Fructosyltransferases

Details Fructosyltransferases Fructosyltransferases

2000-06-28

2003-10-21

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C536S023200

Reexamination Certificate

active

06635460

ABSTRACT:

The present invention is in the field of enzymatic production of biomolecules. The invention is particularly concerned with two novel fructosyltransferases derived from lactobacilli and with a process for recombinant production of the enzymes and for the production of useful levans, in s and fructo-oligosaccharides from sucrose.
BACKGROUND OF THE INVENTION
Lactic acid bacteria (LAB) play an important role in the fermentative production of food and feed. Traditionally, these bacteria have been used for the production of for instance wine, beer, bread, cheese and yogurt and for the preservation of food and feed, e.g. olives, pickles, sausages, sauerkraut and silage, Because of these traditional applications, lactic acid bacteria are food-grade micro-organisms that possess the Generally Recognised As Safe (GRAS) status. Due to the different products which are during fermentation with lactic acid bacteria, these bacteria contribute positively to the taste, smell and preservation of the final product. The group of lactic acid bacteria encloses several genera such as Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, etc.
In recent years also the health promoting properties of lactic acid bacteria have received much attention. They produce an abundant variety of exopolysaccharides EPS's). These polysacharides are thought to contribute to human health by acting as prebiotic substrates, nutraceuticals, cholesterol lowering agents or immunomodulants.
To date high molecular weight polysaccharides produced by plants (such as cellulose, starch and pectin), seaweeds (such as alginate and carrageenan) and bacteria (such as algate, gellan and xanthan) are used in several industrial applications as viscosifying, stabilising, emulsifying, gelling or water binding agents. Although all these polysaccharides are used as food additives, they originate from organisms not having the GRAS status. Thus they are less desirable than the exopolysacciarides of microorganisms, such as lacic acid bacteria, which have the GRAS status.
The exopolysaccharides produced by LAB can be divided in two groups, heteropolysacchrides and homopolysaccharides; these are synthesized by totally different mechanisms. The former consist of repeating units in which residues of different types of sugars are present and the latter consist of one type of monosaccharide. The synthesis of heteropolysaccharides by lactic acid bacteria, including lactobacilli, has been studied extensively in recent years. Considerably less information is available on the synthesis of homopolysaccharides from lactobacilli, although some studies have been performed. Homopolysacoharides with fructose as the constituent sugar can be divided into two groups, inulins and levans. Inulins consist of 2,1-lined &bgr;-fructofuranoside residues, whereas levans consist of 2,6-linked &bgr;-fructofuranoside residues. Both can be linear or branched, The size of bacterial levans can vary from 20 kDa up to several MDa. There is limited information on the synthesis of levans, In most detail this synthesis has been studied in
Zymomonas mobilis
and in Bacillus species, Within lactic acid bacteria, fructosyltransferases have only been studied in streptococci. So far no fructosyltransferases have been reported in lactobacilli.
In a recent report the
Lactobacillus reuteri
strain LB 121 was found to produce both a glucan and a fructan when grown on sucrose, but only a fructan when grown on raffinose (van Geel-Schutten, G. H. et al., Appl. Microbiol. Biotechnol. (1998) 50, 697-703). In another report the glucan and fructan were characterize by their molecular weights (of 3,500 and 150 kDa respectively) and the glucan was reported to be highly branched with a unique structure consisting of a terminal, 4-substituted, 6-substituted, and 4,6-disubstituted &bgr;-glucose in a molar ratio 1.1:2.7:1.5:1.0 (van Geel-Schutten, G. H. et al., Appl. Environ. Microbiol. (1999) 65, 3008-3014). The fructan was identified as a linear (2→6)-&bgr;-D-fructofuranan (also called a levan). This was the first example of fructan synthesis by a Lactobacillus species.
SUMMARY OF THE INVENTION
Two novel genes encoding enzymes having fructosyltransferase activity have now been found in
Lactobacillus reuteri
, and their amino acid sequences have been determined. These are the first two enzymes identified in a Lactobacillus species capable of producing a fructan, One of the eves is an inulosucrase which produces a high molecular weight (>10
7
Da) fructan containing &bgr;(2-1) linked fructosyl units and fructo-oligosaccharides, while the other is a levansucrase which produces a fructan containing &bgr;(2-6) linked fructosyl units. The invention thus pertains to the enzymes, to DNA encoding them, to recombinant cells containing such DNA and to their use in producing carbohydrates, as defined in the appending claims.
DESCRIPTION OF THE INVENTION
It was found according to the invention that one of the novel fructosyltransferases (FTFA; an inulosucrase) produces a high molecular weight inulin with &bgr;(2-1) linked fructosyl units and fructo-oligosaccharides. The fructo-oligosaccharides synthesis was also observed in certain Lactobacillus strains, in particular in certain strains of
Lactobacillus reuteri
. However, the inulin has not been found in
Lactobacillus reuteri
culture supernatants, but only in extracts of
E. coli
cells expressing the above-mentioned fructosyltransferase. This inulosucrase consists of either 798 amino acids (2394 nucleotides) or 789 amino acids (2367 nucleotides) depending on the potential start codon used. The molecular weight (MW) deduced of the amino acid sequence of the latter form is 86 kDa and its isoelectric point is 4.51, at pH 7.
The amino acid sequence of the inulosucrase is shown in SEQ ID No. 1 (
FIG. 1
, amino acid residues 1-789). As mentioned above, the nucleotide sequence contains two putative start codons leading to either a 2394 or 2367 nucleotide form of the inulinsucrase. Both putative start codons are preceded by a putative ribosome binding site, GGGG (located 12 base pairs upstream its start codon) or AGGA (located 14 base pairs upstream its start codon), respectively (see FIG.
1
).
The present invention covers a protein having inulosucrase activity with an amino acid identity of at least 65%, preferably at least 75%, and more preferably at least 85%, compared to the amino acid sequence of SEQ ID No. 1. The invention also covers a part of a protein with at least 15 contiguous amino acids which are identical to the corresponding part of the amino acid sequence of SEQ ID No. 1.
Fructosyltransferases have been found in several bacteria such as
Zymomonas mobilis, Erwinia amylovora, Acetobacter amylovora, Bacillus polymyxa, Bacillus amyloliquefaciens, Bacillus stearothermophilus
, and
Bacillus subtilis
. In lactic acid bacteria this type of enzyme previously has only been found in some streptococci. Most bacterial fructosyltransferases have a molecular mass of 50-100 kDa (with the exception of the fructosyltransferase found in
Streptococcus salivarius
which has a molecular mass of 140 kDa). Amino acid sequence alignment revealed that the novel inulosucrase of lactobacilli has high homology with fructosyltransferases originating from Gram positive bacteria, in particular with Streptococcus enzymes. The highest homology (
FIG. 2
) was found with the SacB enzyme of
Streptococcus mutans
Ingbritt A (62% identity within 539 amino acids).
Certain putative functions based on the alignment and site-directed mutagenesis studies can be ascribed to several amino acids of the novel inulosucrase, Asp-263, Glu-330, Asp-415, Glu-431, Asp-511, Glu-514, Arg-532 and/or Asp-551 of the amino acid sequence of SEQ ID No. 1 are identified as putative catalytic residues. Noteworthy, a hydrophobicity plot according to Kyte and Doolittle (1982) J. Mol. Biol. 157, 105-132 suggests that the novel inulosucrase contains a putative signal sequence according to the Von Heijne rule. The putative signal peptidase site is located between Gly at position 21 and Ala at

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