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 β-galactosidase (EC 3.2.1.23) usually hydrolyzes lactose to the monosaccharides D-glucose and D-galactose. In the normal enzyme reaction of β-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 lactose concentrations some β-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 β-galactosidase enzymes. Two DNA sequences of B. breve and B. longum β-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 β-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-o-ligosaccharides by-the use of β-galactosidases has been reported in several papers. For example, β-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.