Glucan incorporating 4-, 6-, and 4, 6- linked...

Details Glucan incorporating 4-, 6-, and 4, 6- linked... Glucan incorporating 4-, 6-, and 4, 6- linked...

2000-06-28

2002-11-26

Wilson, James O. (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S055100

Reexamination Certificate

active

06486314

ABSTRACT:

The present invention is in the field of enzymatic production of biomolecules. The invention is particularly concerned with a novel type of glucosyltransferase derived from lactobacilli and with a process for production of the enzyme and for the production of useful glucans and gluco-oligosaccharides from sucrose. Furthermore, the invention pertains to the produced glucans and gluco-oligosaccharides.
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 yoghurt, 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 posses the Generally Recognised As Safe (GRAS) status. Due to the different products which are formed 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 polysaccharides 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 alginate, 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 exopolysaccharides of microorganisms, such as lactic acid bacteria, which have the GRAS status. The exopolysaccharides produced by lactic acid bacteria can be divided in two groups, heteropolysaccharides 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. Considerable less information is present on the synthesis of homopolysaccharides from lactobacilli, although some studies have been performed. The information on the synthesis of homopolysaccharides in lactobacilli is mainly limited to the synthesis of glucans and only two reports, written by the present inventors, exist on the synthesis of fructans. In one of these reports 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 the other report was found that
Lactobacillus reuteri
strain LB 35-5, a spontaneous mutant of
Lactobacillus reuteri
strain LB 121, only produced a glucan when grown on sucrose (van Geel-Schutten, G. H. et al.,
Appl. Environ. Microbiol
. (1999) 65, 3008-3014). In the other report the soluble glucan and fructan were also characterised 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 &agr;-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). These reports are incorporated herein by reference. No structurally identification of a similar glucan produced by a
Lactobacillus
had been reported before. The fructan was identified as a (2→6)-&bgr;-D-fructofuranan (also called a levan). This was the first example of levan synthesis by a
Lactobacillus
species.
SUMMARY OF THE INVENTION
A novel enzyme having glucosyltransferase activity using sucrose as a substrate has now been found in
Lactobacillus reuteri
, and its amino acid sequence and other structural properties have been determined. The enzyme is unique in that it is capable of producing a highly branched glucan with &agr;-1,4- and &agr;-1,6 glucosidic links. The invention thus pertains to an enzyme, to DNA encoding it, to cells containing such DNA and to their use in producing carbohydrates, as defined in the appending claims. The invention also pertains to glucans, oligosaccharides and chemically derivatised glucans, containing the unique structure mentioned above.
DESCRIPTION OF THE INVENTION
It was found according to the invention that the glucans are produced by certain
Lactobacillus strains
, in particular by certain strains of
Lactobacillus reuteri
, as a result of the activity of a single glucosyltransferase (glucansucrase).
The nucleotide and amino acid sequences of the novel glucosyltransferase are shown in FIG.
3
. As mentioned above, the nucleotide sequence contains two putative start condons leading to either a 3834 or a 3753 nucleotide form of the glucosyltransferase. Both putative start codons are preceded by a putative ribosome binding site, GCAGG (located 4 base pairs upstream its start codon) or AGAAG (located 14 base pairs upstream its start codon), respectively.
This glucosyltransferase consists of either 1278 amino acids (3834 nucleotides) or 1251 amino acids (3753 nucleotides) depending on the potential start codon used. The molecular weight (MW) deduced of the amino acid sequences of these forms is 143 or 140 kDa, respectively. The molecular weight indicated by SDS-PAGE is 180 kDa. The isoelectric point deduced of the amino acid sequence is 4.73 (for the higher MW protein) or 4.71 (for the lower MW protein), at pH 7.
The present invention covers a protein having glucosyltransferase activity with sucrose as substrate with an amino acid identify of at least 50%, preferably at least 60%, and more preferably at least 70%, compared to the amino acid sequence of SEG 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. The novel glucosyltransferase has homology with several other proteins as revealed by amino acid sequence alignment. A high homology (
FIG. 5
) was found with an alternansucrase of
Leuconostoc mesenteroides
strain NRRL B-1355 (46% identity, within 1261 amino acids) and a dextransucrase of
Leuconostoc mesenteroides
strain NRRL B-512F (44% identity, within 1270 amino acids). Furthermore, the alignment revealed the presence of various domains also found in the other glucosyltransferases, such as an N-terminal variable domain, a catalytic domain and a C-terminal glucan binding domain. The N-terminal domain shows almost no identity with the N-terminal domains of other glucosyltransferases and an N-terminal signal peptide could not be detected.
The invention also covers a protein comprising an amino acid sequence of at least 100 amino acids, exhibiting at least 55%, preferably at least 65% amino acid identity with the corresponding part of the amino acid sequence 442-984 (catalytic domain) of SEQ ID No. 1. The catalytic domain shows a high level of homology (about 50% identity) with other known
streptcoccal
and
Leuconostoc
glucosyltransferases and putative functions based on the alignment can be ascribed to several amino acids within this catalytic domain (FIG.
4
). Asp-494, Glu-531 and Asp-603 are putative catalytic residues, Asp-454 is a putative calcium binding residue and Arg-492 a putative chloride binding residue. His-602 and Gln-984 may stabilize the transition state and the residues Asp-497, Asn-498, Asp-532 and Trp-533 may play a role in bi

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