WO2000014249A1

WO2000014249A1 - Nucleic acid molecules encoding an amylosucrase

Info

Publication number: WO2000014249A1
Application number: PCT/EP1998/005573
Authority: WO
Inventors: Martin Quanz; Nicholas Provart
Original assignee: Planttec Biotechnologie Gmbh
Priority date: 1998-09-02
Filing date: 1998-09-02
Publication date: 2000-03-16
Also published as: AU9535798A; HUP0103414A3; HUP0103414A2; CA2342124A1; JP2002524080A; EP1109916A1

Abstract

Described are nucleic acid molecules which encode an amylosucrase as well as methods for the production of α-1,4 glucans and fructose using such nucleic acid molecules or the encoded proteins. Furthermore, described are host cells transformed with the described nucleic acid molecules.

Description

Nucleic acid molecules encoding an amylosucrase

The present invention relates to nucleic acid molecules encoding a protein having amylosucrase activity and to vectors containing such molecules. Furthermore, the invention relates to the production of α-1 ,4 glucans and fructose using the described nucleic acid molecules or the encoded proteins.

Linear α-1 ,4 glucans are polysaccharides consisting of glucose monomers, the latter being exclusively linked to each other by α-1 ,4 glycosidic bonds. The most frequently occurring natural α-1,4 glucan is amylose, a component of plant starch. Recently, more and more importance has been attached to the commercial use of linear α-1 ,4 glucans. Due to its physico-chemical properties amylose can be used to produce films that are colorless, odorless and flavorless, non-toxic and biologically degradable. Already today, there are various possibilities of application, e.g., in the food industry, the textile industry, the glass fiber industry and in the production of paper.

One has also succeeded in producing fibers from amylose whose properties are similar to those of natural cellulose fibers and which allow to partially or even completely replace them in the production of paper. Being the most important representative of the linear α-1 ,4 glucans, amylose is particularly used as binder for the production of tablets, as thickener of puddings and creams, as gelatin substitute, as binder in the production of sound-insulating wall panels and to improve the flow properties of waxy oils. Another property of the α-1 ,4 glucans, which recently has gained increasing attention, is the capability of these molecules to form inclusion compounds with organic complexers due to their helical structure. This property allows to use the α-1 ,4 glucans for a wide variety of applications. Present considerations relate to their use for the molecular encapsulation of vitamins, pharmaceutical compounds and aromatic substances, as well as their use for the chromatographic separation of mixtures of substances over immobilized linear α-1 ,4 glucans. Amylose also serves as starting material for the production of so-called cyclodextrins (also referred to as cycloamyloses, cyclomaltoses) which in turn are widely used in the pharmaceutical industry, food processing technology, cosmetic industry and analytic separation technology. These cyclodextrins are cyclic maltooligosaccharides from 6-8 monosaccharide units, which are freely soluble in water but have a hydrophobic cavity which can be utilized to form inclusion compounds.

Today, α-1 ,4 glucans, in particular linear α-1 ,4 glucans, are obtained in the form of amylose from starch. Starch itself consists of two components. One component forms the amylose as an unbranched chain of α-1 ,4 linked glucose units. The other component forms the amylopectin, a highly branched polymer from glucose units in which in addition to the α-1 ,4 links the glucose chains can also be branched via α-1 ,6 links. Due to their different structure and the resulting physico-chemical properties, the two components are also used for different fields of application. In order to be able to directly utilize the properties of the individual components, it is necessary to obtain them in pure form. Both components can be obtained from starch, the process, however, requiring several purification steps and involving considerable time and money. Therefore, there is a need to find possibilities of obtaining both components of the starch in a uniform manner. It is known that certain bacteria, in particular those of the genus Neisseria produce enzymes capable of synthesizing linear α-1 ,4 glucans from sucrose. In order to be able to use such enzymes for the efficient production of α-1 ,4 glucans, it is necessary to isolate and characterize the corresponding DNA sequences.

The technical problem underlying the present invention is therefore to provide nucleic acid molecules and processes that allow the production of α-1 ,4 glucans.

The solution of this technical problem is achieved by the present invention by providing the embodiments characterized in the claims. The invention therefore relates to nucleic acid molecules encoding a protein having the enzymatic activity of an amylosucrase selected from the group consisting of

(a) nucleic acid molecules encoding a protein comprising the amino acid sequence as depicted in SEQ ID NO: 2;

(b) nucleic acid molecules comprising the nucleotide sequence of the coding region as indicated in SEQ ID NO: 1 ;

(c) nucleic acid molecules encoding an analogue of the polypeptide having the amino acid sequence as depicted under SEQ ID NO: 2; and

(d) nucleic acid molecules, the sequence of which differs from the sequence of a nucleic acid molecule as defined in (c) due to the degeneracy of the genetic code.

The nucleic acid sequence of the coding region depicted in SEQ ID NO: 1 encodes a protein of Neisseria polysaccharea having the enzymatic activity of an amylosucrase. With the help of the nucleic acid molecules of the present invention it is possible to produce microorganisms and fungi, particularly yeasts, that are capable of producing an enzyme catalyzing the synthesis of α-1 ,4 glucans from sucrose. It is furthermore possible to produce at low production costs α-1 ,4 glucans, in particular linear α-1 ,4 glucans, as well as pure fructose syrup with the help of the DNA sequences of the invention or of the proteins encoded by them.

Nucleotide sequences which encodes an analogue of the polypeptide as depicted in SEQ ID NO: 2 are understood in the scope of the present invention as nucleotide sequence which encode a polypeptide having the following characteristics:

(a) it has amylosucrase activity; and preferably,

(b) it furthermore shows an identity on the amino acid sequence level of at least 80%, more preferably of at least 85%, even more preferably of at least 90% and particularly preferred of at least 95%, to the amino acid sequence as depicted in SEQ ID NO: 2 over its complete length.

Thus, the present invention also relates to nucleic acid molecules encoding a polypeptide the sequence of which differs at one or more positions from the amino acid sequence as depicted in SEQ ID NO: 2 and which still has amylosucrase activity. The differences in the amino acid sequence may be due to replacements of amino acid residues by other amino acid residues, to the addition of amino acid residues, preferably at the N- or C-terminus of the polypeptide, or to deletions of one or more amino acid residues, preferably at the N- or C-terminus of the protein. The generation of nucleic acid molecules encoding such analogues of the described protein is well within the common general knowledge of the person skilled in the art.

The present invention also relates to nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule as defined above and which encode a polypeptide having the enzymatic activity of an amylosucrase.

In this invention the term "hybridization" means a hybridization under stringent conditions as described for example in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). "Stringent conditions" mean that there is a sequence identity of at least 80% of the complete coding sequence, preferably an identity of at least 90%, more preferably of at least 95% and particularly preferred of at least 99%. Nucleic acid molecules hybridizing to the molecules according to the invention may be isolated e.g. from genomic or from cDNA libraries produced from organism expressing an amylosucrase, for example, from microorganisms, in particular from bacteria of the genus Neisseria. The identification and isolation of such nucleic acid molecules may take place by using the molecules according to the invention or parts of these molecules or, as the case may be, the reverse complement strands of these molecules, e.g. by hybridization according to standard methods (see e.g. Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

As a probe for hybridization e.g. nucleic acid molecules may be used which exactly or basically contain the nucleotide sequence of the coding region indicated under SEQ ID NO. 1 or parts thereof. The fragments used as hybridization probe may also be synthetic fragments which were produced by means of the conventional synthesizing methods and the sequence of which is basically identical with that of a nucleic acid molecule according to the invention. After identifying and isolating the genes hybridizing to the nucleic acid sequences according to the invention, the sequence has to be determined and the properties of the proteins encoded by this sequence have to be analyzed.

The molecules hybridizing to the nucleic acid molecules of the invention also comprise fragments, derivatives and allelic variants of the above-described nucleic acid molecules which encode a protein having the enzymatic activity of an amylosucrase. Thereby, fragments are defined as parts of the nucleic acid molecules, which are long enough in order to encode a protein still having the enzymatic activity. This includes also parts of nucleic acid molecules according to the invention which lack the nucleotide sequence encoding the signal peptide responsible for the secretion of the protein. The term derivatives means that the sequences of these molecules differ from the sequences of the above-mentioned nucleic acid molecules at one or more positions and that they exhibit a high degree of homology to these sequences. Hereby, homology means a sequence identity of at least 80%, in particular an identity of at least 90%, preferably of more than 95% and still more preferably a sequence identity of more than 98%. The deviations occurring when comparing with the above-described nucleic acid molecules might have been caused by deletion, substitution, insertion or recombination.

Moreover, homology means that functional and/or structural equivalence exists between the respective nucleic acid molecules or the proteins they encode. The nucleic acid molecules, which are homologous to the above-described molecules and represent derivatives of these molecules, are generally variations of these molecules, that constitute modifications which exert the same biological function. These variations may be naturally occurring variations, for example sequences derived from other organisms, or mutations, whereby these mutations may have occurred naturally or they may have been introduced by means of a specific mutagenesis. Moreover the variations may be synthetically produced sequences. The allelic variants may be naturally occurring as well as synthetically produced variants or variants produced by recombinant DNA techniques. The proteins encoded by the various variants of the nucleic acid molecules according to the invention exhibit certain common characteristics. Enzyme activity, molecular weight, immunologic reactivity, conformation etc. may belong to these characteristics as well as physical properties such as the mobility in gel electrophoresis, chromatographic characteristics, sedimentation coefficients, solubility, spectroscopic properties, stability, pH-optimum, temperature-optimum etc.

An amylosucrase (also referred to as sucrose: 1 ,4-α glucan 4-α-glucosyltransferase, E.C. 2.4.1.4.) is an enzyme for which the following reaction scheme is suggested:

sucrose + (α-1 ,4-D-glucosyl)_n → D-fructose + (α-1 ,4-D-glucosyl)_n+-|

This reaction is a transglucosylation. The transglucosylation can take place in the presence or absence of acceptor molecules. Such acceptor molecules can be polysaccharides, such as maltooligosaccharides, dextrin, glycogen etc. When such an acceptor molecule is a linear, oligomeric α-1 ,4-glucan, the resulting product is a polymeric linear α-1 ,4-glucan. When the transglucosylation catalyzed by the amylosucrase is carried out in the absence of such acceptor molecule, a glucan is obtained which comprises a terminal fructose molecule. All the products obtainable by transglycosylation with the help of an amylosucrase in the absence or presence of an acceptor molecule are referred to in the scope of the present invention as α-1 ,4 glucans.

The reaction mechanism for a transglucosylation by an amylosucrase in the absence of an acceptor molecule can be described as follows:

G-F+n(G-F) → G_n-G-F+πF, wherein G-F is sucrose. G is glucose, F is fructose and G_n-G-F is an α-1 ,4 glucan.

The reaction mechanism in the presence of an acceptor molecule can be described as follows: mG-F+G_n — > G_π+_m+mF, wherein G_n is a polysaccharide acceptor molecule, G_n+m is the polysaccharide plus α-

1 ,4 glucan chains added thereto by amylosucrase, G-F is sucrose, F is fructose and

G is glucose. The products of the reaction catalyzed by an amylosucrase are the above described α-1 ,4 glucans and fructose. Cofactors are not required. Amylosucrase activity so far has been found only in few bacteria species, among them particularly the species Neisseria (MacKenzie et al., Can. J. Microbiol. 24 (1978), 357-362) and the enzyme has been examined only for its enzymatic activity. According to Okada et al., the partially purified enzyme from Neisseria perflava upon addition of sucrose results in the synthesis of glycogen-like polysaccharides which are branched to a small extent (Okada et al., J. Biol. Chem. 249 (1974), 126-135). Likewise, the intra- or extracellularly synthesized glucans of Neisseria perflava and Neisseria polysaccharea exhibit a certain degree of branching (Riou et al., Can. J. Microbiol. 32 (1986), 909-911). Whether these branches are introduced by the amylosucrase or via another enzyme that is present in the purified amylosucrase preparations as contamination, has so far not been elucidated. Since an enzyme introducing branching has so far not been found, it is assumed that both the polymerization and the branching reactions are catalyzed by amylosucrase (Okada et al., loc. cit).

The enzyme that is expressed in a constitutive manner in Neisseria is extremely stable, binds very strongly to the polymerization products and is competitively inhibited by the product fructose (MacKenzie et al., Can. J. Microbiol. 23 (1977), 1303-1307). The Neisseria species Neisseπa polysaccharea secretes the amylosucrase (Riou et al., loc. cit.) while in the other Neisseria species it remains in the cell. Enzymes having amylosucrase activity could only be detected in microorganisms. Plants are not known to have amylosucrases. The detection of the enzymatic activity of the amylosucrase can be achieved by detecting the synthesized glucans, as is described in Example 3, below. Detection is usually carried out by using a iodine stain. It is possible to identify bacterial colonies expressing amylosucrase by, e.g., treatment with iodine vapor. Colonies synthesizing the α-1 ,4 glucans are stained blue.

The enzyme activity of the purified enzyme can be detected on, e.g., sucrose- containing agarose plates. If the protein is applied to such a plate and incubated for about 1 h or more at 37°C, it diffuses into the agarose and catalyzes the synthesis of glucans. The latter can be detected by treatment with iodine vapor. Furthermore, the protein can be detected in native polyacrylamide gels. After a native polyacrylamide gel electrophoresis, the gel is equilibrated in sodium citrate buffer (50 mM, pH 6.5) and incubated over night in a sucrose solution (5% in sodium citrate buffer). If the gel is subsequently stained with Lugol's solution, areas in which proteins having amylosucrase activity are localized are stained blue due to the synthesis of α-1 ,4 glucans.

The protein encoded by a nucleic acid molecule according to the invention preferably has a molecular weight of 63± 20kDa, more preferably of 63± 15kDa and even more preferably of 63± 10kDa when determined in an SDS-PAGE.

In a preferred embodiment, the invention relates to nucleic acid molecules encoding an amylosucrase from a microorganism, particularly a gram negative microorganism, preferably from a bacterium of the species Neisseria and particularly preferred from

Neisseήa polysaccharea.

The nucleic acid molecules according to the invention can be any kind if nucleic acid molecule, for example, RNA or DNA, in particular cDNA or genomic DNA. They can be synthetic, partly synthetic or isolated from natural sources.

Furthermore, the present invention relates to vectors, for example, plasmids, phages, cosmids, phagemids or artificial chromosomes, containing a nucleic acid molecule according to the invention. The invention particularly relates to vectors in which the nucleic acid molecule of the invention is linked to sequences ensuring expression of the nucleic acid molecule in prokaryotic or eukaryotic host cells. Expression in this regard means transcription, preferably transcription and translation. Expression vectors have been extensively described in the art. In addition to a selection marker gene and a replication origin allowing replication in the selected host they normally contain a promoter active in the host cell and a transcription termination signal. Between promoter and termination signal there is normally at least one restriction site or one polylinker which allows insertion of a coding DNA sequence. As promoter sequence the DNA sequence which normally controls transcription of the corresponding gene can be used as long as it is active in the selected organism. This sequence can be replaced by other promoter sequences. Promoters can be used that effect constitutive expression of the gene or inducible promoters that allow a selective regulation of the expression of the gene downstream thereof. Bacterial and viral promoter sequences for the expression in prokaryotic host cells have been extensively described in the art. Promoters allowing a particularly strong expression of the gene downstream thereof, are, e.g., the T7 promoter (Studier et al., in Methods in Enzymology 185 (1990), 60-89), lacuvδ, trp, trp-lacUV5 (DeBoer et al., in Rodriguez, R.L. and Chamberlin, M.J., (Eds.), Promoters, Structure and Function; Praeger, New York, 1982, pp. 462-481 ; DeBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25), lp-| , rac (Boros et al., Gene 42 (1986), 97-100) or the ompF promoter. Vectors for the expression of heterologous genes in yeasts have also been described (e.g., Bitter et al., Methods in Enzymology 153 (1987), 516-544). These vectors, in addition to a selection marker gene and a replication origin for the propagation in bacteria, contain at least one further selection marker gene that allows identification of transformed yeast cells, a DNA sequence allowing replication in yeasts and a polylinker for the insertion of the desired expression cassette. The expression cassette is constructed from promoter, DNA sequence to be expressed and a DNA sequence allowing transcriptional termination and polyadenylation of the transcript. Promoters and transcriptional termination signals from Saccharomyces have also been described and are available. An expression vector can be introduced into yeast cells by transformation according to standard techniques (Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990). Cells containing the vector are selected and propagated on appropriate selection media. Yeasts furthermore allow to integrate the expression cassette via homologous recombination into the genome of a cell using an appropriate vector, leading to a stable inheritance of the feature.

Furthermore, the present invention relates to host cells transformed with a nucleic acid molecule or with a vector according to the invention. Suitable host cells are prokaryotic cells, such as microorganisms, e.g. bacteria, such as E. coli, Bacillus, Streptococcus etc., or eukaryotic cells, e.g. fungal cells, such as Saccharomyces cerevisiae; plant cells or animal cells, e.g. insect cells, CHO cells etc. Moreover, the present invention relates to a process for producing a protein having amylosucrase activity comprising culturing a host cell according to the invention under conditions allowing expression of the protein and recovering the protein from the cells and/or the culture medium.

The present invention also relates to a protein having the enzymatic activity of an amylosucrase which is encoded by a nucleic acid molecule according to the invention, or which is obtainable by the process according to the invention.

In another aspect the present invention relates to a process for producing α-1 ,4 glucans and/or fructose comprising

(a) culturing a host cell according to the invention which secrets the amylosucrase into the culture medium in a medium which contains sucrose and under conditions allowing expression and secretion of the amylosucr-ϋse; and

(b) recovering the produced α-1 ,4 glucans and/or the fructose from the culture medium.

The above described process now allows to produce pure α-1 ,4 glucans in vitro. The amylosucrase expressed by Neisseπa polysaccharea is an extracellular enzyme which synthesizes linear α-1 ,4 glucans outside of the cells on the basis of sucrose. Unlike in the most pathways of synthesis for polysaccharides that proceed within the cell, neither activated glucose derivatives nor cofactors are required. The energy that is required for the formation of the α-1 ,4 glucosidic link between the condensed glucose residues is directly obtained from the hydrolysis of the link between the glucose and the fructose unit in the sucrose molecule.

It is therefore possible to cultivate amylosucrase-secreting host cells in a sucrose- containing medium, with the secreted amylosucrase leading to a synthesis of α-1 ,4 glucans from sucrose in the medium. These glucans can be isolated from the culture medium. Furthermore, the process according to the invention allows to produce in an inexpensive manner pure fructose syrup. Conventional methods for the production of fructose either contemplate the enzymatic hydrolysis of sucrose using an invertase or the degradation of starch into glucose units, often by acidolysis, and subsequent enzymatic conversion of the glucose into fructose by glucose isomerase. Both methods result in mixtures of glucose and fructose. The two components have to be separated from each other by chromatographic processes which are time consuming and expensive.

In the process according of the invention, the separation of the substrate, sucrose, from the two reaction products, fructose and α-1 ,4 glucans, or separation of the two reaction products can be achieved by, e.g., using membranes allowing the permeation of fructose but not of sucrose or glucans. If the fructose is continuously removed via such a membrane, the sucrose is converted more or less completely into fructose and linear glucans.

Also the amylosucrase producing cells can preferably be immobilized on a carrier material located between two membranes, one of which allows the permeation of fructose but not of sucrose or glucans and the other allows the permeation of sucrose but not of glucans. The substrate is supplied through the membrane which allows the permeation of sucrose. The synthesized glucans remain in the space between the two membranes and the released fructose can continuously be removed from the reaction equilibrium through the membrane which allows only the permeation of fructose. Such a set-up allows an efficient separation of the reaction products and thus inter alia the production of pure fructose.

The use of amylosucrases for the production of pure fructose offers the advantage that the comparably inexpensive substrate sucrose can be used as starting material and furthermore that the fructose can be isolated from the reaction mixture in a simple manner without chromatographic processes.

In a preferred embodiment the host cells used in the process is a microorganism, such as Saccharomyces cerevisiae or E. coli, and even more preferably the host cell is immobilized. Immobilization generally is achieved by inclusion of the cells in an appropriate material such as, e.g., alginate, polyacrylamide, gelatin, cellulose or chitosan. It is, however, also possible to adsorb or covalently bind the cells to a carrier material (Brodelius and Mosbach, in Methods in Enzymology, Vol. 135:173- 175). An advantage of the immobilization of cells is that considerably higher cell densities can be achieved than by cultivation in a liquid culture, resulting in a higher productivity. Also the costs for agitation and ventilation of the culture as well as for the measures for maintaining sterility are reduced. An important aspect is that immobilization allows a continuous production so that long unproductive phases which usually occur in fermentation processes can be avoided or can at least be considerably reduced. As mentioned above, yeast cells expressing an amylosucrase can be used as a microorganism in the process. Cultivation methods for yeasts have been sufficiently described (Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990). Immobilization of the yeasts is also possible and is already used in the commercial production of ethanol (Nagashima et al., in Methods in Enzymology 136, 394-405; Nojima and Yamada, in Methods in Enzymology 136, 380-394).

However, the use of yeasts secreting amylosucrase for the synthesis of α-1 ,4 glucans in sucrose-containing media is not readily possible as yeasts secrete an invertase that hydrolyzes extracellular sucrose. The yeasts import the resulting hexoses via a hexose transporter. Gozalbo and Hohmann (Current Genetics 17 (1990), 77-79), however, describe a yeast strain that carries a defective suc2 gene and that therefore cannot secrete invertase. Also, these yeast cells do not contain a transport system for importing sucrose into the cells. If such a strain is modified with the nucleic acid molecule of the invention such that it secretes an amylosucrase into the culture medium, α-1 ,4 glucans are synthesized by the amylosucrase if the culture medium contains sucrose. The fructose being formed as reaction product may subsequently be imported by the yeasts.

Furthermore, the present invention relates to a process for the production of α-1 ,4 glucans and/or fructose in vitro comprising the step of bringing a protein according to the invention into contact with a sucrose-containing solution under conditions allowing the conversion of sucrose to α-1 ,4 glucans and fructose and recovering the produced α-1 ,4 glucans and/or fructose from the solution.

In particular, it is possible to synthesize α-1 ,4 glucans in vitro with the help of a cell- free enzyme preparation. This may be obtained, for example, by cultivating amylosucrase-secreting host cells in a sucrose-free medium allowing expression of the amylosucrase until the stationary growth phase is reached. After removal of the cells from the growth medium by centrifugation the secreted enzyme can be obtained from the supernatant. The enzyme can then be added to sucrose-containing solutions to synthesize α-1 ,4 glucans and fructose. As compared to the synthesis of α-1 ,4 glucans directly in a sucrose-containing growth medium this method is advantageous in that the reaction conditions can be better controlled and that the reaction products are substantially purer and can more easily be further purified. The enzyme can be purified from the culture medium by conventional purification techniques such as precipitation, ion exchange chromatography, affinity chromatography, gel filtration, HPLC reverse phase chromatography, etc. It is furthermore possible to express a polypeptide by modification of the DNA sequence inserted into the expression vector leading to a polypeptide which can be isolated more easily from the culture medium due to certain properties. It is possible to express the enzyme as a fusion protein along with another polypeptide sequence whose specific binding properties allow isolation of the fusion protein via affinity chromatography.

Known techniques are, e.g., expression as fusion protein along with glutathion S transferase and subsequent purification via affinity chromatography on a glutathion column, making use of the affinity of the glutathion S transferase to glutathion (Smith and Johnson, Gene 67 (1988), 31-40). Another known technique is the expression as fusion protein along with the maltose binding protein (MBP) and subsequent purification on an amylose column (Guan et al., Gene 67 (1988), 21-30; Maina et al., Gene 74 (1988), 365-373).

In a preferred embodiment, the amylosucrase in such a process is immobilized. In addition to the possibility of directly adding the purified enzyme to a sucrose- containing solution to synthesize α-1 ,4 glucans, there is the alternative of immobilizing the enzyme on a carrier material. Such immobilization offers the advantage that the enzyme as synthesis catalyst can easily be retrieved and can be used several times. Since the purification of enzymes usually is very time and cost intensive, an immobilization and reuse of the enzyme contributes to a considerable reduction of the costs. Another advantage is the high degree of purity of the reaction products which inter alia is due to the fact that the reaction conditions can be better controlled when immobilized enzymes are used. The insoluble linear glucans yielded as reaction products can then be easily purified further.

There are many carrier materials available for the immobilization of proteins which can be coupled to the carrier material either by covalent or non-covalent links (for an overview see: Methods in Enzymology Vol. 135, 136 and 137). Widely used carrier materials are, e.g., agarose, cellulose, polyacrylamide, silica or nylon.

A further possibility of the use of proteins having amylosucrase activity is to use them for the production of cyclodextrins. Cyclodextrins are produced by the degradation of starch by the enzyme cyclodextrin transglycosyiase (EC 2.4.1.19) which is obtained from the bacterium Bacillus macerans. Due to the branching of starch only about 40% of the glucose units can be converted to cyclodextrins using this system. By providing substantially pure proteins having amylosucrase activity it is possible to synthesize cyclodextrins on the basis of sucrose under the simultaneous action of amylosucrase and cyclodextrin transglycosyiase, with the amylosucrase catalyzing the synthesis of linear glucans from sucrose and the cyclodextrin transglycosyiase catalyzing the conversion of these glucans into cyclodextrins.

Abbreviations used

IPTG isopropyl β-D-thiogalacto-pyranoside

Media and solutions used

YT medium 8 g bacto-tryptone 5 g yeast extract

5 g NaCI ad 1000 ml with ddH2θ

YT plates YT medium with 15 g bacto-agar/

1000 ml

Lugol's solution 12 g Kl

6 g l₂ ad 1.8 l with ddH2θ

The examples serve to illustrate the invention.

Example 1

Isolation of a genomic DNA sequence coding for an amylosucrase activity from Neisseria polysaccharea

For the isolation of a DNA sequence coding for an amylosuciase activity from Neisseria polysaccharea first a genomic DNA library was established. Neisseria polysaccharea cells were cultured on "Columbia blood agar" (Difco) for 2 days at 37°C. The resulting colonies were harvested from the plates. Genomic DNA was isolated according to the method of Ausubel et al. (in: Current Protocols in Molecular Biology (1987), J. Wiley & Sons, NY) and processed. The DNA thus obtained was partially digested with the restriction endonuclease Sau3A. The resulting DNA fragments were ligated into the BamH\ digested vector pBluescript SK(-). The ligation products were transformed in £ co//XL1-Blue cells. For their selection, the cells were plated onto YT plates with ampicillin as selection marker. The selection medium additionally contained 5% sucrose and 1 mM IPTG. After incubation over night at 37°C the bacterial colonies that had formed were stained with iodine by placing crystalline iodine into the lid of a petri dish and placing the culture dishes with the bacteria colonies for 10 min each conversely onto the petri dish. The iodine which evaporated at room temperature stained some regions of the culture dishes that contained amylose-like glucans blue. From bacteria colonies that showed a blue corona plasmid DNA was isolated according to the method of Birnboim & Doly (Nucleic Acids Res. 7 (1979), 1513-1523). Said DNA was retransformed in E. coli SURE cells. The transformed cells were plated onto YT plates with ampicillin as selection marker. Positive clones were isolated.

Example 2

Sequence analysis of the genomic DNA insert of the plasmid pNB2

From an E. coli clone obtained according to working example 1 a recombinant plasmid was isolated. Restriction analyses showed that said plasmid was a ligation product consisting of two vector molecules and an approx. 4.2 kb long genomic fragment. The plasmid was digested with the restriction endonuclease Pst\ and the genomic fragment was isolated (GeneClean, Bio101). The fragment thus obtained was ligated into a pBluescript II SK vector linearized with Pst\, resulting in a duplication of the Psfl and Smal restriction sites. The ligation product was transformed in E. coli cells and the latter were plated on ampicillin plates for selection. Positive clones were isolated. From one of these clones a plasmid was isolated and part of the sequence of its genomic DNA insert was determined by standard techniques using the dideoxy method (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467). The entire insert is approx. 4.2 kbp long. The nucleotide sequence was determined and is indicated in SEQ ID NO. 1.

Example 3

Expression of an extracellular amylosucrase activity in transformed E. coli cells

(a) Detection of an amylosucrase activity during growth on YT plates For the expression of an extracellular amylosucrase activity, E. coli cells were transformed with the isolated plasmid vector according to standard techniques. A colony of the transformed strain was incubated on YT plates (1.5% agar; 100 μg/ml ampicillin; 5% sucrose; 0.2 mM IPTG) over night at 37°C. The amylosucrase activity was detected by subjecting the colonies to iodine vapor. Amylosucrase-expressing colonies exhibit a blue corona. Amylosucrase activity can be observed even if no IPTG was present, probably due to the activity of the endogenous amylosucrase promoters.

(b) Detection of an amylosucrase activity during growth in YT medium

For the expression of an extracellular amylosucrase activity, E. coli were transformed with the isolated plasmid vector according to standard techniques. YT medium (100 μg/ml ampicillin; 5% sucrose) was inoculated with a colony of the transformed strain. The cells were incubated over night at 37°C under constant agitation (rotation mixer; 150-200 rpm). The products of the reaction catalyzed by amylosucrase were detected by adding Lugol's solution to the culture supernatant, leading to blue staining.

(c) Detection of the amylosucrase activity in the culture supernatants of transformed E. coli cells which were cultivated without sucrose

For the expression of an extracellular amylosucrase activity, E. coli cells were transformed with the isolated plasmid vector according to standard techniques. YT medium (100 μg/ml ampicillin) was inoculated with a colony of the transformed strain. The cells were incubated over night at 37°C under constant agitation (rotation mixer; 150-200 rpm). Then the cells were removed by centrifugation (30 min, 4°C, 5500 rpm, JA10 Beckmann rotor). The supernatant was filtered through a 0.2 μm filter (Schleicher & Schuell) under sterile conditions. Detection of an amylosucrase activity was carried out by (i) incubating the supernatant on a sucrose-containing agar plate. 40 μl of the supernatant were placed in a whole punched into an agar plate (5% sucrose in 50 mM sodium citrate buffer pH 6.5) and incubated at least for one hour at 37°C. The products of the reaction catalyzed by amylosucrase were detected by staining with iodine vapor. Presence of the reaction products produces a blue stain.

(ii) or by gel electrophoretic separation of the proteins of the supernatant in a native gel and detection of the reaction products in the gel after incubation with sucrose. 40-80μl of the supernatant were separated by gel electrophoresis on an 8% native polyacrylamide gel (0.375 M Tris pH 8.8) at a voltage of 100 V. The gel was then twice equilibrated 15 min with approx. 100 ml 50 mM sodium citrate buffer (pH 6.5) and incubated over night at 37°C in sodium citrate buffer pH 6.5/5% sucrose. In order to make the reaction product of the reaction catalyzed by amylosucrase visible, the gel was rinsed with Lugol's solution. Bands having amylosucrase activity were stained blue.

Example 4

In vitro production of glucans with partially purified amylosucrase

For the expression of an extracellular amylosucrase activity, E. coli cells were transformed with the isolated plasmid vector according to standard techniques. YT medium (100 μg/ml ampicillin) was inoculated with a colony of the transformed strain. The cells were incubated over night at 37°C under constant agitation (rotation mixer; 150-200 rpm). Then the cells were removed by centrifugation (30 min, 4°C, 5500 rpm, JA10 Beckmann rotor). The supernatant was filtered through a 0.2 μm filter (Schleicher & Schuell) under sterile conditions.

The supernatant was then concentrated by 200 times using an Amicon chamber (YM30 membrane having an exclusion size of 30 kDa, company Amicon) under pressure (p=3 bar). The concentrated supernatant was added to 50 ml of a sucrose solution (5% sucrose in 50 mM sodium citrate buffer pH 6.5). The entire solution was incubated at 37°C. Whitish insoluble polysaccharides are formed.

Example 5

Characterization of the reaction products synthesized by amylosucrase from Example 4

The insoluble reaction products described in Example 4 are soluble in 1 M NaOH. The reaction products were characterized by measuring the absorption maximum. Approx. 100 mg of the isolated reaction products (wet weight) were dissolved in 200 μl 1 M NaOH and diluted with H2O 1 :10. 900 μl of 0.1 M NaOH and 1 ml Lugol's solution were added to 100 μl of this dilution. The absorption spectrum was measured between 400 and 700 nm. The maximum is 605 nm (absorption maximum of amylose: approx. 614 nm).

HPLC analysis of the reaction mixture of Example 4 on a CARBOPAC PA1 column (DIONEX) showed that in addition to the insoluble products soluble products were also formed. These soluble products are short-chained polysaccharides. The chain length was between approx. 5 and approx. 60 glucose units. To a smaller extent, however, even shorter or longer molecules could be detected. With the available analytical methods it was not possible to detect branching in the synthesis products.

Example 6

Expression of an intracellular amylosucrase activity in transformed E. coli cells

Using a polymerase chain reaction (PCR) a fragment was amplified from the isolated plasmid vector which comprises the nucleotides 981 to 2871 of the sequence depicted in SEQ ID NO. 1. The following oligonucleotides were used as primers: TPN2 5' - CTC ACC ATG GGC ATC TTG GAC ATC - 3'

(SEQ ID NO. 3)

TPC1 5' - CTG CCA TGG TTC AGA CGG CAT TTG G - 3'

(SEQ ID NO. 4)

The resulting fragment contains the coding region for amylosucrase except for the nucleotides coding for the 16 N-terminal amino acids. These amino acids comprise the sequences that appear to be necessary for the secretion of the enzyme from the cell. Furthermore, this PCR fragment contains 88 bp of the 3' untranslated region. By way of the primers used Λ/coi restriction sites were introduced into both ends of the fragment.

After digestion with the restriction endonuclease Λ/col the resulting fragment was ligated with the A/col digested expression vector pMex 7. The ligation products were transformed in E. coli cells and transformed clones were selected. Positive clones were incubated over night at 37°C on YT plates (1.5% agar; 100 μg/ml ampicillin; 5% sucrose; 0.2 mM IPTG). After subjecting the plates to iodine vapor no blue staining could be observed in the area surrounding the bacteria colonies, but the intracellular production of glycogen could be detected (brown staining of transformed cells in contrast to no staining in nontransformed XL1-Blue cells). In order to examine the functionality of the protein, transformed cells cultivated on YT medium were broken up by ultrasound and the obtained crude extract was pipetted onto sucrose- containing agar plates. After subjecting the plates to iodine vapor a blue stain could be observed.

Claims

1. A nucleic acid molecule encoding a protein having the enzymatic activity of an amylosucrase, selected from the group consisting of

(a) nucleic acid molecules encoding a protein comprising the amino acid sequence depicted under SEQ ID NO. 2;

(b) nucleic acid molecules comprising the coding region depicted under SEQ ID NO. 1 ;

(d) nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule as defined in (c) due to the degeneracy of the genetic code.

2. The nucleic acid molecule of claim 1 which is genomic DNA.

3. A vector containing a nucleic acid molecule of claim 1 or 2.

4. The vector of claim 3, in which the nucleic acid molecule encoding a protein having the enzymatic activity of an amylosucrase is functionally linked to sequences allowing expression in prokaryotic or eukaryotic host cells.

5. A host cell transformed with a nucleic acid molecule of claim 1 or 2 or with a vector of claim 3 or 4.

6. A process for producing a protein with the enzymatic activity of an amylosucrase comprising culturing the host cell of claim 5 under conditions allowing expression of the amylosucrase and recovering the protein from the cells and/or the culture medium.

7. A protein having the enzymatic activity of an amylosucrase which is encoded by a nucleic acid molecule of claim 1 or 2 or which is obtainable by the process of claim 6.

8. A process for the production of ╬▒-1 ,4 glucans and/or fructose comprising

(a) culturing a host cell of claim 5 which secrets the amyiosucrase into the culture medium in a sucrose-containing culture medium under conditions allowing expression and secretion of the amylosucrase; and

(b) recovering the produced ╬▒-1 ,4 glucans and/or fructose from the culture medium.

9. The process of claim 8, wherein the host cell is a microorganism.

10. The process of claim 9 or 10, wherein the host cell is immobilized.

11. A process for the production of ╬▒-1 ,4 glucans and/or fructose in vitro comprising

(a) contacting a sucrose-containing solution with a protein of claim 7 under conditions allowing the conversion of sucrose to ╬▒-1 ,4 glucans and fructose by the amylosucrase; and

(b) recovering the produced ╬▒-1 ,4 glucans and/or fructose from the solution.

12. The process of claim 11 , wherein the protein is immobilized.