Two β-galactosidases from the human isolate Bifidobacterium breve DSM 20213: molecular cloning and expression, biochemical characterization and synthesis of galacto-oligosaccharides.

Sheryl Lozel Arreola,Montira Intanon,Dietmar Haltrich,Roman Kittl,Jasmina Suljic,Thu-Ha Nguyen,Ngoc Hung Pham,Paul Kosma,Inês A Cardoso Pereira

doi:10.1371/journal.pone.0104056

Abstract

Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The k cat/K m values for oNPG and lactose hydrolysis are 722 and 7.4 mM−1s−1 for β-gal I, and 543 and 25 mM−1s−1 for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6)-D-Glc (allolactose) and β-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6) and β-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.

Highlights

The colonic microbiota is composed of more than 400 different species, some of which have been related to health and well-being of the host [1]
The major Bifidobacterium species found in the adult microflora are Bifidobacterium adolescentis and B. longum while B. infantis and B. breve are the predominant bifidobacteria in infant intestinal tracts [4,6]
The resulting clones were cultivated under inducing conditions in MagicMedia to compare the expression yields with and without chaperone co-expression. b-Gal I and b-gal II expressed in the strains with chaperones showed a 30- and 14-fold increase in activity compared to the activity obtained from the strains without chaperones, respectively (Table 1)

Summary

Introduction

The colonic microbiota is composed of more than 400 different species, some of which have been related to health and well-being of the host [1]. Members of the genus Bifidobacterium are one of the most common organisms found in the human gastro-intestinal tract [3,4] These species are considered to be important in maintaining human health as they contribute to carbohydrate fermentations in the colon, and their diversity and number provide a marker for the stability of the human intestinal microflora [5]. The major Bifidobacterium species found in the adult microflora are Bifidobacterium adolescentis and B. longum while B. infantis and B. breve are the predominant bifidobacteria in infant intestinal tracts [4,6]. Activity of these bacteria has been linked to health effects such as increased resistance to infection, stimulation of the immune system activity, protection against cancer, or other prophylactic and therapeutic benefits. Biochemical properties of these enzymes and their potential to produce GOS are presented

Materials and Methods

Results

Discussion