EP3990655A1 - Production enzymatique de fructo-oligosaccharides prébiotiques à base de lévane - Google Patents

Production enzymatique de fructo-oligosaccharides prébiotiques à base de lévane

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Publication number
EP3990655A1
EP3990655A1 EP20734888.9A EP20734888A EP3990655A1 EP 3990655 A1 EP3990655 A1 EP 3990655A1 EP 20734888 A EP20734888 A EP 20734888A EP 3990655 A1 EP3990655 A1 EP 3990655A1
Authority
EP
European Patent Office
Prior art keywords
levansucrase
endolevanase
nucleotide sequence
host organism
encodes
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.)
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Application number
EP20734888.9A
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German (de)
English (en)
Inventor
Uwe Deppenmeier
Marcel HÖVELS
Konrad Kosciow
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.)
Univ Bonn Rheinische Friedrich Wilhelms
Rheinische Friedrich Wilhelms Universitaet Bonn
Original Assignee
Univ Bonn Rheinische Friedrich Wilhelms
Rheinische Friedrich Wilhelms Universitaet Bonn
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Application filed by Univ Bonn Rheinische Friedrich Wilhelms, Rheinische Friedrich Wilhelms Universitaet Bonn filed Critical Univ Bonn Rheinische Friedrich Wilhelms
Publication of EP3990655A1 publication Critical patent/EP3990655A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • 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)
    • C12N9/1055—Levansucrase (2.4.1.10)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/0101—Levansucrase (2.4.1.10)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01065—Levanase (3.2.1.65)

Definitions

  • the present invention relates to a method for preparing levan or levan-based fructooligosaccharides (FOS) using at least one (genetically modified) host organism or enzymes recombinantly expressed in such host organisms. Specifically, the expression of a levansucrase and/or an endolevanase in the host organism allows to convert the substrate sucrose into levan and/or FOS.
  • FOS fructooligosaccharides
  • the invention is directed to levan and/or fructooligosaccharides obtainable by the method according to the invention; a specific expression vector, a specific genetically modified host organism, and a specific cell extract or culture supernatant, and purified or immobilized enzymes which are usable for the production of levan and/or FOS; and a prebiotic or food supplement comprising or consisting of the levan and/or FOS.
  • Levan is a polymer of p-2,6-glycosidically linked fructose units.
  • the structural formula of a monomeric fructose unit of the levan backbone is shown below:
  • levansucrases EC 2.4.1.10
  • the substrate for the enzymatic reaction is preferably the disaccharide sucrose, which is hydrolyzed in an initial step into the monomer subunits glucose and fructose. While the glucose is released from the active center, the remaining fructose is coupled to a new sucrose molecule. This process, in which the sucrose serves as the terminal acceptor, can be repeated cyclically, whereby fructose units from the sucrose are successively added to the growing levan chain.
  • a prebiotic is defined as "a substrate that is selectively utilized by host microorganisms conferring a health benefit" (Gibson et al. (2017) Nat. Rev. Gastroenterol. Hepatol). In earlier definitions, selectivity mostly referred to members of the genera Lactobacillus and Bifidobacterium. Especially the specific stimulation of bifidobacteria (bifidogenesis) was considered to have a prebiotic effect. Meanwhile, further representatives of intestinal microbiota have been identified, which mediate positive effects on host health.
  • SCFAs short chain fatty acids
  • C2 body the short chain fatty acids
  • C3 propionate
  • C4 n- butyrate
  • the physiological effects of these SCFAs on the local and systemic level are versatile and influence, among others, the functionality of colonocytes, intestinal homeostasis, the immune system, composition and number of blood lipids, appetite and renal physiology (Roberfroid et al. (2010) Br. J. Nutr. 104 Suppl:S1 -63, doi: 10.1017/S00071 14510003363; O’Keefe (2016) Nat. Rev. Gastroenterol. Hepatol.
  • the fructose-based polymer levan is also considered a promising prebiotic. As has been verified in numerous in vivo studies (Jang et al. (2003) J. Microbiol. Biotechnol.; Hamdy et al. (2016) Biocatal. Agric. Biotechnol., doi: 10.1016/j.bcab.2017.12.001) and in vitro (Porras-Dominguez et al. (2014) Process Biochem., doi: 10.1016/j.procbio.2014.02.005; Adamberg (2015) PLoS One., doi:
  • levan and levan-type FOS can be selectively fermented by beneficial representatives of our intestinal flora and thus has a beneficial effect on host health.
  • Levan has unique physicochemical properties, including antioxidant (Liu et al. (2012) Food Chem. Toxicol., doi: 10.1016/j.fct.201 1 .1 1 .016), anti-inflammatory (Srikanth et al. (2015) Carbohydr. Polym., doi:10.1016/j.carbpol.2014.12.079), antibacterial (Byun et al. (2014) Int. J. Food. Sci. Technol., doi: 10.1 1 1 1/ijfs.12304) and antiviral (Esawy et al. (201 1) Carbohydr. Polym., doi: 10.1016/j.carbpol.201 1 .05.035) functions.
  • the inventors were able to develop a method to circumvent the problematic and labor- intensive purification of high-molecular levan chains.
  • the combined activity of two enzymes (levansucrase and endolevanase), in particular the levansucrase from G. japonicus LMG 1417 and the endolevanase from Azotobacter (A.) chroococcum DSM 2286 which are newly characterized, allows the production of (short-chain) fructooligosaccharides based on levan, preferably by starting from the renewable and low-cost substrate sucrose.
  • the (short-chain) FOS can be easily purified from industrial process solutions, e.g. by using suitable filtration systems.
  • the invention relates to a method for preparing fructooligosaccharides (FOS) comprising contacting an endolevanase enzyme comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6 or an amino acid sequence that has at least 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity with the amino acid sequence set forth in SEQ ID Nos. 5 or 6 over the full length of the sequence or active fragments thereof with levan under conditions suitable for producing fructooligosaccharides (FOS).
  • an endolevanase enzyme comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6 or an amino acid sequence that has at least 60 %, 70 %, 75 %, 80 %, 85 %,
  • the fragments of the endolevanase comprise or consist of the amino acid sequence of SEQ ID NO:6 but lack the first 35 N-terminal amino acids of SEQ ID NO:6, i.e. start with A36. Accordingly, also comprised are fragments of the above-described endolevanase that retain at least 75 % of the activity of the full length sequence but lack one or more N-terminal and/or C-terminal amino acids.
  • the present invention is directed to a method for preparing fructooligosaccharides (FOS) comprising or consisting of the following steps:
  • nucleic acid molecule comprising a nucleotide sequence which encodes an endolevanase, preferably comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6 or an amino acid sequence that has at least 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity with the amino acid sequence set forth in SEQ ID Nos.
  • a host organism comprising a nucleotide sequence which encodes an endolevanase comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6 or an amino acid sequence that has at least 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity with the amino acid sequence set forth in SEQ ID Nos. 5 or 6 over the full length of the sequence or active fragments thereof;
  • step b) cultivating the host organism of step b) under conditions which allow the expression of the nucleotide sequence
  • step (d) preparing fructooligosaccharides using the endolevanase expressed in step (c), by subjecting the enzyme to conditions which allow the production of the fructooligosaccharides. These conditions may comprise the contacting of the enzyme with levan as a substrate.
  • the endolevanase is
  • step (i) comprised in a culture supernatant from the culture medium used in step (c), wherein the culture supernatant is subjected to conditions which allow the preparation of said fructooligosaccharides;
  • step (ii) comprised in a cell extract from the host organism cultivated in step (c), wherein the cell extract is subjected to conditions which allow the preparation of said fructooligosaccharides;
  • step (iii) comprised in a host organism or present at the surface of the host organism cultivated in step (c), wherein the host organism is subjected to conditions which allow the preparation of said fructooligosaccharides; and/or
  • the invention relates to a method for preparing fructooligosaccharides (FOS) comprising or consisting of the following steps:
  • nucleic acid molecule comprising a first nucleotide sequence which encodes a levansucrase and/or a second nucleotide sequence which encodes an endolevanase into at least one host organism;
  • a host organism comprising a first nucleotide sequence which encodes a levansucrase and a second nucleotide sequence which encodes an endolevanase;
  • a first host organism comprising a first nucleotide sequence which encodes a levansucrase and a second host organism comprising a second nucleotide sequence which encodes an endolevanase;
  • the host organism comprising the first nucleotide sequence which encodes a levansucrase and the second nucleotide sequence which encodes an endolevanase under conditions which allow the expression of the first and the second nucleotide sequence;
  • step (ii) the first host organism comprising the first nucleotide sequence which encodes a levansucrase and the second host organism comprising the second nucleotide sequence which encodes an endolevanase under conditions which allow the expression of the first and the second nucleotide sequence; and (d) preparing fructooligosaccharides using the levansucrase and the endolevanase expressed in step (c), by subjecting the enzymes to conditions which allow the production of the fructooligosaccharides.
  • step (d) the levansucrase and the endolevanase are, individually or together,
  • step (i) comprised in a culture supernatant from the culture medium used in step (c), wherein the culture supernatant is subjected to conditions which allow the preparation of said fructooligosaccharides;
  • step (ii) comprised in a cell extract from the at least one host organism cultivated in step (c), wherein the cell extract is subjected to conditions which allow the preparation of said fructooligosaccharides;
  • step (iii) comprised in a host organism or at the surface of the host organism cultivated in step (c), wherein the host organism is subjected to conditions which allow the preparation of said fructooligosaccharides;
  • the method according to the invention comprises or consists of the following steps:
  • nucleic acid molecule comprising a first nucleotide sequence which encodes a levansucrase and a second nucleotide sequence which encodes an endolevanase into a host organism;
  • step (d) using the levansucrase and the endolevanase obtained in step (c) and comprised in the cell extract from the host organism or in the culture supernatant from the culture medium after step (c), and subjecting said cell extract or culture supernatant to conditions which allow the preparation of said fructooligosaccharides.
  • the fructooligosaccharides obtained in step (d) of all methods described herein comprise, essentially consist of or consist of compounds of the formulas F m and/or GF n , wherein
  • F is a monomeric fructose unit, preferably D-fructose unit
  • G is a monomeric glucose unit, preferably D-glucose unit
  • m is >3, preferably 3 to 20, more preferably 3 to 15, more preferably 3 to 13, most preferably 3 to 10; n is >2, preferably 2 to 19, more preferably 2 to 14, more preferably 2 to 12, most preferably 2 to 9; m and/or n are the same or different in the individual fructooligosaccharides obtained in step (d); and the fructose units are covalently coupled to each other by b-(2®6) linkages and may further comprise b-(2®1) branching.
  • the fructooligosaccharides obtained in step (d) of the method for preparing fructooligosaccharides according to the invention are preferably also referred to as short-chain fructooligosaccharides.
  • the (amino acid sequence of the) levansucrase comprises or consists of
  • an amino acid sequence which has at least 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity with the amino acid sequence set forth in SEQ ID Nos. 1 or 2 over the full length of the sequence.
  • the levansucrase comprises or consists of the amino acid sequence of SEQ ID NO:2 but lacks the N-terminal M of SEQ ID NO:2. Also comprised are fragments of the above- described levansucrases that retain at least 75 % of the activity of the full length sequence but lack one or more N-terminal and/or C-terminal amino acids.
  • the (amino acid sequence of the) endolevanase comprises or consists of
  • the endolevanase comprises or consists of the amino acid sequence of SEQ ID NO:6 but lacks the first 35 N-terminal amino acids of SEQ ID NO:6, i.e. starts with A36. Accordingly, also comprised are fragments of the above-described endolevanase that retain at least 75 % of the activity of the full length sequence but lack one or more N-terminal and/or C-terminal amino acids.
  • the (first) nucleotide sequence which encodes the levansucrase comprises or consists of
  • nucleotide sequence which has at least 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %,
  • these nucleotide sequences encode levansucrases that comprise or consist of the amino acid sequence set forth in SEQ ID Nos. 1 or 2 or an amino acid sequence which has at least 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity with the amino acid sequence set forth in SEQ ID Nos. 1 or 2 over the full length of the sequence or fragments thereof, as defined above.
  • the (second) nucleotide sequence which encodes the endolevanase comprises or consists of
  • nucleotide sequence which has at least 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity with the nucleotide sequence set forth in SEQ ID Nos. 7 or 8 over the full length of the sequence.
  • these nucleotide sequences encode endolevanases that comprise or consist of the amino acid sequence set forth in SEQ ID Nos.
  • the conditions which allow the preparation of the fructooligosaccharides comprise providing sucrose to the levansucrase and/or levan to the endolevanase used in step (d).
  • the levan may be produced in situ by a levansucrase.
  • the levan is converted to the fructooligosaccharides obtained in step (d), wherein the conversion is catalyzed by the endolevanase.
  • the sucrose is converted to the fructooligosaccharides obtained in step (d), wherein the conversion of sucrose to levan is catalyzed by the levansucrase and the conversion of the levan to the fructooligosaccharides is catalyzed by the endolevanase.
  • the levansucrase hydrolyzes the sucrose and uses the released fructose to form fructan- polymers, also referred to as levan, preferably with up to 100.000 fructose units, and then the endolevanase hydrolyzes the fructan-polymers to prepare the fructooligosaccharides obtained in step (d).
  • the method comprises after step (c) and before step (d) a further step to separate the culture medium from the cells (e.g. by centrifugation). Additionally, a further step of cell disruption and/or filtration and/or purification and/or immobilization and/or lyophilization, without being limited to these methods, can be present between step (c) and (d) of the method according to the invention.
  • the method comprises after step (d) a further step (e) for purifying the fructooligosaccharides obtained in step (d).
  • the purification step (e) may be a chromatography step or a filtration step, without being limited to these methods.
  • the at least one host organism is a bacterial or yeast organism, preferably a bacterial organism, for example an Escherichia coli or a Gluconobacter, Lactobacillus, Bifidobacterium, Zymomonas, Bacillus, Rahnella, Leuconostoc, Acetobacter, Azotobacter or Erwinia species, preferably Escherichia coli or an Azotobacter, Gluconobacter, Lactobacillus or Bifidobacterium species, more preferably an Escherichia coli or an Azotobacter (e.g.
  • Azotobacter chroococcum in particular Azotobacter chroococcum DSM 2286
  • Gluconobacter species more preferably an Escherichia coli or a Gluconobacter species, such as Gluconobacter japonicus, Gluconobacter cerinus or Gluconobacter oxydans, in particular Gluconobacter japonicus.
  • the host organism may be Escherichia coli BL21 or E. coli BL21 DE3, or Gluconobacter japonicus LMG 1417.
  • Suitable yeast organisms include, but are not limited to those of the genus Saccharomyces and Pichia, such as Saccharomyces cerevisiae and Pichia pastoris.
  • the at least one nucleic acid molecule is an expression vector.
  • the first nucleotide sequence which encodes the levansucrase and/or the second nucleotide sequence which encodes the endolevanase is
  • the method for preparing fructooligosaccharides comprising contacting an endolevanase enzyme with levan, as described herein, may be an in vitro method.
  • the isolated and/or purified enzyme that may be recombinantly produced can be contacted with its substrate under pure in vitro conditions, i.e. in the absence of any (host) organism that produces the enzyme, the substrate or both.
  • Such methods may also be multi-step methods, where in a first step levan is produced by a levansucrase and in a second step said levan is then subjected to enzymatic hydrolysis catalyzed by the endolevanase.
  • Such methods may have the advantage that undesired side reactions may be suppressed.
  • the enzymes used in such methods are those described above in connection with the methods using a host organism. It is similarly possible to perform one of these steps by using a host organism and the other by using a purified/isolated enzyme.
  • the substrate may be continuously produced by a living organism and (then) contacted with a free/isolated enzyme in solution. All these hybrid methods where one or more steps of the reaction are carried out in vitro by purified/isolated and optionally immobilized or substrate-bound enzymes are also contemplated to fall within the scope of the present invention. This particularly applies to all methods where the endolevanase described herein, i.e. having the amino acid sequence of SEQ ID NO:5 or 6, or a variant/fragment thereof is used.
  • the invention relates to fructooligosaccharides obtainable by the method according to the invention.
  • the invention relates to an expression vector comprising a first nucleotide sequence which encodes a levansucrase and/or a second nucleotide sequence which encodes an endolevanase; wherein the first nucleotide sequence which encodes the levansucrase comprises or consists of a nucleotide sequence
  • the second nucleotide sequence which encodes the endolevanase, comprises or consists of a nucleotide sequence
  • the invention is directed to a polypeptide having endolevanase activity, preferably the endolevanase enzyme described herein, said polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6 or an amino acid sequence that has at least 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity with the amino acid sequence set forth in SEQ ID Nos.
  • the invention is directed to a polypeptide having levansucrase enzyme activity, preferably the levansucrase enzyme described herein, comprising or consisting of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 or an amino acid sequence that has at least 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity with the amino acid sequence set forth in SEQ ID Nos. 1 or 2 over the full length of the sequence or active fragments thereof, preferably in isolated or purified form.
  • the invention is directed to a kit or composition that comprises both of the afore-described enzymes.
  • the invention relates to a genetically modified host organism comprising
  • the host organism may comprise these vectors, nucleic acids and/or enzymes as heterologous molecules, i.e. not produce or contain them naturally, but may be specifically engineered to produce them.
  • the invention relates to a cell extract or culture supernatant comprising the levansucrase and/or the endolevanase according to the invention.
  • the invention relates to a prebiotic or food supplement comprising or (essentially) consisting of the fructooligosaccharides according to the present invention.
  • the prebiotic or food supplement comprising or consisting of the fructooligosaccharides obtainable by the method for preparing fructooligosaccharides according to the invention can be comprised, without being limited to it, in general food products, baby food and animal food.
  • the invention relates to a method for preparing levan comprising or consisting of the following steps:
  • step (d) using the levansucrase obtained in step (c) and subjecting it to conditions which allow the preparation of levan.
  • the levansucrase is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-asucrase
  • step (i) comprised in a culture supernatant from the culture medium used in step (c), wherein the culture supernatant is subjected to conditions which allow the preparation of levan;
  • step (ii) comprised in a cell extract from the host organism cultivated in step (c), wherein the cell extract is subjected to conditions which allow the preparation of levan;
  • step (iii) comprised in a host organism or at the surface of the host organism cultivated in step (c), wherein the host organism is subjected to conditions which allow the preparation of levan;
  • FIG. 1 Macroscopic image of different G. strains on yeast mannitol agar (A) and yeast sucrose agar (B). Photographic documentation of the plated strains G. japonicus LMG 1417 (1), G. cerinus LMG 1425 (2), G. oxydans LMG 1385 (3), G. oxydans DSM 2003 (4), G. oxydans DSM 3504 (5) and G. oxydans 621 H (6) was performed after 24 hours of incubation at 28 °C. The incubation of G. japonicus LMG 1417 was additionally shown in time lapse (C).
  • FIG 3. FTIR spectra of EPS (black) produced by G.japonicus LMG 1417 and commercial levan (grey) produced by Erwinia herbicola (Blake et al. (1982) J. Bacteriol). The measurement was performed in absorption mode in a Bruker Tensor 27 FT-IR.
  • Figure 4. Plasmid map of the overexpression plasmid pASK5 JevS1417. Amp R , ampicillin resistance cassette; ori, origin of replication.
  • FIG. 1 Silver stained SDS-PAGE (A), pH profile (B) and Michaelis-Menten kinetics (C) of the purified recombinant levansucrase from G. japonicus LMG 1417.
  • the protein was purified by affinity chromatography after heterologous overproduction in E. coli DH5a. Marker of the SDS-PAGE: PageRuler Prestained Protein Ladder (ThermoFisher).
  • Figure 7 Plasmid map of the modified broad host range vector pBBR1-p264-streplong (Zeiser et al. (2014) Appl. Microbiol. Biotechnol., doi: 10.1007/s00253-013-5016-5).
  • Kan R kanamycin resistance cassette
  • rep replication protein
  • mob mob gene
  • p264 strong G. -specific promoter
  • MCS multiple cloning site.
  • FIG. 8 Plasmid map of the overexpression plasmid pBBR1_p264 _levS1417.
  • Kan R kanamycin resistance cassette
  • rep replication protein
  • mob mob gene
  • p264 strong Gluconobacter- specific promoter
  • MCS multiple cloning site.
  • Figure 9 Schematic representation of the process solution for cell-free levan production.
  • the total volume of the reaction was 10 mL.
  • the reaction solution was incubated at 30 °C.
  • FIG. 10 Reaction kinetics of the cell-free levan production based on G. japonicus LMG 1417 (A). To simplify the visualization of the less concentrated fructooligosaccharides, the scaling of the Y-axis was adapted (B). All products with a concentration of more than 10 mM are shown.
  • FIG. 11 Reaction kinetics of cell-free levan production based on the genetically modified strain G. japonicus LMG 1417 pBBR1_p264 _levS1417 (A). To simplify the visualization of the less concentrated fructooligosaccharides, the scaling of the Y-axis was adapted (B). All products with a concentration of more than 10 mM are shown.
  • FIG. 12 Comparison of the yields of different processes for levan production. The levan concentration that can be generated per L reaction solution is shown on the left. Shown on the right is the maximum yield of levan that can be obtained with one liter of the corresponding culture.
  • FIG 13 Plasmid map of the created overexpression plasmid pASK5_/evB2286. Amp R , ampicillin resistance cassette; ori, origin of replication. Figure 14. Comparison of the specific activities of different endolevanases. The quantification of the activities for the endolevanases from A. chroococcum DSM 2286, Bacteroides thetaiotaomicron DSM 2079 and Bacillus licheniformis DSM 13 was performed by HPLC.
  • FIG. 15 Plasmid map of the created overexpression plasmid pASK5 _levS1417_levB2286. Amp R , ampicillin resistance cassette; ori, origin of replication.
  • FIG. 16 Protein biochemical detection of recombinant levansucrase LevS1417 and endolevanase LevB2286 after heterologous production in E. coli and subsequent purification by streptactin affinity chromatography. The visualization was performed by western blotting (A) and silver staining (B).
  • FIG. 1 Schematic representation of the production of E. coli cell extract for the enzymatic production of levan-based FOS.
  • FIG. 18 Reaction kinetics of extract-based production of levan-type FOS from sucrose.
  • Cell extract of the strain E. coli BL21 pASK5 _levS1417_levB2286 was used for the illustrated reaction.
  • FIG. 19 Product spectrum of different endolevanases.
  • the chromatographic detection of the reaction products formed over time by the endolevanases from Azotobacter chroococcum DSM 2286 (A), Bacillus licheniformis IB1t (B) and Bacteroides thetaiotaomicron DSM 2079 (C) is shown.
  • the enzymes were used for the assays after heterologous production in Escherichia coli DH5a and subsequent purification by streptactin affinity chromatography.
  • the substrate was commercial levan (Megazyme, Bray, Ireland), which was purified from Timothy grass.
  • F fructose
  • 2 - 8 degree of polymerization of the generated FOS.
  • FIG. 20 Quantitative representation of the product spectra of various endolevanases (Fig.19). Shown are the total products generated overtime ( ⁇ ), FOS with a DP > 3 (A) as well as fructose and levanbiose (combined; ⁇ ). The product concentrations are expressed in fructose equivalents.
  • the endolevanases from Azotobacter chroococcum DSM 2286 (A), Bacillus licheniformis DSM 13 (B) and Bacteroides thetaiotaomicron DSM 2079 (C) were used.
  • FIG. 21 Michaelis-Menten kinetics of the endolevanase from A. chroococcum DSM 2286, which was produced recombinantly in Escherichia coli DH5a and purified by streptactin affinity chromatography. Commercial levan, purified from Timothy grass, served as substrate for the reaction shown.
  • the terms“one or more” or“at least one”, as interchangeably used herein, relate to at least 1 , or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25 or a plurality of species, e.g. nucleic acid molecules.
  • the term“plurality” means more than one, preferably 2 or more, such as up to 1000.
  • fructooligosaccharides may comprise little amounts of further compounds of different formulas in addition to the mentioned formulas Fm and/or GF n .
  • the further compounds are comprised in amounts of less than 10 wt.-%, preferably less than 5 wt.-%, more preferably less than 1 wt.-%, more preferably less than 0.1 wt.-%, more preferably less than 0.01 wt.-%, more preferably less than 0.001 wt.-%, wherein most preferably the fructooligosaccharides consist of the compounds of the mentioned formulas, if not explicitly stated otherwise.
  • the terms as used interchangeably herein, relate to oligomers of at least 3 monosaccharide units that are glycosidically linked to each other.
  • Such oligomers typically comprise 2 or more and up to 100 fructose units.
  • the oligomers may be linear or branched.
  • the linkage between the individual units may be beta 2-6 glycosidic and/or beta 1 -2 glycosidic.
  • the oligomers comprise at least 2 fructose units and are typically comprised of at least 65 mol.-% fructose units. If other monosaccharide units are present, these are for example glucose units and located on the terminus/termini of the fructose oligomer.
  • fructose means D-fructose and glucose means D-glucose.
  • glucose means D-glucose.
  • Levan relates to a polymer of p-2,6-glycosidically linked fructose units with the structural formula of a monomeric fructose unit of the levan backbone being shown below:
  • Levan may comprise a terminal glucose unit and may be branched by fructose units that are linked b- 2,1 -glycosidically.
  • levansucrase and/or endolevanase, as used herein, relate to functional enzymes that have the designated functionality of producing levan (from sucrose) and cleaving levan into FOS, respectively.
  • the levansucrase is preferably an enzyme of EC 2.4.1 .10, for example a prokaryotic or prokaryotic- derived enzyme, for example an enzyme originating from or derived from a bacterium of the genus Gluconobacter, Zymomonas, Bacillus, Rahnella, Leuconostoc, Acetobacter or Erwinia, in particular Gluconobacter, such as Gluconobacter japonicus, Gluconobacter cerinus or Gluconobacter oxydans, in particular Gluconobacter japonicus.
  • Gluconobacter such as Gluconobacter japonicus, Gluconobacter cerinus or Gluconobacter oxydans, in particular Gluconobacter japonicus.
  • the endolevanase is preferably an enzyme of EC 3.2.1 .65, for example a prokaryotic or prokaryotic-derived enzyme, for example an enzyme originating from or derived from a bacterium of the genus Azotobacter, Bacteroides or Bacillus, in particular Azotobacter, such as Azotobacter chroococcum. In some embodiments, it is derived from Azotobacter chroococcum DSM 2286 and may have the amino acid sequence set forth in SEQ ID NO:5 or 6 or a variant or active fragment thereof.
  • Activity fragment refers to a fragment that retains the enzymatic activity of the full length protein to an extent of at least 75%, preferably at least 80%, at least 90% or at least 95%, as determined by suitable activity assays.
  • isolated or“purified”, as used herein, relate to molecules that have been at least partially separated from molecules that accompany them in their natural environment, typically a cellular environment. These other molecules from which they are separated comprise other proteins, nucleic acids, cellular debris and cell components in general. Various methods for isolating and/or purifying proteins, such as those described herein, are known in the art and routinely practiced by the person skilled in the art.
  • the invention is based, inter alia, on the inventors’ surprising finding that the endolevanase described herein and being derived from Azotobacter chroococcum DSM 2286 (SEQ ID NO:5 or 6) possesses unique properties which distinguish the enzyme from endolevanases described in the literature and provides for significantly improved properties that allow cost- and time-efficient production of levan- based dietary fiber.
  • the invention in one aspect therefore relates to a method for preparing fructooligosaccharides (FOS) comprising contacting an endolevanase enzyme comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6 or an amino acid sequence that has at least 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity with the amino acid sequence set forth in SEQ ID Nos. 5 or 6 over the full length of the sequence or active fragments thereof with levan under conditions suitable for producing fructooligosaccharides (FOS).
  • Another aspect relates to the enzyme as such, for example in isolated/purified form. Said enzyme may also form part of a composition or kit, in which it may be combined with other components, such as the levansucrase described herein.
  • the fragments of the endolevanase comprise or consist of the amino acid sequence of SEQ ID NO:6 but lack the first 35 N-terminal amino acids of SEQ ID NO:6, i.e. start with A36. Accordingly, also comprised are fragments of the above-described endolevanase that retain at least 75 % of the activity of the full length sequence but lack one or more N-terminal and/or C-terminal amino acids.
  • the present invention is directed to a method for preparing fructooligosaccharides (FOS) comprising or consisting of the following steps:
  • nucleic acid molecule comprising a nucleotide sequence which encodes an endolevanase, preferably comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6 or an amino acid sequence that has at least 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity with the amino acid sequence set forth in SEQ ID Nos. 5 or 6 over the full length of the sequence or active fragments thereof, into at least one host organism;
  • step (c) cultivating the host organism of step b) under conditions which allow the expression of the nucleotide sequence; and (d) preparing fructooligosaccharides using the endolevanase expressed in step (c), by subjecting the enzyme to conditions which allow the production of the fructooligosaccharides.
  • These conditions may comprise the contacting of the enzyme with levan as a substrate.
  • the endolevanase is
  • step (i) comprised in a culture supernatant from the culture medium used in step (c), wherein the culture supernatant is subjected to conditions which allow the preparation of said fructooligosaccharides;
  • step (ii) comprised in a cell extract from the host organism cultivated in step (c), wherein the cell extract is subjected to conditions which allow the preparation of said fructooligosaccharides;
  • step (iii) comprised in a host organism or present at the surface of the host organism cultivated in step (c), wherein the host organism is subjected to conditions which allow the preparation of said fructooligosaccharides; and/or
  • the invention relates to a method for preparing fructooligosaccharides comprising or consisting of the following steps:
  • nucleic acid molecule comprising a first nucleotide sequence which encodes a levansucrase and/or a second nucleotide sequence which encodes an endolevanase into at least one host organism;
  • a host organism comprising a first nucleotide sequence which encodes a levansucrase and a second nucleotide sequence which encodes an endolevanase;
  • a first host organism comprising a first nucleotide sequence which encodes a levansucrase and a second host organism comprising the second nucleotide sequence which encodes an endolevanase;
  • the host organism comprising the first nucleotide sequence which encodes a levansucrase and the second nucleotide sequence which encodes an endolevanase under conditions which allow the expression of the first and the second nucleotide sequence;
  • the first host organism comprising the first nucleotide sequence which encodes a levansucrase and the second host organism comprising the second nucleotide sequence which encodes an endolevanase under conditions which allow the expression of the first and the second nucleotide sequence;
  • step (d) preparing fructooligosaccharides using the levansucrase and the endolevanase expressed in step (c), by subjecting the enzymes to conditions which allow the production of the fructooligosaccharides.
  • the at least one nucleic acid molecule is an expression vector.
  • the terms“(expression) vector” and“(expression) plasmid” can be used synonymously and commonly refer to a circular DNA sequence which is used to transfer (foreign) genetic material into a target cell, if not stated otherwise.
  • the expression vector preferably comprises at least one insert (sequence of interest), more preferably the first and/or the second nucleotide sequence as defined in the present invention.
  • the purpose of the vector is to multiply or express the insert in the target cell, preferably to express the at least one insert, more preferably the first and/or the second nucleotide sequence, to obtain the at least one enzyme, in particular the levansucrase and/or the endolevanase which is encoded by the first nucleotide sequence and/or by the second nucleotide sequence.
  • the person skilled in the art knows (expression) vectors which are suitable for the application described.
  • a suitable expression vector, typically without an insert is commercially available, e.g. from IBA GmbH.
  • the at least one insert can be inserted later into the vector by typical (cloning) methods, which are known to the person skilled in the art. Examples of expression vectors comprising at least one insert and which can be used in the methods according to the invention, are illustrated in Figures 4, 8, 13 and 16.
  • the (first) nucleotide sequence which encodes the levansucrase and/or the (second) nucleotide sequence which encodes the endolevanase according to the present invention is/are
  • the (first) nucleotide sequence encoding the levansucrase and/or the (second) nucleotide sequence encoding the endolevanase according to the invention is/are integrated in the genome, preferably in the chromosomal DNA, of the host organism provided in step (b) of the method according to the invention.
  • the (first) nucleotide sequence encoding the levansucrase and the (second) nucleotide sequence encoding the endolevanase are integrated in the genome, preferably in the chromosomal DNA, of the host organism provided in step (b) of the method according to the invention.
  • step (d) of all methods described herein the levansucrase and/or the endolevanase, depending on the method concerned, are, individually or together,
  • step (i) comprised in a culture supernatant from the culture medium used in step (c), wherein the culture supernatant is subjected to conditions which allow the preparation of said fructooligosaccharides;
  • step (ii) comprised in a cell extract from the at least one host organism cultivated in step (c), wherein the cell extract is subjected to conditions which allow the preparation of said fructooligosaccharides;
  • step (iii) comprised in a host organism or at the surface of the host organism cultivated in step (c), wherein the host organism is subjected to conditions which allow the preparation of said fructooligosaccharides;
  • the levansucrase and/or the endolevanase are released from the levansucrase and/or endolevanase producing host organism(s) during the cultivation process of step (c) and accumulate in the cell culture medium.
  • the culture medium can be separated from the cells after step
  • step (c) (e.g. by centrifugation) to obtain the “culture supernatant” comprising the levansucrase and/or endolevanase.
  • the culture supernatant is cell-free.
  • the culture supernatant can be used in step (d) of the method according to the invention by subjecting the“culture supernatant” to conditions which allow the preparation of FOS.
  • step (d) this generally means the step of the actual production of the FOS by the enzyme(s). This reference to step
  • step (d) is thus to be understood to be by means of example only and not to exclude those methods where said production step of FOS by enzymatic action is not explicitly referred to as step (d) but rather in more general terms.
  • the levansucrase and/or endolevanase producing host organism(s) are separated from the cell culture medium by centrifugation after step (c).
  • the resulting cell pellet is in various embodiments subjected to cell disrupting methods to set free the contained cell components including the levansucrase and/or endolevanase. Suitable methods for cell disruption are known to the person skilled in the art (e.g. chemical or enzymatic lysis or mechanical methods, e.g. sonification).
  • the composition obtained may be centrifuged and/or filtered and/or lyophilized before use in step (d) of the method according to the invention.
  • the final composition is referred to as“cell extract” or“crude cell extract”.
  • the cell extract is cell-free.
  • the levansucrase and/or the endolevanase comprised in the cell extract of the host organism of step (c) are purified from the cell extract (e.g. by chromatography methods). Afterwards the purified enzymes can be used in step (d) of the method according to the invention to prepare said fructooligosaccharides.
  • the purified enzymes can also be immobilized or lyophilized before using them in step (d). The person skilled in the art knows suitable immobilization techniques.
  • the levansucrase and the endolevanase do not have to be used in the same form in step (d) of the method according to the invention.
  • the levansucrase may be used as a cell extract and the endolevanase as a purified enzyme, or the levansucrase is used in its purified form and the endolevanase is used as a cell extract in step (d).
  • the method according to the invention comprises or consists of the following steps:
  • nucleic acid molecule comprising a first nucleotide sequence which encodes a levansucrase and a second nucleotide sequence which encodes an endolevanase into a host organism;
  • step (c) cultivating the host organism comprising the first nucleotide sequence which encodes a levansucrase and the second nucleotide sequence which encodes an endolevanase under conditions which allow the expression of the first and the second nucleotide sequence; and (d) preparing said fructooligosaccharides using the levansucrase and the endolevanase expressed in step (c) and comprised in the cell extract from the host organism after step (c), and subjecting said cell extract to conditions which allow the preparation of said fructooligosaccharides.
  • the method according to the invention comprises or consists of the following steps:
  • nucleic acid molecule comprising a first nucleotide sequence which encodes a levansucrase and a second nucleotide sequence which encodes an endolevanase into a host organism;
  • step (d) preparing said fructooligosaccharides using the levansucrase and the endolevanase expressed in step (c) and comprised in the culture supernatant from the culture medium after step (c), and subjecting said culture supernatant to conditions which allow the preparation of said fructooligosaccharides.
  • the method for preparing fructooligosaccharides comprises or consists of the following steps:
  • nucleic acid molecule comprising a first nucleotide sequence which encodes a levansucrase and a second nucleotide sequence which encodes an endolevanase into one host organism, preferably the first nucleotide sequence and the second nucleotide sequence are comprised in one expression vector, which is introduced into the host organism;
  • step (d) preparing said fructooligosaccharides using the levansucrase and the endolevanase expressed in step (c) and comprised in the host organism after step (c), and subjecting said host organism to conditions which allow the preparation of said fructooligosaccharides.
  • the method for preparing fructooligosaccharides comprises or consists of the following steps:
  • nucleic acid molecule comprising a (first) nucleotide sequence which encodes a levansucrase and/or a (second) nucleic acid molecule comprising a second nucleotide sequence which encodes an endolevanase into at least one host organism, wherein the (first) nucleotide sequence encoding the levansucrase may be comprised in one expression vector and the (second) nucleotide sequence encoding the endolevanase may be comprised in a second expression vector, wherein the first and the second vectors are introduced into the same host organism or into different host organisms;
  • the host organism comprising a (first) nucleotide sequence which encodes a levansucrase and a (second) nucleotide sequence which encodes an endolevanase;
  • the first host organism comprising the (first) nucleotide sequence which encodes a levansucrase and the second host organism comprising the (second) nucleotide sequence which encodes an endolevanase;
  • the host organism comprising the (first) nucleotide sequence which encodes a levansucrase and the (second) nucleotide sequence which encodes an endolevanase under conditions which allow the expression of the both nucleotide sequences;
  • the first host organism comprising the (first) nucleotide sequence which encodes a levansucrase and the second host organism comprising the (second) nucleotide sequence which encodes an endolevanase under conditions which allow the expression of both nucleotide sequences;
  • step (d) preparing said fructooligosaccharides using the levansucrase and the endolevanase expressed in step (c) and comprised in the at least one host organism after step (c), and subjecting said at least one host organism to conditions which allow the preparation of said fructooligosaccharides.
  • “Comprised in the host organism” preferably means that the whole cells of the host organism, which comprise the levansucrase and/or the endolevanase, are used in step (d) without actively breaking or disrupting the cells.
  • the cells of the host organism are separated from the culture medium (e.g. by centrifugation) before they are used in step (d) of the method according to the invention.
  • the at least one host organism is a prokaryotic or eukaryotic organism, preferably a bacterial or yeast organism, more preferably a bacterial organism, more preferably Escherichia coli or a Gluconobacter species, most preferably Escherichia coli BL21 or Gluconobacter japonicus LMG 1417.
  • the term“one nucleic acid molecule” or“at least one nucleic acid molecule” means one type or at least one type of nucleic acid molecule, but does not define the amount of the molecule.
  • the term“at least one nucleic acid molecule comprising a first nucleotide sequence which encodes a levansucrase and/or a second nucleotide sequence which encodes an endolevanase” means that the first nucleotide sequence and the second nucleotide sequence can be combined in one nucleic acid molecule. However, it also means that the first nucleotide sequence can be comprised in one nucleic acid molecule and the second nucleotide sequence can be comprised in a second nucleic acid molecule.
  • the first and the second nucleic acid molecules are expression vectors, also referred to as expression plasmids.
  • the first nucleotide sequence and the second nucleotide sequence are comprised in one expression vector, which is introduced into the host organism.
  • the first nucleotide sequence is comprised in one expression vector and the second nucleotide sequence is comprised in a second expression vector, wherein the first and the second vectors are introduced into the same host organism or into different host organisms.
  • Conditions which allow the expression of the first and the second nucleotide sequence, are typical cultivation conditions, preferably used for Gluconobacter or Escherichia coli species, more preferably for Escherichia coli BL21 or Gluconobacter japonicus LMG 1417. Typically, these conditions allow the transcription of the first and/or second nucleotide sequence into the corresponding mRNA. Afterwards, it allows the translation of the formed mRNA into the respective corresponding amino acid chain and its folding to the corresponding enzyme. The production of the levansucrase and the endolevanase can take place in the same host organism or in different host organisms.
  • the levansucrase is produced in Gluconobacter japonicus and the endolevanase is produced in Escherichia coli BL21 .
  • the cultivation conditions for the two host organisms can be different in step (c) to allow the production of the single enzymes.
  • the levansucrase and the endolevanase are produced in the same host organism, preferably in Gluconobacter japonicus LMG 1417 or Escherichia coli BL21 .
  • the host organism is selected such that either the levansucrase or the endolevanase or both are heterologous to the host organism, i.e. the host organism does not naturally express these enzymes.
  • the host organism is genetically engineered to express the heterologous enzyme(s).
  • the cultivation of step (c) of the method according to the invention is carried out at 20 to 45°C, or up to 40°C or up to 37°C, preferably at 25 to 35 °C, more preferably at 28 °C or 30 °C. In various embodiments, the cultivation is carried out for up to 72 hours, preferably for up to 48 hours, more preferably for up to 24 hours.
  • step (a) at least one nucleic acid molecule, preferably one nucleic acid molecule, comprising a first nucleotide sequence which encodes a levansucrase and a second nucleotide sequence which encodes an endolevanase is introduced into one host organism.
  • the host organism is a Gluconobacter or Escherichia coli species, more preferably Escherichia coli BL21 .
  • step (b) one host organism comprising the first nucleotide sequence which encodes a levansucrase and the second nucleotide sequence which encodes an endolevanase is provided.
  • the host organism preferably Escherichia coli BL21
  • step (c) is cultivated in step (c) under conditions which allow the expression of the first and the second nucleotide sequence (the term“expression” typically comprises every step that is needed to produce the levansucrase and endolevanase in the host organism).
  • step (d) the host organism of step (c), which comprises the levansucrase and the endolevanase, is subjected to condition which allow the preparation of fructooligosaccharides. That means that the whole cells of the host organism comprising the levansucrase and the endolevanase are subjected to conditions which allow the production of fructooligosaccharides.
  • a cell extract is prepared from the host organism after step (c).
  • the cell extract comprises the levansucrase and the endolevanase, which were produced in the host organism in step (c).
  • This cell extract is used in step (d) to obtain the fructooligosaccharides by subjecting the cell extract to conditions which allow the preparation of said fructooligosaccharides.
  • the levansucrase and the endolevanase can be purified from the cell extract of the host organism.
  • the purified enzymes or alternatively the purified immobilized enzymes can be subjected to conditions in step (d) which allow the preparation of said fructooligosaccharides.
  • the culture supernatant comprising the levansucrase and the endolevanase is used in step (d) of the method according to the invention to obtain the fructooligosaccharides by subjecting the culture supernatant to conditions which allow the preparation of said FOS.
  • the host organism is a Gluconobacter species, preferably Gluconobacter japonicas, more preferably Gluconobacter japonicus LMG 1417, which comprises the first nucleotide sequence which encodes for the levansucrase naturally in its genome.
  • the wildtype host organism can be used to produce the levansucrase. It is not necessary to introduce a nucleic acid molecule comprising the first nucleotide sequence which encodes the levansucrase into the host organism.
  • the nucleic acid molecule comprising the second nucleotide sequence which encodes the endolevanase can be introduced into the same host organism or into another host organism before starting step (c).
  • the host organism preferably a Gluconobacter species, more preferably Gluconobacter japonicus, most preferably Gluconobacter japonicus LMG 1417, comprises the first nucleotide sequence which encodes for the levansucrase in its genome, preferably in its chromosomal DNA and, additionally, comprises at least one nucleic acid molecule, preferably an expression vector, comprising the first nucleotide sequence which encodes the levansucrase.
  • the levansucrase production will be increased in the cell in comparison to the wildtype host organism, which comprises the first nucleotide sequence only in its genome.
  • the host organism may comprise the second nucleotide sequence, which encodes the endolevanases, in the afore-mentioned nucleic acid molecule or in a second nucleic acid molecule.
  • the host organism may comprise the nucleotide sequence encoding the endolevanse in its genome, preferably in its chromosomal DNA and, additionally, comprises at least one nucleic acid molecule, preferably an expression vector, comprising the nucleotide sequence which encodes the endolevanase.
  • the host organism comprises the first nucleotide sequence which encodes the levansucrase and the second nucleotide sequence which encodes the endolevanase in its genome, preferably in its chromosomal DNA.
  • This host organism is provided in step (b) of the method according to the invention.
  • the sequences were previously integrated in the host’s chromosomal DNA by biotechnological methods, which are known to the person skilled in the art.
  • the host organism is a Gluconobacter or Escherichia coli species, preferably an Escherichia coli species.
  • the host organism comprises the first nucleotide sequence which encodes the levansucrase in its genome, preferably in its chromosomal DNA.
  • the host organism comprises the first nucleotide sequence which encodes the levansucrase naturally in its genome, preferably in its chromosomal DNA.
  • the second nucleotide sequence which encodes the endolevanase is comprised in an expression vector in the host organism. This host organism is provided in step (b) of the method according to the invention.
  • the host organism comprises the second nucleotide sequence which encodes the endolevanase in its genome.
  • the first nucleotide sequence which encodes the levansucrase is comprised in an expression vector in the host organism. This host organism is provided in step (b) of the method according to the invention.
  • the levansucrase can be used in step (d)
  • the endolevanase can be used in step (d)
  • the levansucrase used in step (d) is comprised in a cell extract from the host organism.
  • the cell extract may comprise only the levansucrase, or the levansucrase and the endolevanase.
  • the endolevanase used in step (d) is comprised in a cell extract from the host organism.
  • the cell extract may comprise only the endolevanase, or the levansucrase and the endolevanase.
  • the levansucrase used in step (d) is comprised in a culture supernatant from the culture medium after step (c).
  • the culture supernatant may comprise only the levansucrase, or the levansucrase and the endolevanase.
  • the endolevanase used in step (d) is comprised in a culture supernatant from the culture medium after step (c).
  • the culture supernatant may comprise only the endolevanase, or the levansucrase and the endolevanase.
  • the second enzyme may have to be added in step (d) of the method for preparing FOS according to the invention, as well, preferably in purified or immobilized form or comprised in a (second) cell extract, in a (second) culture supernatant or comprised in the host organism or at the surface of the host organism.
  • the enzyme used in the method is the endolevanase, in particular the endolevanase described herein, the need of using a second enzyme, i.e. the levansucrase, may be avoided by using levan as a substrate.
  • step (d) it is possible, that the two enzymes are used (or applied or added) in step (d) simultaneously or sequentially.
  • the levansucrase is added in step (d) to produce levan from a substrate, preferably from the substrate sucrose.
  • the endolevanase is added to hydrolyze the levan to the fructooligosaccharides.
  • the enzymes are used sequentially.
  • the levansucrase and the endolevanase are used (or applied or added) simultaneously in step (d) to form the suitable fructooligosaccharides.
  • the two enzymes can be used (or applied or added) in purified form or comprised in a cell extract from the host organisms, in a culture supernatant from the culture medium or comprised in the host organism or at the surface of the host organism.
  • Suitable methods to use (or apply or add) the levansucrase and the endolevanase in step (d) are known to the person skilled in the art. Some of these methods have already been described above and may be comprised in at least one further step, which is comprised in the method according to the invention between step (c) and step (d).
  • the method according to the invention comprises after step (d) a further step (e) for purifying the fructooligosaccharides obtained in step (d).
  • step (e) is a chromatography step or a filtration step, without being limited to these methods.
  • the person skilled in the art knows which techniques are most appropriate.
  • the conditions which allow the preparation of the fructooligosaccharides comprise providing sucrose to the levansucrase, or to the levansucrase and endolevanase.
  • the sucrose is converted to the fructooligosaccharides obtained in step (d), wherein the conversion is catalyzed by the levansucrase and the endolevanase.
  • the levansucrase hydrolyzes the sucrose to form fructan-polymers, also referred to as levan, preferably, the fructan-polymers comprise up to 100.000 fructose units,
  • a mixture of (short-chain) fructooligosaccharides of different lengths is obtained in (e.g. step (d) of) the method for preparing fructooligosaccharides according to the invention.
  • the (short-chain) fructooligosaccharides obtained in the methods described herein, for example obtained in step (d), comprise, essentially consist of or consist of compounds of the formulas F m and/or GF n , wherein
  • F is a monomeric fructose unit, preferably D-fructose unit
  • G is a monomeric glucose unit, preferably D-glucose unit
  • n is >3, preferably 3 to 20, more preferably 3 to 15, more preferably 3 to 13, most preferably 3 to 10; n is >2, preferably 2 to 19, more preferably 2 to 14, more preferably 2 to 12, most preferably 2 to 9; m and/or n are the same or different in the individual fructooligosaccharides obtained in step (d); and the fructose units are covalently coupled to each other by b-(2®6) linkages and may further comprise b-(2®1) branching.
  • the mixture comprises, essentially consists of or consists of compounds of the formulas F m and GF n of different lengths as defined above.
  • the mixture of (short-chain) fructooliosaccharides of different lengths further comprises compounds of the formula F m , wherein m is 1 and/or 2 (free fructose and/or levanbiose).
  • the amount of compounds of the formula F m , wherein m is 1 and/or 2, preferably free fructose and/or levanbiose is reduced in the fructooligosaccharides obtained in step (d) of the method for preparing fructooligosaccharides according to the invention (catalyzed by the levansucrase and endolevanase as defined according to the invention) or in the levan obtained in step (d) of the method for preparing levan according to the invention (catalyzed by the levansucrase as defined according to the invention), in comparison to methods which use different enzymes or enzyme combinations.
  • the amount of compounds of the formula F m , wherein m is 1 and/or 2 is less than 12 %, preferably less than 8 % based on the amount of fructose units which are comprised in the sucrose which is added in step (d) of the methods according to the invention.
  • Fructooligosaccharides of the formula F m as obtainable by the method according to the invention typically only consist of monomeric fructose units which are linked to each other, preferably by beta-2, 6- glycosidic bonds.
  • a monomeric fructose unit is illustrated in the following formula:
  • Fructooligosaccharides of the formula GF m as obtainable by the method according to the invention typically consist of one monomeric glucose unit which is linked to a terminal fructose unit of a fructose chain, wherein the fructose units of the fructose chain are preferably linked to each other by beta-2, 6- glycosidic bonds.
  • fructooligosaccharides obtained in step (d) are preferably of the levan-type.
  • levan-type or“levan-based” typically comprises oligo- and polysaccharides, which contain two or more fructose units, wherein the single fructose units are (mainly) linked to each other by beta- 2, 6-glycosidic bonds, if not explicitly stated otherwise.
  • the levansucrase preferably converts sucrose to fructan-polymers, preferably with up to 100.000 fructose units, which are linked to each other by beta-2, 6-glycosidic bonds.
  • This intermediate can be hydrolyzed by the endolevanase to form smaller fructooligosaccharide compounds of the formula Fm and/or GF n , wherein m is >3, preferably 3 to 20, more preferably 3 to 15, more preferably 3 to 13, most preferably 3 to 10; and n is >2, preferably 2 to 19, more preferably 2 to 14, more preferably 2 to 12, most preferably 2 to 9; wherein m and/or n may be different in the respective fructooligosaccharides obtained in step (d).
  • the fructooligosaccharides obtainable by the method according to the invention have a molecular weight of up to 3258 g mol ⁇ 1 , more preferably up to 2448 g mol ⁇ 1 , and most preferably up to 1638 g mol ⁇ 1 .
  • fructooligosaccharides In another preferred embodiment of the method for preparing fructooligosaccharides according to the invention, at least 30 % or 40 % or 50 % or 60 % or 70 % or 75 % or 80 % or 85 % or 86 % or 87 % or 88 % or 89 % or 90 % or 91 % or 92 % or 93 % or 94 % or 95 % or 96 % or 97 % or 98 % or 99 % or 99,9 % of the sucrose is converted to fructooligosaccharides, preferably to fructooligosaccharides of the formulas F m and/or GF n , wherein F, G, m and n are as defined above, based on the sucrose concentration and the amount of levansucrase and endolevanase which is added in step (d) of the method according to the invention.
  • the sucrose is converted within 600 hours, more preferably within 500 hours, more preferably within 490 hours, more preferably within 480 hours, more preferably within 400 hours, more preferably within 300 hours, more preferably within 260 hours, more preferably within 200 hours, more preferably within 100 hours, more preferably within 60 hours, more preferably within 50 hours, more preferably within 49 hours, more preferably within 48 hours, more preferably within 40 hours, more preferably within 30 hours, more preferably within 26 hours, more preferably within 20 hours, more preferably within 15 hours, more preferably within 10 hours, more preferably within 5 hours, based on the sucrose concentration and the amount of levansucrase and endolevanase which is added in step (d) of the method according to the invention.
  • the time of conversion from sucrose to fructooligosaccharides according to the present invention can be reduced by adding increased amounts of levansucrase and endolevanase in step (d) of the method according to the invention.
  • the levansucrase and the endolevanase are comprised in culture supernatant or cell extract or whole cells of the host organism.
  • increased amounts of culture supernatant or cell extract or whole cells comprising the levansucrase and the endolevanase result in a faster conversion from sucrose to FOS in step (d) of the method according to the invention.
  • sucrose is added in step (d) in amounts of up to up to 2.5 mol L ⁇ 1 , up to 2 mol L ⁇ 1 , up to 1 .5 mol L ⁇ 1 , up to 1 mol L ⁇ 1 , up to 0.5 mol L ⁇ 1 , up to 0.25 mol L ⁇ 1 , up to 0.2 mol L ⁇ 1 , up to 0.15 mol L ⁇ 1 , up to 0.1 mol L ⁇ 1 , up to 0.05 mol L ⁇ 1 or up to 0.01 mol L ⁇ 1 .
  • sucrose is added in step (d) in amounts of 5 mol L ⁇ 1 , 3 mol L 1 , 2.5 mol L ⁇ 1 , 2 mol L ⁇ 1 , 1 .5 mol L ⁇ 1 , 1 mol L ⁇ 1 , 0.5 mol L ⁇ 1 , 0.25 mol L ⁇ 1 , 0.2 mol L ⁇ 1 , 0.15 mol L ⁇ 1 , 0.1 mol L ⁇ 1 , 0.05 mol L ⁇ 1 or 0.01 mol L ⁇ 1 .
  • the conversion of sucrose to FOS is carried out at 20 to 37 °C, preferably at 25 to 35 °C, more preferably at 28 °C or 30 °C.
  • the levansucrase of the invention or used in the methods of the invention comprises or consists of
  • an amino acid sequence which has at least 50 %, at least 60 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % sequence identity with the amino acid sequence set forth in SEQ ID Nos. 1 or 2 over the full length of the sequence.
  • the levansucrase comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 or a fragment thereof lacking the N-terminal methionine (M) residue.
  • suitable fragments of the above-described levansucrases include, but are not limited to those that retain at least 75 % of the activity of the full length sequence but lack one or more N-terminal and/or C-terminal amino acids.
  • the (first) nucleotide sequence which encodes the levansucrase, is originated from Gluconobacter japonicus LMG 1417.
  • the (first) nucleotide sequence which encodes the levansucrase, comprises or consists of a nucleotide sequence
  • nucleotide sequences encode levansucrases that comprise or consist of the amino acid sequence set forth in SEQ ID Nos.
  • the (first) nucleotide sequence which encodes the levansucrase, comprises or consists of a nucleotide sequence set forth in SEQ ID NO:3.
  • the (first) nucleotide sequence which encodes the levansucrase, comprises or consists of a nucleotide sequence set forth in SEQ ID NO:4.
  • the endolevanase of the invention or used in the methods of the invention comprises or consists of
  • an amino acid sequence which has at least 50 %, at least 60 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % sequence identity with the amino acid sequence set forth in SEQ ID Nos. 5 or 6 over the full length of the sequence.
  • the endolevanase comprises or consists of the amino acid sequence set forth in SEQ ID NO:6 or a fragment thereof that lacks the first 35 N-terminal amino acids and starts with A36.
  • suitable fragments of the above-described levansucrases include, but are not limited to those that retain at least 75 % of the activity of the full length sequence but lack one or more N-terminal and/or C-terminal amino acids.
  • Preferred fragments include those of SEQ ID NO:6 that lack one or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 ,13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34 or up to 35 amino acids from the N-terminus.
  • the (second) nucleotide sequence which encodes the endolevanase, is originated from Azotobacter chroococcum DSM 2286.
  • the (second) nucleotide sequence which encodes the endolevanase, comprises or consists of a nucleotide sequence
  • nucleotide sequences encode endolevanases that comprise or consist of the amino acid sequence set forth in SEQ ID Nos.
  • the (second) nucleotide sequence which encodes the endolevanase comprises or consists of a nucleotide sequence set forth in SEQ ID NO:7 or 8.
  • sequence identity typically refers to amino acid sequences that share identical amino acids at corresponding positions or nucleotide sequences sharing identical nucleotides at corresponding positions, if not explicitly stated otherwise.
  • Amino acid sequences with a sequence identity of less than 100 % typically relate to amino acid sequences which have one or more amino acids added, deleted, substituted or otherwise modified in comparison to another amino acid sequence that serves as a reference.
  • the given sequence identity refers to the sequence identity over the entire length of the reference sequence.
  • any query sequence that needs to have a sequence identity of, for example, 70 % needs to have at least 70 identical amino acids in corresponding positions over the 100 amino acid long stretch of the reference sequence when both are properly aligned. These 70 identical amino acids may be contiguous but do not need to be contiguous. This also means that the query sequence is at least 70 amino acids in length. The remaining 30 amino acids may differ between both sequences.
  • sequence identity applies to nucleotide sequences. Here the identity refers to identical nucleotides in corresponding positions.
  • the determination of percent identity described herein between two amino acid or nucleotide sequences can be accomplished using a mathematical algorithm.
  • a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the BLASTN and BLASTX programs and can be accessed, for example, at the National Center for Biotechnology Information (NCBI) world wide web site having the universal resource locator "www.ncbi.nlm.nih.gov/BLAST".
  • NCBI National Center for Biotechnology Information
  • Blast nucleotide searches can be performed with BLASTN program, whereas BLAST protein searches can be performed with BLASTX program or the NCBI "blastp" program.
  • Another algorithm available in the art is the FASTA algorithm. Sequence comparisons (alignments), in particular multiple sequence comparisons, can be generated using computer programs. Commonly used are for example the Clustal series (See, e.g. ,Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31 , 3497-3500), T-Coffee (See, e.g., Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. J. Mol. Biol.
  • sequence comparisons are possible with the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA, USA) with the pre-set standard parameters, the AlignX-module of which is based on ClustalW. If not explicitly defined otherwise, sequence identity is determined using the BLAST algorithm.
  • the levansucrase has a specific activity of at least 1000 U/mg, preferably of at least 2000 U/mg, more preferably of at least 2500 U/mg, most preferably of at least 3000 U/mg, measured based on Michaelis-Menten kinetics.
  • the levansucrase is from Gluconobacter japonicus LMG 1417, produced in Escherichia coli, and purified by affinity chromatography. More preferably, the specific activity is measured at 20 to 37 °C, preferably at 25 to 35 °C, most preferably at approx. 30 °C.
  • the pH is preferably between 5.0 and 6.0, more preferably between 5.2 and 6.8, most preferably the pH is approx. 5.4.
  • the endolevanase has a specific activity of at least 500 U/mg, preferably of at least 1500 U/mg, more preferably of at least 2500 U/mg, more preferably of at least 3000 U/mg, most preferably of at least 5000 U/mg, measured based on Michaelis-Menten kinetics.
  • the endolevanase is from Azotobacter chroococcum DSM 2286, produced in Escherichia coli, and purified by affinity chromatography. More preferably, the specific activity is measured at 20 to 37 °C, preferably at 25 to 35 °C, most preferably at approx. 30 °C.
  • the pH is preferably between 5.5 and 6.5, more preferably between 5.7 and 6.3, most preferably the pH is approx. 6.0.
  • the invention relates to a method for preparing levan comprising or consisting of the following steps:
  • step (d) preparing levan using the levansucrase expressed in step (c), and subjecting it to conditions which allow the preparation of levan.
  • step (d) of the method for preparing levan according to the invention the levansucrase is
  • step (i) comprised in a culture supernatant of the host organism cultivated in step (c), wherein the culture supernatant is subjected to conditions which allow the preparation of levan;
  • step (ii) comprised in a cell extract from the host organism cultivated in step (c), wherein the cell extract is subjected to conditions which allow the preparation of levan;
  • step (iii) comprised in a host organism or at the surface of the host organism cultivated in step (c), wherein the host organism is subjected to conditions which allow the preparation of levan;
  • the first nucleotide sequence which encodes a levansucrase, originates from a Gluconobacter species. In various embodiments, it may comprise or consist of the nucleotide sequences encoding levansucrase disclosed herein.
  • the host organism may be a Gluconobacter species as well, preferably Gluconobacter japonicus such as Gluconobacter japonicus LMG 1417. This host organism comprises the first nucleotide sequence in its genome, preferably in its chromosomal DNA.
  • nucleic acid molecule comprising the first nucleotide sequence, which encodes the levansucrase, is additionally introduced in the host organism to increase expression of the levansucrase.
  • the host organism would still be genetically engineered although the introduced coding sequence is homologous.
  • the nucleic acid molecule is an expression vector/plasmid.
  • Such an expression vector/plasmid may comprise sequence elements, for example regulatory elements, such as promotors and the like, that are heterologous to the host organism.
  • the cultivation process of step (c) of the method for preparing levan according to the invention is carried out at 20 to 37 °C, preferably at 25 to 35 °C, more preferably at 28 °C or 30 °C. In various embodiments, the cultivation is carried out for up to 72 hours, preferably for up to 48 hours, more preferably for up to 24 hours.
  • the culture supernatant or the cell extract comprising the levansucrase is added in step (d) of the method according to the invention to form levan from sucrose.
  • the levansucrase of step (d) is subjected to sucrose to catalyze the conversion of sucrose to fructan-polymers.
  • Levan may comprise up to 100.000 fructose units which are (mainly) linked to each other by beta-2, 6-glycosidic bonds.
  • At least 30 % or 40 % or 50 % or 60 % or 70 % or 75 % or 80 % or 85 % or 86 % or 87 % or 88 % or 89 % or 90 % or 91 % or 92 % or 93 % or 94 % or 95 % or 96 % or 97 % or 98 % or 99 % or 99,9 % of the sucrose is converted to levan, based on the sucrose concentration and the amount of levansucrase which is added in step (d) of the method according to the invention.
  • the sucrose is converted within 600 hours, more preferably within 500 hours, more preferably within 490 hours, more preferably within 480 hours, more preferably within 400 hours, more preferably within 300 hours, more preferably within 260 hours, more preferably within 200 hours, more preferably within 100 hours, more preferably within 60 hours, more preferably within 50 hours, more preferably within 49 hours, more preferably within 48 hours, more preferably within 40 hours, more preferably within 30 hours, more preferably within 26 hours, more preferably within 20 hours, more preferably within 15 hours, more preferably within 10 hours, more preferably within 5 hours, based on the sucrose concentration and the amount of levansucrase which is added in step (d) of the method according to the invention.
  • the time of conversion from sucrose to levan can be reduced by adding increased amounts of levansucrase in step (d) of the method according to the invention.
  • the levansucrase is comprised in culture supernatant or cell extract or whole cells of the host organism.
  • increased amounts of culture supernatant or cell extract or whole cells comprising the levansucrase and the endolevanase result in a faster conversion from sucrose to levan in step (d) of the method according to the invention.
  • the conversion time can be reduced to approx one tenth if the enzyme is added in a 10-fold concentration.
  • sucrose is added in step (d) in amounts of up to 5 mol L ⁇ 1 or up to 3 mol L ⁇ 1 or up to 2.5 mol L ⁇ 1 or up to 2 mol L 1 or up to 1 .5 mol L 1 or up to 1 mol L ⁇ 1 or up to 0.5 mol L ⁇ 1 or up to 0.25 mol L ⁇ 1 or up to 0.2 mol L 1 or up to 0.15 mol L 1 or up to 0.1 mol L ⁇ 1 or up to 0.05 mol L ⁇ 1 or up to 0.01 mol L ⁇ 1 .
  • sucrose is added in step (d) in amounts of 5 mol L ⁇ 1 or 3 mol L ⁇ 1 or 2.5 mol L ⁇ 1 or 2 mol L ⁇ 1 or 1 .5 mol L ⁇ 1 or 1 mol L ⁇ 1 or 0.5 mol L ⁇ 1 or 0.25 mol L ⁇ 1 or 0.2 mol L ⁇ 1 or 0.15 mol L ⁇ 1 or 0.1 mol L ⁇ 1 or 0.05 mol L ⁇ 1 or 0.01 mol L ⁇ 1 .
  • the conversion from sucrose to levan is carried out at 20 to 37 °C, preferably at 25 to 35 °C, more preferably at 28 °C or 30 °C.
  • sucrose which are preferably compounds of the formula F m and/or GF n , wherein F, G, m and n are as defined above.
  • F, G, m and n are as defined above.
  • the invention relates to a method for preparing fructoligosaccharides from levan comprising or consisting of the following steps:
  • step (d) preparing fructooligosaccharides from levan using the endolevanase expressed in step (c), and subjecting it to conditions which allow the production of fructooligosaccharides.
  • step (d) of the method for preparing FOS according to the invention the endolevanase is
  • step (i) comprised in a culture supernatant of the host organism cultivated in step (c), wherein the culture supernatant is subjected to conditions which allow the preparation of FOS;
  • step (ii) comprised in a cell extract from the host organism cultivated in step (c), wherein the cell extract is subjected to conditions which allow the preparation of FOS;
  • step (iii) comprised in a host organism or at the surface of the host organism cultivated in step (c), wherein the host organism is subjected to conditions which allow the preparation of FOS; or
  • the (second) nucleotide sequence which encodes an endolevanase, originates from an Azotobacter species. In various embodiments, it may comprise or consist of the nucleotide sequences encoding endolevanase disclosed herein.
  • the host organism may be Azotobacter chroococcum DSM 2286. This host organism comprises the second nucleotide sequence in its genome, preferably in its chromosomal DNA. However, in such embodiments, it may be preferred that a nucleic acid molecule comprising the second nucleotide sequence, which encodes the endolevanase, is additionally introduced in the host organism to increase expression of the endolevanase.
  • the host organism would still be genetically engineered although the introduced coding sequence is homologous.
  • the nucleic acid molecule is an expression vector/plasmid.
  • Such an expression vector/plasmid may comprise sequence elements, for example regulatory elements, such as promotors and the like, that are heterologous to the host organism.
  • the invention relates to fructooligosaccharides (FOS) obtainable by the method for preparing fructooligosaccharides according to the invention.
  • FOS fructooligosaccharides
  • the invention relates to at least one expression vector comprising a first nucleotide sequence which encodes a levansucrase and/or a second nucleotide sequence which encodes an endolevanase;
  • the (first) nucleotide sequence which encodes the levansucrase comprises or consists of a nucleotide sequence
  • the (second) nucleotide sequence which encodes the endolevanase comprises or consists of a nucleotide sequence
  • the invention is directed to the endolevanase enzyme comprising or consisting of the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6 or an amino acid sequence that has at least 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity with the amino acid sequence set forth in SEQ ID Nos. 5 or 6 over the full length of the sequence or active fragments thereof, preferably in isolated or purified form.
  • the amino acids in the positions that correspond to positions 180-182 and 229-232 of SEQ ID NO:5 are invariable and therefore also retained in the variants and fragments disclosed herein.
  • the invention is directed to the levansucrase enzyme comprising or consisting of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 or an amino acid sequence that has at least 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity with the amino acid sequence set forth in SEQ ID Nos. 1 or 2 over the full length of the sequence or active fragments thereof, preferably in isolated or purified form.
  • the levansucrase or endolevanase of the invention and described herein may be encoded by the expression vectors of the invention.
  • the invention is directed to a kit or composition that comprises the endolevanase and/or levansucrase of the invention, preferably both.
  • these enzymes may be provided in spatially separated form, e.g. two separate compositions each containing one of the enzymes.
  • the invention relates to a genetically modified host organism comprising
  • the term“genetically modified host organism” typically comprises organisms which comprise foreign DNA, preferably at least one nucleic acid molecule according to the invention (e.g. an expression vector), and/or which are modified in their genome sequence, if not explicitly stated otherwise.
  • the host organism is, in various embodiments, selected such that at least one of the introduced nucleotide sequences is heterologous relative to the host organism. For example, if the host organism is Gluconobacter japonicus LMG 1417, the introduced sequences may comprise the sequence encoding the endolevanase derived from Azotobacter chroococcum DSM 2286 and vice versa.
  • the term“host organism” as used in steps (b) and (c) (and partly in step (d)) of the methods according to the invention refers to the“genetically modified host organism” as described in aspect four.
  • the invention relates to a cell extract or a culture supernatant comprising the levansucrase and/or the endolevanase according to the invention.
  • the invention is directed to a prebiotic or a food supplement comprising or (essentially) consisting of the fructooligosaccharides obtainable by the method for preparing fructooligosaccharides according to the invention.
  • a prebiotic or food supplement of the present invention can be comprised, for example, without being limited to it, in general food products, baby food and animal food.
  • Gluconobacter strains were plated on yeast extract agar to which either mannitol or sucrose was added as a carbon source.
  • the colony morphology after 24 hours of incubation at 28 °C was documented photographically and is shown in Figure 1 .
  • the production of extracellular polymeric substances (EPS) by microorganisms leads to a slimy colony morphology.
  • EPS extracellular polymeric substances
  • the polymer was isolated from the corresponding culture supernatant by ethanol precipitation.
  • G. japonicus LMG 1417 was cultured in a complex medium consisting of yeast extract (6 g/L), sucrose (200 mM) and mannitol (5 mM).
  • a potassium phosphate buffer pH 6.9 in a final concentration of 100 mM was added to the medium. After 24 hours of cultivation at 28 °C and 180 rpm shaking speed, the cells were separated by centrifugation and the culture supernatant mixed with three parts of 96 % ethanol.
  • Ethanol precipitation is a cost-effective and reliable method for the precipitation of microbial polysaccharides (Smith et al. (2007) J. Chem. Technol. Biotechnol. 32:1 19-129., doi: 10.1002/jctb.50303201 16).
  • the precipitate was air-dried and then analyzed by 13 C-NMR analysis and FTIR spectroscopy.
  • commercial levan was used, which was produced by Erwinia herbicola and obtained from Sigma-Aldrich (Steinheim, Germany) (Blake et al. (1982) J. Bacteriol.).
  • the spectra of the 13 C-NMR analysis are shown in Figure 2.
  • the two levan preparations were additionally analyzed by FTIR spectroscopy.
  • the functional groups of the analyzed molecules are stimulated by long-wave infrared radiation, resulting in substance-specific absorption spectra.
  • the two recorded spectra are shown in Figure 3.
  • the FTIR spectra of the levan preparations confirmed the assumption that the EPS formed by G. japonicus LMG 1417 is levan.
  • the two spectra were also evaluated according to the publication of Barone and Medynets, in which an FTIR analysis of levan was carried out for the first time (Barone et al. (2007) Carbohydr. Polym., doi: 10.1016/j.carbpol.2007.01.017).
  • the gene sequence coding for the enzyme (GenBank accession: KXV23964.1) was introduced into the overexpression vector pASK-IBA5plus (IBA GmbH).
  • the native nucleotide sequence of the gene encoding for the levansucrase (LevS1417) from G. japonicus LMG 1417 is set forth in SEQ ID NO:4.
  • the corresponding amino acid sequence of the native levansucrase from G. japonicas LMG 1417 is set forth in SEQ ID NO:2.
  • the primer sequences of p5_levS1417_ior and p5_levS1417_rev are set forth in SEQ ID Nos. 9 and 10.
  • the insertion was carried out using the endonucleases Sacll and Xho I. Due to the chosen cloning strategy, the N-terminus of the levansucrase was fused to a Strep-Tag II. This affinity tag enables efficient chromatographic purification of the recombinant protein from the total cell extract of an E. coli overexpression culture.
  • the plasmid map of pASK-IBA5plus (IBA GmbH) is illustrated together with the components of the vector, for example, under: https://search.cosmobio.co.jp/cosmo_search_p/search_gate2/docs/IBA_/21404000.20060609.pdf.
  • the vector pASK5-IBA5plus (IBA GmbH) is designed for heterologous overproduction of proteins in E. coli.
  • the Tet promoter upstream of the multiple cloning site (MCS) allows strong transcription of any gene insert.
  • the promoter is regulated and can be activated by the addition of the inductor anhydrotetracycline.
  • the final plasmid pASK5_levS1417 is shown in Figure 4.
  • the native sequence of the gene, coding for the levansucrase was altered by the cloning strategy and the associated modification of the 5’-end.
  • the open-reading frame (ORF) coding for the modified levansucrase (Strep-Tag II, linker) is set forth in SEQ ID NO:3.
  • the amino acid sequence of the modified levansucrase variant (N-terminal modification: Strep-Tag II, linker) is set forth in SEQ ID NO:1 .
  • the levansucrase was purified from the total cell extract via the fused N-terminal Strep Tag II.
  • the Strep-Tactin®XT Superflow ® 50 % suspension (IBA GmbH) served as the matrix for the chromatographic purification.
  • the generated elution fraction was then separated by SDS-PAGE and proteins in the fraction were visualized by silver staining (Blum et al. 1987).
  • a pH profile and Michaelis Menten kinetics were prepared for the recombinant enzyme. The images resulting from this characterization are shown in Figure 5.
  • the silver staining of the SDS gel confirmed the successful and reliable plasmid-mediated production of the recombinant levansucrase LevS1417 in E. coli DH5a.
  • the protein which has a predicted size of 51 .2 kDa, was clearly visualized without any impurities.
  • the pH profile shows that the investigated levansucrase is adapted to a slightly acidic environment and works optimally at a pH of 5. This observation coincides with the physiology of members of the genus Gluconobacter, which are adapted to an acidic habitat (Matsushita et al. (1989) Agric. Biol. Chem., doi: 10.1080/00021369.1989.10869793).
  • the enzyme behaves kinetically according to Michaelis-Menten and shows a Vmax of 3064 ⁇ 103 U mg 1 and a K m value of 147 ⁇ 16 mM at 30 °C. At 50 °C a specific activity of 5190 ⁇ 886,9 U mg 1 was measured.
  • LevS1417 from G. japonicus LMG 1417 is the most active levansucrase described to date in the literature.
  • the enzyme is suitable for industrial applications due to its enormously high activity and was therefore selected as the basis for the intended production of levan-based FOS.
  • a mutant strain based on G. japonicus LMG 1417 was generated, which is capable of a plasmid-mediated homologous overproduction of the investigated levansucrase LevS1417.
  • the vector pBBR1 -p264-streplong served as the platform for the overexpression (Zeiser et al. (2014) Appl. Microbiol. Biotechnol., doi: 10.1007/s00253-013-5016-5).
  • This modified variant of the broad host range vector pBBR1 MCS-2 was extended upstream of the MCS by a Gluconobacter- specific promoter region (p264).
  • An additional insert downstream of the MCS contains the sequence coding for Strep-Tag II and the termination sequence of the pASK-IBA3 vector (IBA GmbH).
  • the plasmid map of the generated vector is shown in Figure 7.
  • the mentioned promoter region is the upstream region of the gene gox0264 encoded in the genome of Gluconobacter oxydans 621 H. A strong, constitutive promoter is located in this area.
  • the gene sequence coding forthe levansucrase from G.japonicus LMG 1417 was amplified using the primers listed in Table 3.
  • levS1417_EcoRV_1or and levS1417_Asc ⁇ _rev are set forth in SEQ ID Nos. 1 1 and 12.
  • the amplificate was inserted via the restriction endonucleases EcoRV and Ascl into the complementarily digested vector pBBR1 -p264-streplong.
  • the insert was cloned off-frame to the Strep-Tag II coding sequence downstream of the MCS.
  • the resulting plasmid pBBR1_p264 JevS1417 is shown in Figure 8.
  • the constructed plasmid was transformed by electroporation into electrocompetent cells of the strain G. japonicus LMG 1417.
  • the transformation was carried out according to the protocol of Mostafa and colleagues (Mostafa et al. (2002) Appl. Environ. Microbiol., doi: 10.1 128/AEM.68.5.2619-2623.2002).
  • the wild type G. japonicus LMG 1417 and the mutant G. japonicus LMG 1417 pBBR1_p264 _levS1417 were successfully used for cell-free levan production. Therefore, the two strains were first cultivated in the following medium up to an ODeoonm of 2. YPSM-50/5:
  • MES buffer pH 6.5 in a final concentration of 100 mM was supplemented.
  • the cells were harvested after 24 hours of incubation at 28 °C and 180 rpm shaking speed. The cultures were centrifuged for 25 minutes at room temperature and 20,000 rpm. The resulting supernatant served as the starting point for cell-free levan production, which is shown schematically in Figure 9.
  • G. japonicus LMG 1417 secretes the levansucrase LevS1417 via an unknown secretory system into the culture supernatant. The supernatant can therefore be used directly for levan production without additional purification processes.
  • HPLC high-performance liquid chromatography
  • the described overexpression mutant G. japonicus LMG 1417 pBBR1_p264 JevS1417 was constructed.
  • the plasmid-mediated overproduction was intended to elevate the amounts of secreted levansucrase and thus enable faster conversion of the supplemented sucrose.
  • the reaction course of the cell-free levan production based on the culture supernatant of the mutant strain is shown in Figure 1 1 .
  • Figure 12 shows the most productive processes based on Zymomonas mobilis CCT 4494 (Lorenzetti et al. (2015) J. Food Process Eng. 38:31-36., doi: 10.1 1 1 1/jfpe.12123), Bacillus subtilis ( natto ) CCT7712 (Dos Santos et al. (2013) Rom. Biotechnol. Lett.) and Bacillus methylotrophicus SK 21 .002 (Zhang et al. (2014) Carbohydr. Polym., doi: 10.1016/j.carbpol.2013.10.045).
  • G. japonicus LMG 1417 e.g. wild type or overexpression mutant
  • Enzyme class EC 3.2.1 .65 endolevanases are capable of hydrolyzing levan into short-chain FOS.
  • a total of three endolevanases were cloned into independent overexpression vectors.
  • the vector pASK-IBA5plus (IBA GmbH) served as the basis for the heterologous production of the desired endolevanases in E. coli.
  • particular focus will be placed on the previously uncharacterized endolevanase from A. chroococcum DSM 2286.
  • the gene sequence coding for the endolevanase was amplified using the oligonucleotide primers listed in Table 4. The selected cloning strategy deleted the native N-terminal signal peptide of the endolevanase.
  • the primer sequences of levB2286_p5_i and levB2286_ p5_r are set forth in SEQ ID Nos. 13 and 14.
  • the amplificate was introduced via the Bsal restriction sites into the complementarily digested vector pASK-IBA5plus.
  • the native sequence of the endolevanase was altered by the cloning strategy.
  • the openreading frame (ORF) coding for the modified endolevanase has the sequence set forth in SEQ ID NO:7 (modification: sequence coding for Strep-Tag II; linker).
  • the corresponding amino acid sequence of the modified endolevanase variant is set forth in SEQ ID NO:5.
  • the native amino acid sequence is set forth in SEQ ID NO:6.
  • the recombinant protein was purified from the cell extract by streptactin affinity chromatography. The successful production and purification of the recombinant protein was verified after the separation of the elution fraction via SDS-PAGE. The 59.2 kDa protein was clearly visualized by silver staining. Characterization of the purified enzyme was carried out, with particular attention to the activity and the specific product spectrum. For the intended process, the investigated endolevanase should ideally generate FOS with a degree of polymerization (DP) of > 3.
  • DP degree of polymerization
  • carbohydrate polymers consisting of three or more monomeric units can be declared as fiber. Dietary fibers are significantly more valuable than di- or monosaccharides and are therefore desirable reaction products.
  • EU European Parliament
  • carbohydrate polymers consisting of three or more monomeric units can be declared as fiber. Dietary fibers are significantly more valuable than di- or monosaccharides and are therefore desirable reaction products.
  • the endolevanase from A. chroococcum DSM 2286 is the fastest endolevanase, which has been described within the enzyme class EC 3.2.1 .65 to date. At 30 °C, the enzyme has a maximal specific activity of 6685 ⁇ 224 U mg 1 .
  • Hexaose makes up the largest part of the reaction products.
  • the hydrolysis using the endolevanases from B. licheniformis DSM 13 and B. thetaiotaomicron DSM 2079 is significantly slower, while at the same time relevant amounts of fructose and levanbiose are formed, especially with elongated incubation.
  • endolevanases described in the literature Jensen et al. J. Biol. Macromol. (2016), 85:514-521 ; Porras-Dominguez et al. (2014) Process Biochem., doi: 10.1016/j.procbio.2014.02.005; Mardo et al. (2017) PLoS One 12, doi:
  • the investigated endolevanase from A. chroococcum DSM 2286 is the only endolevanase described so far that guarantees a unique combination of high activity and high product stability.
  • the enzyme is therefore ideally suited for the industrial production of levan-based dietary fibers.
  • an overexpression plasmid was generated that carries the coding genes of both proteins.
  • the plasmid pASK5 JevS1417 which was constructed for the overexpression of the levansucrase from G. japonicus LMG 1417, served as the starting point for the cloning strategy.
  • the gene sequence coding for the levansucrase was amplified together with the flanking regulatory elements (promoter region and termination sequence).
  • the amplificate was inserted by using the NEBuilder® HiFi DNA Assembly Master Mix into the plasmid pASK5_/evB2286, which was previously enzymatically linearized via the endonuclease Mscl.
  • the overexpression plasmid was introduced into E. coli BL21 , the recombinant proteins were purified by affinity chromatography after heterologous production. The proteins in the elution fraction were visualized by SDS-PAGE and silver staining.
  • sucrose In order to validate whether sucrose can be enzymatically converted into levan-based FOS in a single reaction, a suitable assay based on the E. coli cell extract was performed. A saturated sucrose solution ( ⁇ 2.5 M) was adjusted to pH 5 by adding a sodium acetate buffer (40 mM), which ensures the functionality of both enzymes. The enzymatic reaction was started by adding the cell extract. The added extract volume corresponded to 3.85 ml_ per L reaction. The educts and products were again quantified by HPLC ( Figure 18).
  • sucrose was almost completely converted within 55 hours. Only 7.8 ⁇ 0.2 % of the fructose units contained in the sucrose were released in the form of free fructose. Most of the fructose was introduced into the levan polymer by the recombinant levansucrase, which was then hydrolyzed to short-chain FOS by the catalytic activity of the recombinant endolevanase. After 55 hours of incubation, a FOS yield of 371 .7 ⁇ 10.2 g L-1 was detected. The described process converted the fructose units contained in sucrose to FOS with a DP of > 3 with an efficiency of 88.4 %.
  • the fructose concentration at this time (tss h ) was 173 ⁇ 6 mM. A concentration of 43 ⁇ 9 mM could be determined for levanbiose. The loss of fructose units that were not incorporated into FOS with a DP of > 3 was thus 1 1 .6 %.
  • the two described enzymes (levansucrase and endolevanase) were heterologously overproduced in Escherichia coli DH5a and subsequently purified by affinity chromatography.
  • the purification was carried out via the N-terminal Strep-Tag II using Strep-Tactin®XT Superflow® (IBA GmbH).
  • the assays to determine the specific levansucrase activity were performed at 30 °C.
  • the reaction was buffered by a sodium acetate buffer pH 5.4 at a final concentration of 100 mM.
  • Sucrose in different concentrations (0-1500 mM) served as the substrate for the reactions.
  • the specific activity of the endolevanase from Azotobacter chroococcum DSM 2286 was measured at 30 °C.
  • the reaction was buffered by a Mcllvaine buffer pH 6.
  • the endolevanase of the invention thus combines an exceptionally high conversion rate with advantageous hydrolytic properties. This enables rapid production of levan-based FOS, which maintain a consistently high product titer even with longer incubation times and when using higher enzyme concentrations.

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Abstract

La présente invention concerne un procédé de préparation de fructo-oligosaccharides (FOS) (à base de lévane) à l'aide d'au moins un organisme hôte (génétiquement modifié). Grâce à la production d'une levansucrase et d'une endoévanase chez l'organisme hôte, les enzymes peuvent être utilisées pour convertir le saccharose du substrat en FOS. Par ailleurs, l'invention concerne des fructo-oligosaccharides pouvant être obtenus par le procédé selon l'invention; un vecteur d'expression spécifique, un organisme hôte génétiquement modifié spécifique, et un extrait cellulaire spécifique ou un surnageant de culture, qui sont utilisables pour la production de FOS; et un prébiotique ou un complément alimentaire comprenant ou constitué du FOS. L'invention concerne enfin un procédé de préparation de lévane au moyen de lévansucrase.
EP20734888.9A 2019-06-27 2020-06-23 Production enzymatique de fructo-oligosaccharides prébiotiques à base de lévane Pending EP3990655A1 (fr)

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EP19182814.4A EP3757209A1 (fr) 2019-06-27 2019-06-27 Production enzymatique de fructooligosaccharides prébiotiques à base de levan
PCT/EP2020/067447 WO2020260249A1 (fr) 2019-06-27 2020-06-23 Production enzymatique de fructo-oligosaccharides prébiotiques à base de lévane

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