucleic acid molecule according to the invention are genetically modified in that they are at least in addition to their natural genome contain a nucleic acid molecule according to the invention. This can be freely given in the cell if present as a self-replicating molecule, or it can stably enter the genome of the host cell are integrated.

The host cells are preferably microorganisms. Among them are the In the present application, all bacteria and all protists (e.g. fungi, especially yeast and Algae) understood, as they z. B. in Schlegel "General Microbiology" (Georg Thieme Verlag (1985), 1-2) are defined.

The present invention further relates to methods for producing a branching enzyme from bacteria of the genus Neisseria, in the host cells according to the invention under conditions cultured which allow expression of the protein and the protein from the culture, i.e. H. is obtained from the cells and / or the culture medium. A is preferably used Host organism used that secretes the branching enzyme.

The present invention further relates to a method for producing a Branching enzyme from bacteria of the genus Neisseria, the protein in an in vitro Transcription and translation system using an inventive Nucleic acid molecule is made. Such systems are familiar to the person skilled in the art.

The invention also relates to proteins by the nucleic acid molecules according to the invention are encoded, or which are obtainable by a method according to the invention.

The present invention further relates to antibodies which specifically contain an inventive  Detect protein. These can e.g. B. be monoclonal or polyclonal antibodies. It can also be fragments of antibodies that recognize proteins according to the invention. Methods for producing such antibodies or fragments are known to the person skilled in the art.

Furthermore, the present invention relates to the use of an inventive Branching enzyme for the production of α-1,6-branched α-1,4-glucans in in vitro Systems.

The present invention particularly relates to transgenic plant cells that contain nucleic acid molecules or vectors according to the invention. Preferably, the cells according to the invention characterized in that the introduced invention Nucleic acid molecule is stably integrated into the genome and under the control of an in plant cell active promoter.

A large number of promoters are available for the expression of a nucleic acid molecule according to the invention in plant cells. In principle, any promoter that is functional in the plants selected for the transformation can be used. The promoter can be homologous or heterologous with respect to the plant species used. For example, the 355 promoter of the Cauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812) is suitable, which ensures constitutive expression in all tissues of a plant and the promoter construct described in WO / 9401571. Another example is the promoters of maize polyubiquitin genes (Christensen et al., Loc. Cit.). However, promoters can also be used which are only activated at a point in time determined by external influences (see for example WO / 9307279). Promoters of heat shock proteins that allow simple induction can be of particular interest. It is also possible to use the promoters which lead to expression of downstream sequences in a particular tissue of the plant (see, for example, Stockhaus et al., EMBO J. 8 (1989), 2245-2251), for example the ST-LS1 Promoter that is only active in photosynthetically active tissue (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947). Also to be mentioned are promoters which are active in the starch-storing organs of plants to be transformed. These are e.g. B. in maize the corn kernels, while in the potato it is the tubers. The tuber-specific B33 promoter (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) can be used, for example, to overexpress the nucleic acid molecules according to the invention in the potato. Seed-specific promoters have already been described for various plant species. So z. B. the USP promoter from Vicia faba, which ensures seed-specific expression in V. faba and other plants (Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679; Bäumlein et al., Mol Gen. Genet. 225: 459-467 (1991)). In maize, for example, promoters of the zein genes ensure specific expression in the endosperm of the maize kernel (Pedersen et al., Cell 29 ( 1982 ), 1015-1026; Quattrocchio et al., Plant Mol. Biol. 15 (1990), 81-93 ).

It is thus possible to express the nucleic acid molecules according to the invention in plants Cells.

The present invention thus also relates to a method for producing transgenic Plant cells, comprising the introduction of a nucleic acid molecule according to the invention or Vector in plant cells. Different plant transformation systems are available Expert available, e.g. B. is the use of T-DNA for the transformation of Plant cells intensively examined and described in EP-A-120 S 16; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4, 1-46 and An, EMBO J. 4 (1985), 277-287.

Plant explants can expediently be used for the transfer of the DNA into the plant cell can be cocultured with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material (e.g. leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) can then in a suitable medium, which Antibiotics or biocides for the selection of transformed cells can contain whole Plants are regenerated. The plants thus obtained can then on the presence of imported DNA are examined. Other ways of introducing foreign DNA using the biolistic method or by protoplast transformation known (see e.g. Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm, G. Reed, A. Pühler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge).

Alternative systems for the transformation of monocotyledonous plants are transformation by means of the biolistic approach, the electrically or chemically induced DNA uptake in Protoplasts, the electroporation of partially permeabilized cells, the macro injection of DNA in inflorescences, the microinjection of DNA in microspores and pro-embryos that DNA uptake by germinating pollen and DNA uptake in embryos  Swelling (see e.g. Lusardi, Plant J. 5 (1994), 571-582; Paszkowski, Biotechnology 24: 387-392 (1992)).

During the transformation of dicotyledonous plants via Ti plasmid vector systems with the help of Agrobacterium tumefaciens is well established, recent work suggests that too monocot plants of transformation by means of vectors based on Agrobacterium are well accessible (Chan, Plant Mol. Biol. 22 (1993), 491-506; Hiei, Plant J. 6 (1994), 271- 282; Bytebier, Proc. Natl. Acad. Sci. USA 84: 5345-5349 (1987); Raineri, Bio / Technology 8 (1990), 33-38; Gould, Plant Physiol. 95: 426-434 (1991); Mooney, Plant, Cell Tiss. & Org. Cult. 25: 209-218 (1991); Li, Plant Mol. Biol. 20: 1037-1048 (1992).

In the past, three of the above-mentioned transformation systems could be established for different cereals: the electroporation of tissue, the transformation of protoplasts and the DNA transfer by particle bombardment into regenerable tissue and cells (for an overview: Jähne, Euphytica 85 (1995), 35-44). The transformation of wheat is described in various ways in the literature (for an overview: Maheshwari, Critical Reviews in Plant Science 14 ( 2 ) (1995), 149-178).

In the expression of the nucleic acid molecules according to the invention in plants basically the possibility that the synthesized protein in any compartment the plant cell can be localized. To localize in a particular To reach the compartment, the coding region may have to contain DNA sequences linked to ensure localization in the respective compartment. Such sequences are known (see for example Braun, EMBO J. 11 (1992), 3219-3227; Wolter, Proc. Natl. Acad. Sci. USA 85: 846-850 (1988); Sonnewald, Plant J. 1 (1991), 95-106; Rocha-Sosa, EMBO J. 8 (1989) 23-29).

The present invention thus also relates to transgenic plant cells which have one or several nucleic acid molecule (s) according to the invention have been transformed, and transgenic Plant cells derived from cells transformed in this way. Such cells contain one or more nucleic acid molecule (s) according to the invention, these (s) preferably with regulatory DNA elements that are linked to transcription in plant cells ensure, especially with a promoter. Such cells can be obtained from na differentiate naturally occurring plant cells in that they have at least one contain nucleic acid molecule according to the invention.

The transgenic plant cells can be whole according to techniques known to those skilled in the art  Plants are regenerated. The regeneration of the invention Plants obtainable from transgenic plant cells are also the subject of the present invention Invention. The invention further relates to plants which are those described above contain transgenic plant cells. In principle, the transgenic plants can be Trade plants of any plant species, d. H. both monocot and dicot Plants. They are preferably useful plants, in particular starch-storing plants Plants such as B. grain, rice, corn, cassava or potato.

The invention also relates to propagation material and crop products of the invention Plants, for example fruits, seeds, tubers, rhizomes, seedlings, cuttings etc.

In another aspect, the present invention relates to a regulatory region that naturally the transcription of an invention described above Nucleic acid molecule which encodes a branching enzyme from bacteria of the genus Neisseria, controlled in bacterial cells.

In the context of the present invention, the term "regulatory region" includes a Region understood that the specificity and / or the extent of expression of a gene sequence influenced, for example in such a way that expression in response to certain external stimuli or occurs at a specific time. Such regulatory regions are in the Usually in an area called a promoter. The term "promoter" includes in Within the scope of the present invention nucleotide sequences necessary for the initiation of transcription are necessary, d. H. for the binding of the RNA polymerase, and can e.g. B. also the TATA Include box (s).

In a preferred embodiment, the regulatory region according to the invention comprises a nucleotide sequence selected from the group consisting of:

• a) Nucleotide sequences which contain nucleotides 1 to 169, which are described in Seq ID No. 1 shown Include nucleotide sequence;
• b) the nucleotide sequence of the regulatory region involved in the insertion of the plasmid DSM 12425 is included, or parts thereof; and
• c) nucleotide sequences which are identical to the sequences mentioned under (a) or (b) hybridize stringent conditions.

Nucleotides 1 to 169 of the in Seq ID No. 1 sequence belong to the  regulatory region of the branching enzyme gene from Neisseria denitrificans. Putative promoter sequences are located at positions 36 to 44, 51 to 55 and 157 to 162, where the sequence "GGGAGA" may be a shine Dalgarno sequence.

The present invention also relates to those regulatory regions that have such a high Homology to the above-mentioned regulatory regions shows that they have at least hybridize one of these regions, preferably under stringent conditions. Especially preferred are regulatory regions that are at least 80%, preferably at least 90% and particularly preferably at least 95% sequence identity to one of the above regulatory regions, in particular to that in Seq ID No. 1 shown.

These also include those regulatory regions that e.g. B. due to deletion (s), Insertion (s), substitution (s), addition (s) and / or recombination (s) and / or Modification (s) compared to the above-mentioned regulatory regions have been changed.

Procedures to introduce such changes in regulatory regions are the Expert familiar. It is also known to those skilled in the art that the inventive regulatory regions with additional elements that support transcription in bacterial cells influence, can be connected, e.g. B. with enhancer elements.

The present invention also relates to recombinant DNA molecules that have a include regulatory region of the invention.

The regulatory region is preferably included in such a recombinant DNA molecule linked to a heterologous DNA sequence. The expression "heterologous" means that a such sequence is naturally not linked to the regulatory region. Further can a recombinant DNA molecule according to the invention further regulatory elements contain that are of importance for transcription and / or translation in bacterial cells are, e.g. B. transcription or translation enhancers.

Furthermore, the present invention relates to host cells with an inventive regulatory region, a recombinant DNA molecule or a vector are.

The present invention further relates to vectors that are regulatory according to the invention Region or contain a recombinant DNA molecule according to the invention. Such vectors  include e.g. B. also plasmids, cosmids, bacteriophages, viruses, etc., the conventionally used for molecular genetic methods.

The plasmid pBB48 produced in the context of the present invention was used in the as international depository recognized German collection of microorganisms and Cell cultures (DSMZ) in Braunschweig, Federal Republic of Germany, according to the Requirements of the Budapest Treaty on September 25, 1998, under the Filing number DSM 12425.

Fig. 1 shows schematically the construction of plasmid pBB48 (DSM 12425)

Fig. 2 shows a number of different highly branched α-1,6 α-1,4-glucans produced by the novel process and that were subsequently stained with Lugol's solution.

From left to right: Amylosucrase (left), Amylosucrase + falling amounts Branching enzyme activity. The absorption maxima of the corresponding samples were: 615 nm, 483 nm, 500 nm, 526 nm, 534 nm, 560 nm, 577 nm.

FIG. 3 shows an HPLC chromatogram of a highly branched process product (A) branched with isoamylase and a rat liver glycogen sample (B) branched with isoamylase.

Fig. 4 shows the scheme of the methylation analysis.

Fig. 5 shows a graph of the analysis results of the sample 7 as described in Example 9 and 10 after one and after two methylation steps. The values for the 2, 3, 6-methylation are 96.12% and 96.36%, respectively.

Fig. 6 shows a graphical representation of units of terminal ( "2346 Me") and 6- linked ( "23 Me") of glucose units of the glucan samples examined.

FIGS. 7 and 8 show gas chromatograms of the samples described in Examples 3 and 7.

The following examples illustrate the invention

materials

Digestion buffer: 100 mM Tris / HCl pH 8.5; 5 mM Na ₂

EDTA; 2mM DTT; 1 mM Pefabloc®
Wash buffer: 50 mM Tris / HCl pH 8.5; 5 mM Na ₂

EDTA; 10% glycerol)
HIC buffer: 50 mM potassium phosphate buffer pH 7.0; 5mM EDTA; 2mM DTT; 10% glycerol
Oyster Glycogen Type II from Oyster (Sigma G8751)

example 1 Isolation of a genomic DNA sequence coding for branching enzymes Neisseria denitrificans

A genomic DNA library was first created to isolate the branching enzyme from Neisseria denitrificans. For this purpose, Neisseria denitrificans cells of the strain which is deposited with ATCC as ATCC 14686 were cultivated on Columbia blood agar plates and then harvested. The genomic DNA was extracted using the method of Ausubel et al. (in: Current Protocolls in Molecular Biology (1987); J. Wiley & Sons, NY) isolated and purified. After a partial restriction digest with the restriction endonuclease Sau3A, ligation with BamH I cut phage vector DNA (lamdaZAPExpress from Stratagene) was carried out. After the in vivo excision of the phage library, the plasmids obtained were transformed into the E. coli mutant (PGM-) (Adhya and Schwartz, J. Bacteriol. 108 (1971), 621-626). This mutant forms linear polysaccharides when grown on maltose, which appear blue after staining with iodine. 60,000 transformants were plated on YT agar plates with IPTG (1 mM), kanamycin (12.5 mg / l) and maltose (1%) and, after 16 hours of incubation at 37 ° C., evaporated with iodine. 60 bacterial colonies, which were red, brown or yellow in color after iodine evaporation, were selected and plasmid DNA was isolated from them (Birmboim-Doly, Nucleic Acid Res. 7, 1513-1523). The isolated plasmids were then used to retransform the same E. coli (PGM) mutant (Adhya and Schwartz, J. Bacteriol. 108 (1971), 621-626). After repeated plating and iodine evaporation, the clones were reduced from 60 to four isolates. A restriction analysis of these four plasmids was carried out, which showed an equally large EcoRI fragment (1.6 kb) in all four plasmids ( FIG. 1).

Example 2 Sequence analysis of the genomic fragment of plasmid pBB48

The 1.6 kb EcoR I fragment was isolated (Geneclean, Bio101) from a clone (pBB48) obtained according to Example 1, which had an approximately 3.9 kb insertion in the vector pBK-CMV (Stratgene), and for the purpose of DNA sequencing cloned into the vector pBluescript cut with EcoR I. The plasmid thus obtained was sequenced. The complete branching enzyme-coding DNA sequence and the sequence of flanking regions were then determined with the aid of the starting plasmid pBB48 (Seq ID No. 1). The plasmid pBB48 is shown in FIG. 1. The plasmid is deposited under the number DSM 12425.

Example 3 Expression of the branching enzyme in recombinant E. coli cells

In the E. coli laboratory strains there is generally an endogenous branching enzyme (glgB) expressed. For this reason, the E. coli G6MD2 mutant used. The strain E. coli Hfr G6MD2 (E. coli Genetic Stock Center, Yale University, CGSC # 5080) carries an extensive deletion in the field of Glucan synthesis genes (glgA, glgB, glgC). For the detection of branching enzyme activity this mutant was transformed with the plasmid pBB48 and one of the multiplied cells Prepares raw extract. The proteins of this crude extract were in a polyacrylamide gel electrophoretically separated and subsequently to demonstrate the branching enzyme activity and incubated without rabbit phosphorylase b (100 mM Na citrate pH 7.0; AMP, glucose-1 Phosphate). Only in the gel stimulated by phosphorylase did violet bands appear, indicating a indicates strong vigorous enzyme activity.

Example 4 In vitro production of α-1,6-branched α-1,4-glucans with crude protein extracts in cell-free system

The mutant E. coli G6MD2 with the plasmid was used for the expression of the branching enzyme pBB48 transformed. The cells were washed with YT medium with kanamycin (12.5 mg / l) for 16 hours Shake attracted in the Erlenmeyer flask. After centrifugation (5000xg) the obtained pellet washed with 100 mM Tris / HCl pH 7.5, 1 mM DTT and after suspension in same cells, the cells are disrupted with an ultrasound probe. Another Centrifugation (10000xg) separated the cell debris from the soluble proteins and gave a yellowish supernatant with a protein concentration of approx. 10 mg / ml.  

Various amounts were then obtained from this crude protein extract obtained in this way (100 µl, 10 µl, 1 µl, 0.1 µl, 0.01 µl, 0.001 µl) together with a constant amount an amylosucrase in 50 ml 100 mM Na citrate pH 7.0 with 20% sucrose and 0.02% Na azide given. The first turbidities in the reaction mixture were observed after a few hours become. After three days, centrifugation was carried out and the resulting products were deionized Washed water.

The products are soluble in DMSO and can be characterized by measuring an absorption spectrum with Lugol's solution. This makes it possible to estimate the degree of branching of the products produced. For this purpose, the DMSO solution was strongly diluted with water, Lugol's solution was added and the spectrum from 400 nm to 700 nm was immediately measured in a Beckmann spectrophotometer (see FIG. 2).

Separation of the side chains cleaved with isoamylase on a Carbopak PA100 column using HPLC (DIONEX; eluent: 150 mM NaOH with 1M Na acetate gradient) shows the same pattern for a highly branched product as for a rat liver glycogen debranched with isoamylase ( Fig . 3).

Cleavage of side chains after incubation with a pullulanase occurred only to a very small extent Measurements on.

Example 5 Purification of the branching enzyme and N-terminal sequencing of the protein

To isolate the Neisseria denitrificans branching enzyme from recombinant Hfr G6MD2-E. coli cells (see above) which had been transformed with the pBB48, an overnight culture of these cells was first centrifuged. The cell precipitate was then suspended in 3 volumes of digestion buffer and digested in the French press at a pressure of approximately 16,000-17,000 psi. After centrifugation at 10,000 g for one hour, the supernatant was diluted to 4 times the volume by adding wash buffer, bound to DEAE cellulose DE52 in a batch process and poured into a chromatography column which was washed with 2 to 3 column volumes of wash buffer. A linear 1M NaCl gradient was then applied for elution. The fractions with branching enzyme activity (see Example 8) were combined, mixed with (NH ₄ ) ₂ SO ₄ (final concentration 20% (w / v)) and applied to a TSK Butyl-Toyopearl 650M column. After washing with 2 to 3 column volumes of HIC buffer, which had previously been additionally mixed with an ammonium sulfate solution with a degree of saturation of 20% (114 g ammonium sulfate per liter), the branching enzyme was used using an ammonium sulfate gradient falling linearly from 20% to 0% eluted in the HIC buffer. Fractions with branching enzyme activity were pooled. To concentrate the protein, the purification step was then repeated with the combined fractions using a small TSK Butyl-Toyopearl 650M column (Tose Haas (Montgomery Ville, Pensylvania)). The purified protein was then applied to a polyacrylamide gel, blotted on a PVDF membrane, then brought back into solution and sequenced by the company WITA GmbH, Teltow, Germany, N-terminally using the Edman method. The sequence obtained was: MNRNXH (Seq ID No. 3).

Example 6 Cleaning an amylosucrase

For the production of an amylosucrase, E. coli cells were used which were equipped with a DNA, coding an amylosucrase from Neisseria polysaccharea. The DNA has those under Seq ID No. 4 nucleotide sequence shown and comes from a genomic N. polysaccharea library.

An overnight culture of these E. coli cells which secrete the amylosucrase from Neisseria polysaccharea was centrifuged and in about ¹ / _20th volume of 50 mM sodium citrate buffer (pH 6, S), 10 mM DTT (dithiothreitol), 1 mM PMSF (phenylmethylsulfonyl fluoride) resuspended. The cells were then disrupted twice with a French press at 16,000 psi. Thereafter, 1 mM MgCl _{2 was} added to the cell extract and benzonase (from Merck; 100,000 units; 250 units µl ^-1 ) in a final concentration of 12.5 units ml ^-1 . The mixture was then incubated at 37 ° C. with gentle stirring for at least 30 min. The extract was left on ice for at least 1.5 hours. The mixture was then centrifuged at 4 ° C. at 40,000 g for 30 min until the supernatant was relatively clear.

A prefiltration was carried out with a PVDF membrane (Millipore "Durapore", or the like) which had a pore diameter of 0.45 μm. The extract was left at 4 ° C overnight. Before performing the HI (hydrophobic interaction) chromatography, the extract was mixed with solid NaCl and adjusted to a concentration of 2 M NaCl. The mixture was then centrifuged again at 4 ° C. and about 40,000 mg for 30 min. The extract was then freed of the last residues of E. coli by filtering with a PVDF membrane (Millipore "Durapore", or the like) which had a pore diameter of 0.22 μm. The filtered extract was separated on a Butyl Sepharose 4B column (Pharmacia) (volume of the column: 93 ml, length: 17.5 cm). Approx. 50 ml of extract with an amylosucrase activity of 1 to 5 units µl ^-1 were added to the column. Subsequently, non-binding proteins from the column (buffer B: 50 mM sodium citrate pH 6.5, 2 M NaCl) were washed with 150 ml of buffer B. The amylosucrase was finally eluted using a falling linear NaCl gradient (from 2 M to 0 M NaCl in 50 mM sodium citrate in a volume of 433 ml at an inflow rate of 1.5 ml min-1), which was carried out using a automatic pumping system (FPLC, Pharmacia) was generated. The amylosucrase was eluted between 0.7 M and 0.1 M NaCl. The fractions were collected, desalted on a PD10 Sephadex column (Pharmacia), stabilized with 8.7% glycerol, checked for amylosucrase activity and finally frozen in storage buffer (8.7% glycerol, 50 mM citrate).

Example 7 Determination of amylosucrase activity

The amylosucrase activity is determined by adding purified protein or crude protein extract in different dilutions to 1 ml batches containing 5% sucrose, 0.1% dextrin and 100 mM citrate pH 6.5 and incubating at 37 ° C. After 0 min. 30 min. 60 min. 120 min. 180 min. 240 min. 300 .mu.l and 300 .mu.l are removed from this batch in each case and the enzymatic activity of the amylosucrase is ended by immediate heating to 95.degree. The proportion of fructose released by the amylosucrase is then determined in the coupled photometric test. For this purpose, 1 ul to 10 ul of the inactivated sample in 1 ml 50 mM imidazole buffer pH 6.9, 2 mM MgCl ₂ , 1 mM ATP, 0.4 mM NAD ⁺ and 0.5 U / ml hexokinase are added. After sequential addition of glucose-6-phosphate dehydrogenase (from Leuconostoc mesenteroides) and phosphoglucose isomerase, the change in absorption is measured at 340 nm. The amount of fructose released is then calculated using the Lambert-Beerschen law.

If one relates the value obtained to the time of sampling, then the number of units (1U = µmol fructose / min) (per µl protein extract or µg purified Protein).  

Example 8 Determination of the enzyme activity of a branching enzyme from Neisseria denitrificans

The determination of the enzymatic activity of the branching enzyme was based on a method described in the literature (Krisman et al., Analytical Biochemistry 147 (1985), 491-496; Brown and Brown, Meth. Enzymol. 8 (1966), 395-403). The The method is based on the principle of reduced iodine binding affinity of branched Glucans compared to unbranched α-1,4-glucans.

In order to determine the enzymatic activity of the branching enzyme, a series of samples of various dilutions of the branching enzyme were placed in a cooled microtiter plate. The reaction was then started by adding 190 μl of amylose reaction mixture (preparation see below) and incubated at 37 ° C. in the incubator. After exactly 30 minutes, the reaction was stopped by adding 100 μl of Lugol's solution (0.5 mM) and the samples were measured in a microtiter plate reader (Molecular Devices) at 650 nm. A batch without amylose served as a control. The reference sample with the maximum extinction value, but which contained no branching enzyme in amylose, had an OD ₆₅₀ = 1.2.

For better comparability of independent assays, only the sample dilution is used for the evaluation, which leads to a decrease of the OD ₆₅₀ by 0.5 units during the incubation period of 30 min.

Defining an activity unit (U) of the branching enzyme

Half a unit of the branching enzyme is the amount of enzyme which, in the test described, causes the OD ₆₅₀ to decrease by 0.5 unit from 1.2 to 0.7 in 30 minutes.

Preparation of the amylose reaction mixture

1 ml 0.5% amylose solution (manufacturer: Fluka; amylose from potato) w / v in DMSO are stirred in 10 ml Na citrate buffer (100 mM, pH 6.5, 0.02% w / v NaN ₃ ) given. The clear stock solution is again diluted 1: 4 to 1: 8 with Na citrate buffer for the measurement. The absorption with Lugol's solution should be 1.2 in the test with the reference sample used (maximum).

Example 9 Production of α-1,6-branched α-1,4-glucans with different degrees of branching

For the production of α-1,6-branched α-1,4-glucans with different degrees of branching, a 20% sucrose solution (w / v) in a reaction volume of 10.86 ml with a purified amylosucrase from Neisseria polysaccharea (see Example 6) and a purified branching enzyme from Neisseria denitrificans (see Example 5). Depending on the experimental approach, these two enzymes were used in different protein activity ratios (determination of amylosucrase: see example 7; determination of branching enzyme: see example 8) to one another (see table 1):
Amylosucrase preparation: 6.2 U / mg; 1.8 mg / ml
Branching enzyme preparation: 75 U / mg; 6.9 mg / ml

Table 1

Example 10 Determination of the degree of branching using methylation analysis

The degree of branching of the glucans obtained was then determined using a Methylation analysis determined.

1. Investigations carried out

• - Methylation of all free OH groups of the glucan samples, double determinations in each case
• Hydrolysis of the permethylated polymers, followed by reduction at C-1 and Acetylation of the monomer mixture
• - Gas chromatographic analysis and quantification of the reaction products

The degree of branching of the glucan samples was clarified by means of a methylation analysis (see FIG. 4). The free OH groups of the polymer are marked by conversion to methyl ether in 08748 00070 552 001000280000000200012000285910863700040 0002019846635 00004 08629ng.

The degradation to the monomers takes place under acid hydrolysis and leads to partially methylated glucose molecules which are present in pyranosider / furanoside form and as α- and β-glucosides. These variants are focused by reduction with NaBH ₄ in the respective partially methylated sorbitol derivative. The reaction products can be examined by gas chromatography through the final acetylation of free OH groups.

The following table shows the consistency and the DMSO solubility of the glucans obtained.

Table 2

2. Experimental part a) Preparation of the DMSO solutions

1% (w / v) solutions were prepared in DMSO. The samples weren't all at Well soluble at room temperature: 1, 3 and 13 had to be heated to 110 ° C for 30 minutes. Except for solutions 1 and 3, which were slightly cloudy, there were optically clear solutions (see table 2).

b) methylation

2 ml of the DMSO solution (ie 20 mg of polymer) were transferred to a 50 ml nitrogen flask, 5 equivalents / OH (eq / OH) of freshly prepared dimsyl solution were added in a stream of N _{2 and the} mixture was stirred for 30 minutes. The solutions became cloudy and viscous. The contents of the flask were frozen in an ice bath, 10 eq / OH methyl iodide were added and, after thawing, the mixture was stirred for at least 2 h. Excess methyl iodide was removed in vacuo before the second deprotonation and methylation step.

Working up was carried out after removing the excess methyl iodide by adding 50 ml of water and extracting once with 10 ml of dichloromethane. The organic phase was then freed from traces of DMSO by extraction three times with water, dried with CaCl ₂ , filtered and concentrated. The products were in the form of clear, slightly yellowish films.

On the basis of sample 7, it was first checked how many methylation steps are necessary for permethylation of the hydroxyl groups. After the first methylation, half of the mixture was worked up and the other half was methylated again. After both samples had been broken down, the results of the GC analyzes were compared. It was found that the reaction was almost quantitative after only one methylation step (see FIG. 5). In order to verify any branching at C-3, which can also be simulated by undermethylation at this position, a second methylation was connected in each case.

Fig. 5 shows a graph of the analysis results of the sample 7 after one and after two methylation steps; the values for 2,3,6-methylation are 96.12% and 96.36%

c) hydrolysis

2 mg of the methylated sample were weighed into a 1 ml pressure glass, 0.9 ml of 2 M trifluoroacetic acid were added and the mixture was stirred at 120 ° C. for 2.5 h. After the glass had cooled, the mixture was concentrated in a stream of N ₂ . To remove traces of acid, toluene was added three times and blown off.

Table 3

Methylation data

d) reduction

The residue from the previous reaction step was treated with 0.5 ml of a 0.5 M ammoniacal NaBD4 solution added and stirred for 1 h at 60 ° C. The reagent was carefully destroyed with a few drops of glacial acetic acid, the borate formed five times  Add 15% methanolic acetic acid and then blow off as Removed boric acid trimethyl ester.

e) acetylation

The residue from the previous reaction step was mixed with 50 ul pyridine and 250 ul acetic anhydride and stirred at 95 ° C for 2 h. After cooling, the reaction mixture was dropped into 10 ml of saturated NaHCO ₃ solution and extracted five times with dichloromethane. The reaction products in the organic phase were examined by gas chromatography (for products see FIG. 4)

f) gas chromatography

The gas chromatographic investigations were carried out on a device from Carlo Erba GC 6000 Vega Series 2 with on-column inlet and FID detector. The Separations took place on a fused silica capillary column Supelco SPB5 (inner diameter 0.2 mm, length 30 m) with hydrogen as a carrier gas and a pressure of 80 kPa. It was The following temperature program is used: 60 ° C (1 min) -25 ° C / min → 130 ° C-4 ° C / min → 280 ° C.

3. Results

The gas chromatograms were evaluated by identifying the peaks and integrating them peak areas and data correction using Sweet et all's ECR concept. (Sweet et al., Carbohydr. Res. 40 (1975), 217).

The 1,6-anhydro compounds observed in samples 1 and 3 are high Degree of branching at C-6. This leads to monomers during the hydrolysis with a free OH group at C-6, which under the reaction conditions to these derivatives can continue to react. The shares must be used in calculating the degree of branching "2.3-Me" value can be added.

Fig. 6 is a graphical representation of the proportions of terminal ("2346Me") and 6-linked ("23" Me) glucose units of the examined glucan samples.

Table 4

Analysis results in mol%: the short names (A, B, etc.) correspond to those in Fig. 1; "16AnhPy" = 1,6-anhydro-4-O-acetyl-2,3-di-O-methyl-D-glucopyranose, "16AnhFu" = 1.6 anhydro-5-O-acetyl-2,3-di -O-methyl-D-glucofuranose; "Me1" and "Me2" denote two independent methylation analyzes of the respective samples

Example 11 Production of α-1,6-branched α-1,4-glucans of different molecular weights

To prepare α-1,6-branched α-1,4-glucans of different molecular weights, a 20% sucrose solution (w / v) in a reaction volume of 10.86 ml with a purified amylosucrase from Neisseria polysaccharea (see Example 6) and a purified branching enzyme from Neisseria denitrificans (see Example 5). Depending on the experimental approach, these two enzymes were used in different protein activity ratios (determination of amylosucrase activity; see Example 7; branching enzyme: see Example 8) to one another (see Table 1). The molecular weights and the radius of inertia Rg were determined by laser scattering (Light Scattering from Polymer solutions, editor: Huglin, MB, Academic Press, London, 1972). For this purpose, the dried samples 1-11 in DMSO. H ₂ O (in a 90:10 ratio) and various dilutions (approx. 2.5 g / l to 025 g / l) in a light scattering meter (SOFICA, Societe francaise d'instruments de controle et dänalyses. Le Mesnil Saint-Denis , France). The data thus obtained were according to Berry (J. Chem. Phys. 44 (1966), 4550ff.).

Table 5

SEQUENCE LOG

Claims (25)

1. In vitro method for the production of α-1,6-branched α-1,4-glucans under Use of the substrate sucrose and an enzyme combination of one Amylosucrase and a branching enzyme. 2. The method of claim 1, wherein the amylosucrase is of prokaryotic origin. 3. The method of claim 2, wherein the prokaryote is Neisseria polysaccharea. 4. The method according to any one of claims 1 to 3, wherein the amylosucrase is purified. 5. The method according to any one of claims 1 to 4, wherein the branching enzyme is of prokaryotic origin. 6. The method of claim 5, wherein the prokaryote is Neisseria denitrificans. 7. The method according to any one of claims 1 to 6, wherein the branching enzyme is purified is. 8. The method according to any one of claims 1 to 7, wherein the amylosucrase and / or that Branching enzyme are produced recombinantly. 9. The method according to any one of claims 1 to 8, wherein the amylosucrase and / or Branching enzyme are immobilized. 10. α-1,6-branched α-1,4-glucans obtainable by a method according to one of the Claims 1 to 9. 11. Nucleic acid molecule encoding a branching enzyme from a bacterium of the genus Neisseria selected from the group consisting of

• a) Nucleic acid molecules which encode a protein which has the properties listed under Seq ID No. 2 includes amino acid sequence indicated;
• b) Nucleic acid molecules which are listed under Seq ID No. 1 encoding region specified;
• c) nucleic acid molecules encoding a protein comprising the amino acid sequence encoded by the insertion in plasmid DSM 12425;
• d) nucleic acid molecules comprising the coding region for a branching enzyme contained in the insertion of the plasmid DSM 12425;
• e) nucleic acid molecules, the complementary strand of which hybridizes with a nucleic acid molecule according to (a), (b), (c) and / or (d) and which encode a branching enzyme from a bacterium of the genus Neisseria; and
• f) nucleic acid molecules whose sequence deviates from the sequence of a nucleic acid molecule according to (e) due to the degeneration of the genetic code.

12. Vector containing a nucleic acid molecule according to claim 11. 13. The vector of claim 12, wherein the nucleic acid molecule in sense orientation with regulatory sequences linked to transcription in prokaryotic or ensure eukaryotic cells. 14. A host cell that is genetically modified with a nucleic acid molecule according to claim 11 or with a vector according to claim 12 or 13. 15. A method for producing a branching enzyme from a bacterium of the genus Neisseria, wherein a host cell according to claim 14 is cultivated under conditions which allow expression of the protein and the protein from the cultured cells and / or the culture medium is isolated. 16. A method for producing a branching enzyme from a bacterium of the genus Neisseria, the protein in an in vitro transcription and translation system using a nucleic acid molecule according to claim 11.   17. Protein encoded by a nucleic acid molecule according to claim 11 or obtainable by a method according to claim 15 or 16. 18. Antibody that specifically recognizes a protein according to claim 17. 19. Use of a protein according to claim 17 for the production of α-1,6-branched α- 1,4-glucans in in vitro systems. 20. Regulatory region that naturally transcribes a Controlled nucleic acid molecule according to claim 11 in bacterial cells. 21. Regulatory region according to claim 20 containing a nucleotide sequence selected from the group consisting of:

• a) Nucleotide sequences which contain the nucleotides 1 to 169 of the in Seq ID No. 1 nucleotide sequence shown;
• b) the nucleotide sequence of the regulatory region contained in the insertion of the plasmid DSM 12425 or parts thereof;
• c) Nucleotide sequences which hybridize with the sequences mentioned under (a) or (b) under stringent conditions.

22. Recombinant DNA molecule comprising a regulatory region according to claim 20 or 21. 23. A recombinant DNA molecule according to claim 22, wherein the regulatory region is functionally linked to a heterologous DNA sequence. 24. Vector containing a regulatory region according to claim 20 or 21 or recombinant DNA molecule according to claim 22 or 23. 25. Host cell that is transformed with a regulatory region according to claim 20 or 21, with a recombinant DNA molecule according to claim 22 or 23 or with one The vector of claim 24.