EP1109916A1 - Molecules d'acide nucleique codant une amylosucrase - Google Patents

Molecules d'acide nucleique codant une amylosucrase

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Publication number
EP1109916A1
EP1109916A1 EP98948899A EP98948899A EP1109916A1 EP 1109916 A1 EP1109916 A1 EP 1109916A1 EP 98948899 A EP98948899 A EP 98948899A EP 98948899 A EP98948899 A EP 98948899A EP 1109916 A1 EP1109916 A1 EP 1109916A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
amylosucrase
glucans
protein
fructose
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98948899A
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German (de)
English (en)
Inventor
Martin Quanz
Nicholas Provart
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer Bioscience GmbH
Original Assignee
Planttec Biotechnologie GmbH Forschung and Entwicklung
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Planttec Biotechnologie GmbH Forschung and Entwicklung filed Critical Planttec Biotechnologie GmbH Forschung and Entwicklung
Publication of EP1109916A1 publication Critical patent/EP1109916A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)

Definitions

  • 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 ⁇ -1 ,4 glucans and fructose using the described nucleic acid molecules or the encoded proteins.
  • Linear ⁇ -1 ,4 glucans are polysaccharides consisting of glucose monomers, the latter being exclusively linked to each other by ⁇ -1 ,4 glycosidic bonds.
  • the most frequently occurring natural ⁇ -1,4 glucan is amylose, a component of plant starch. Recently, more and more importance has been attached to the commercial use of linear ⁇ -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.
  • 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.
  • ⁇ -1 ,4 glucans in particular linear ⁇ -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 ⁇ -1 ,4 linked glucose units. The other component forms the amylopectin, a highly branched polymer from glucose units in which in addition to the ⁇ -1 ,4 links the glucose chains can also be branched via ⁇ -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 ⁇ -1 ,4 glucans from sucrose. In order to be able to use such enzymes for the efficient production of ⁇ -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 ⁇ -1 ,4 glucans.
  • nucleic acid molecules encoding a protein comprising the amino acid sequence as depicted in SEQ ID NO: 2;
  • 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 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • This reaction is a transglucosylation.
  • the transglucosylation can take place in the presence or absence of acceptor molecules.
  • acceptor molecules can be polysaccharides, such as maltooligosaccharides, dextrin, glycogen etc.
  • acceptor molecules can be polysaccharides, such as maltooligosaccharides, dextrin, glycogen etc.
  • the resulting product is a polymeric linear ⁇ -1 ,4-glucan.
  • 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 ⁇ -1 ,4 glucans.
  • G-F+n(G-F) ⁇ G n -G-F+ ⁇ F wherein G-F is sucrose.
  • G glucose
  • F fructose
  • G n -G-F is an ⁇ -1 ,4 glucan.
  • G-F sucrose
  • F fructose
  • G is glucose.
  • the products of the reaction catalyzed by an amylosucrase are the above described ⁇ -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.
  • Neisseria 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 Neisse ⁇ a 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 ⁇ -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 ⁇ -1 ,4 glucans.
  • 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
  • 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.
  • 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.
  • 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.
  • 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), lacuv ⁇ , 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.
  • 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.
  • 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.
  • 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 relates to a process for producing ⁇ -1 ,4 glucans and/or fructose comprising
  • the above described process now allows to produce pure ⁇ -1 ,4 glucans in vitro.
  • the amylosucrase expressed by Neisse ⁇ a polysaccharea is an extracellular enzyme which synthesizes linear ⁇ -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 ⁇ -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.
  • amylosucrase-secreting host cells in a sucrose- containing medium, with the secreted amylosucrase leading to a synthesis of ⁇ -1 ,4 glucans from sucrose in the medium.
  • These glucans can be isolated from the culture medium.
  • 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.
  • the separation of the substrate, sucrose, from the two reaction products, fructose and ⁇ -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.
  • 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.
  • 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.
  • 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).
  • yeasts secreting amylosucrase for the synthesis of ⁇ -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 describe a yeast strain that carries a defective suc2 gene and that therefore cannot secrete invertase.
  • these yeast cells do not contain a transport system for importing sucrose into the cells.
  • ⁇ -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.
  • the present invention relates to a process for the production of ⁇ -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 ⁇ -1 ,4 glucans and fructose and recovering the produced ⁇ -1 ,4 glucans and/or fructose from the solution.
  • 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.
  • fusion protein 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).
  • the amylosucrase in such a process is immobilized.
  • 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.
  • 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.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • 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 ⁇ /coi restriction sites were introduced into both ends of the fragment.
  • 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 ⁇ g/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).
  • 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.

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Abstract

L'invention porte sur des molécules d'acide nucléique qui codent une amylosucrase, et sur des procédés de production d'α-1,4 glycanes et de fructose au moyen de ces molécules d'acide nucléique ou des protéines codées. L'invention porte en outre sur des cellules hôtes transformées par les molécules d'acide nucléique précitées.
EP98948899A 1998-09-02 1998-09-02 Molecules d'acide nucleique codant une amylosucrase Withdrawn EP1109916A1 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP1998/005573 WO2000014249A1 (fr) 1998-09-02 1998-09-02 Molecules d'acide nucleique codant une amylosucrase

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EP1109916A1 true EP1109916A1 (fr) 2001-06-27

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JP (1) JP2002524080A (fr)
AU (1) AU9535798A (fr)
CA (1) CA2342124A1 (fr)
HU (1) HUP0103414A3 (fr)
WO (1) WO2000014249A1 (fr)

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WO2000014249A1 (fr) 2000-03-16
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CA2342124A1 (fr) 2000-03-16
AU9535798A (en) 2000-03-27

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