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Backgroundβ-D-Galactosidases (EC 3.2.1.23) catalyze the hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. Cold-active β-D-galactosidases have recently become a focus of attention of researchers and dairy product manufactures owing to theirs ability to: (i) eliminate of lactose from refrigerated milk for people afflicted with lactose intolerance, (ii) convert lactose to glucose and galactose which increase the sweetness of milk and decreases its hydroscopicity, and (iii) eliminate lactose from dairy industry pollutants associated with environmental problems. Moreover, in contrast to commercially available mesophilic β-D-galactosidase from Kluyveromyces lactis the cold-active counterparts could make it possible both to reduce the risk of mesophiles contamination and save energy during the industrial process connected with lactose hydrolysis.ResultsA genomic DNA library was constructed from soil bacterium Paracoccus sp. 32d. Through screening of the genomic DNA library on LB agar plates supplemented with X-Gal, a novel gene encoding a cold-active β-D-galactosidase was isolated. The in silico analysis of the enzyme amino acid sequence revealed that the β-D-galactosidase Paracoccus sp. 32d is a novel member of Glycoside Hydrolase Family 2. However, owing to the lack of a BGal_small_N domain, the domain characteristic for the LacZ enzymes of the GH2 family, it was decided to call the enzyme under study 'BgaL'. The bgaL gene was cloned and expressed in Escherichia coli using the pBAD Expression System. The purified recombinant BgaL consists of two identical subunits with a combined molecular weight of about 160 kDa. The BgaL was optimally active at 40°C and pH 7.5. Moreover, BgaL was able to hydrolyze both lactose and o-nitrophenyl-β-D-galactopyranoside at 10°C with Km values of 2.94 and 1.17 mM and kcat values 43.23 and 71.81 s-1, respectively. One U of the recombinant BgaL would thus be capable hydrolyzing about 97% of the lactose in 1 ml of milk in 24 h at 10°C.ConclusionsA novel bgaL gene was isolated from Paracoccus sp. 32d encoded a novel cold-active β-D-galactosidase. An E. coli expression system has enabled efficient production of soluble form of BgaL Paracoccus sp. 32d. The amino acid sequence analysis of the BgaL enzyme revealed notable differences in comparison to the result of the amino acid sequences analysis of well-characterized cold-active β-D-galactosidases belonging to Glycoside Hydrolase Family 2. Finally, the enzymatic properties of Paracoccus sp. 32d β-D-galactosidase shows its potential for being applied to development of a new industrial biocatalyst for efficient lactose hydrolysis in milk.

BackgroundCold-active enzymes, sourced from cold-adapted organisms, are characterized by high catalytic efficiencies at low temperatures compared with their mesophilic counterparts, which have poor activity. This property makes them advantageous for biotechnology applications as it: (i) saves energy costs, (ii) shortens the times for processes operated at low temperatures, (iii) protects thermosensitive substrates or products of the enzymatic reaction, (iv) prevents undesired chemical transformations, and (v) prevents the loss of volatile compounds.ResultsA bglMKg gene that encodes a monomeric cold-active glycoside hydrolase family 1 enzyme with an apparent molecular mass of 50 kDa was isolated by the functional screening of a marine metagenomic library. The BglMKg enzyme was expressed in E. coli, purified by FPLC and characterized. The recombinant BglMKg could effectively hydrolyze various chromogenic substrates and β-linked oligosaccharides, and had remarkably high β-galactosidase, β-glucosidase and β-fucosidase activities. Because of the lack of information about the usefulness of β-fucosidases in industry, further characterization of the enzymatic properties of BglMKg was only carried out with substrates specific for β-glucosidase or β-galactosidase. The BglMKg had maximal β-galactosidase and β-glucosidase activities at approximately 40°C and 45°C, respectively. The optimum pH for β-galactosidase activity was 6.5, whereas the optimum pH for β-glucosidase activity was 7.5. In general, the enzyme was stable below 30°C and from pHs 6.0 to 8.0. The results of the kinetic studies revealed that BglMKg more efficiently hydrolyzed β-glucosidase substrates than β-galactosidase ones.ConclusionsBglMKg is a small, monomeric, cold-active β-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications.

A psychrotrophic bacterium producing a cold-adapted beta-galactosidase upon growth at low temperatures was classified as Arthrobacter sp. 20B. A genomic DNA library of strain 20B introduced into Escherichia coli TOP10F' and screening on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside)-containing agar plates led to the isolation of beta-galactosidase gene. The beta-galactosidase gene (bgaS) encoding a protein of 1,053 amino acids, with a calculated molecular mass of 113,695 kDa. Analysis of the amino acid sequence of BgaS protein, deduced from the bgaS ORF, suggested that it is a member of the glycosyl hydrolase family 2. A native cold-adapted beta-galactosidase was purified to homogeneity and characterized. It is a homotetrameric enzyme, each subunit being approximately 116 kDa polypeptide as deduced from native and SDS-PAGE, respectively. The beta-galactosidase was optimally active at pH 6.0-8.0 and 25 degrees Celsius. P-nitrophenyl-beta-D-galactopyranoside (PNPG) is its preferred substrate (three times higher activity than for ONPG-o-nitrophenyl-beta-D-galactopyranoside). The Arthrobacter sp. 20B beta-galactosidase is activated by thiol compounds (53% rise in activity in the presence of 10 mM 2-mercaptoethanol), some metal ions (activity increased by 50% for Na(+), K(+) and by 11% for Mn(2+)) and inactivated by pCMB (4-chloro-mercuribenzoic acid) and heavy metal ions (Pb(2+), Zn(2+), Cu(2+)).