Enzyme isolated from a Bifidobacterium

Details Enzyme isolated from a Bifidobacterium Enzyme isolated from a Bifidobacterium

2001-05-29

2003-04-29

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S193000, C435S201000, C426S582000, C426S583000

Reexamination Certificate

active

06555348

ABSTRACT:

TECHNICAL FIELD OF INVENTION
The present invention concerns improvement of fermented dairy products. In particular, the invention concerns a &bgr;-galactosidase with transgalactosylating activity. More particularly, the invention concerns a &bgr;-galactosidase isolated from
Bifidobacterium bifidum
where the C-terminal end of the protein has been deleted and the resulting truncated enzyme has higher transgalactosylating activity than hydrolase activity. When lactose is used as a substrate, galacto-oligosaccharides are products of the transgalactosylase activity. Galacto-oligosaccharides enhance growth of health-promoting Bifidobacterium that may be used in a number of applications in the dairy industry.
BACKGROUND OF THE INVENTION
The genus Bifidobacterium is one of the most commonly used types of bacteria cultures in the dairy industry for fermenting a variety of dairy products. Ingestion of Bifidobacterium-containing products furthermore has a health-promoting effect. This effect is not only achieved by a lowered pH of the intestinal contents, but also by the ability of Bifidobacterium to repopulate the intestinal flora in individuals who have had their intestinal flora disturbed by, for example, intake of antibiotics. Bifidobacterium furthermore has the potential of outcompeting potential harmful intestinal micro-organisms.
Galacto-oligosaccharides are known to enhance the growth of Bifidobacterium. This effect is likely achieved through the unique ability of Bifidobacterium to exploit galacto-oligosaccharides as a carbon source. Dietary supplement of galacto-oligosaccharides is furthermore thought to have a number of long-term disease protecting effects. For example, galacto-oligosaccharide intake has been shown to be highly protective against development of colorectal cancer in rats (Wijnands, et al., 1999). There is therefore a great interest in developing cheap and efficient methods for producing galacto-oligosaccharides for use in the industry for improving dietary supplements and dairy products.
The enzyme &bgr;-galactosidase (EC 3.2.1.23) usually hydrolyzes lactose to the monosaccharides D-glucose and D-galactose. In the normal enzyme reaction of &bgr;-galactosidases, the enzyme hydrolyzes lactose and transiently binds the galactose monosaccharide in a galactose-enzyme complex that transfers galactose to the hydroxyl group of water, resulting in the liberation of D-galactose and D-glucose. However, at high lactos concentrations, some &bgr;-galactosidases are able to transfer galactose to the hydroxyl groups of D-galactose or D-glucose in a process called transgalactosylation, whereby galacto-oligosaccharides are produced.
Enzymes capable of transgalactosylation have been isolated from a wide range of micro-organisms, including bacteria and yeasts. The observation that galacto-oligosaccharides enhance the growth of health-promoting Bifidobacterium has stimulated investigations of Bifidobacterium and their &bgr;-galactosidase enzymes. Two DNA sequences of
B. breve
and
B. longum
&bgr;-galactosidase genes have been deposited in GeneBank (accession numbers E5040 and AJ242596, respectively). Dumortier et al. (1994) have reported that
B. bifidum
DSM 20215 contains three &bgr;-galactosidases and one of these enzymes has trans-galactosylating properties. However, no identification of the enzyme possessing this activity or any sequence of the enzyme or the corresponding gene from
B. bifidum
DSM 20215 has been published.
Production of galacto-oligosaccharides by the use of &bgr;-galactosidases has been reported in several papers. For example, &bgr;-galactosidase from
E. coli
has been shown to produce oligosaccharides at high lactose concentrations (0.5 M or approximately 20% lactose; Huber et al. 1976). Various thermophilic microorganisms have been shown to produce oligosaccharides at high temperatures and high lactose concentrations, e.g.
Sterigmatomyces elviae
can produce 39% oligosaccharides from 20% lactose at 60° C. (onishi & Tanaka, 1995), and
Saccharopolyspora rectivirgula
can synthesize 41% oligosaccharides in 1.75 M lactose at 70° C. (Nako et al., 1994).
However, the enzymes described above all have the drawbacks of requiring either high temperatures or high lactose concentrations or both in order to exhibit significant transgalactosylase activity. There is thus a need for developing cheaper and more efficient methods of producing galacto-oligosaccharides for use in the industry.
SUMMARY OF THE INVENTION
The present invention describes a new &bgr;-galactosidase from
Bifidobacterium bifidum
. A truncated version of the enzyme has surprisingly been shown to have a high transgalactosylating activity. When the truncated enzyme, or a host cell expressing the recombinant truncated enzyme, is incubate with lactose under appropriate conditions, galacto-oligosaccharides are produced at a high efficiency. Presence of galacto-oligosaccharides in dairy products or other comestible products has the advantage of enhancing the growth of health-promoting Bifidobacterium in the product or in the intestinal flora of the consumer after intake of the product, or both.


REFERENCES:
patent: 5939289 (1999-08-01), Ertesvag et al.
V. Dumortier, et al. “Purification and Properties of a &bgr;-D-Galactosidase FromBifidobacterium bifidumExhibiting A Transgalactosylation Reaction”, Biotechnology. Appl. Biochem., vol. 19, pp. 341-354 (1994).
N. Onishi et al., “Purification and Properties of a Novel Thermostable Galacto-Oligosaccharide-Producing &bgr;-Galactosidase FromSterigmatomyces elviaeCBS8119”, Applied and Envornmental Microbiology, vol. 61, No. 11, pp. 4026-4030, (Nov. 1995).
M. Nakao et al., “Purification and Characterization of a Thermostable &bgr;-Galactosidase with High Transgalactosylation Activity FromSaccharopolyspora rectivirgula”, Appl. Microbiol Biotechnol., vol. 40, pp. 657-663; (1994).
R.E. Huber, et al., “A Quantitation of the Factors which Affect the Hydrolase and Transgalactosylase Activities of &bgr;-Galactosidase (E. coli) on Lactose”, Biochemistry, vol. 15, No. 9, pp. 1994-2001, (1976).
M.V.W. Wijnands, et al., “A Comparison of the Effects of Dietary Cellulose and Fermentable Galacto-Oligsaccharide, in a Rat Model of Colorectal Carcinogenesis: Fermentable Fibre Confers Greater Protection Then Non-Fermentable Fibre in Both High and Low Fat Backgrounds”, Carcinogenesis, vol. 20, No. 4, pp. 651-656; (1999).

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