| Summary: | <p>Abstract</p> <p>Background</p> <p>β-<smcaps>D</smcaps>-Galactosidases (EC 3.2.1.23) catalyze the hydrolysis of terminal non-reducing β-<smcaps>D</smcaps>-galactose residues in β-<smcaps>D</smcaps>-galactosides. Cold-active β-<smcaps>D</smcaps>-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-<smcaps>D</smcaps>-galactosidase from <it>Kluyveromyces lactis </it>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.</p> <p>Results</p> <p>A genomic DNA library was constructed from soil bacterium <it>Paracoccus </it>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-<smcaps>D</smcaps>-galactosidase was isolated. The <it>in silico </it>analysis of the enzyme amino acid sequence revealed that the β-<smcaps>D</smcaps>-galactosidase <it>Paracoccus </it>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 <it>bgaL </it>gene was cloned and expressed in <it>Escherichia coli </it>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 <it>o</it>-nitrophenyl-β-<smcaps>D</smcaps>-galactopyranoside at 10°C with <it>K</it><sub>m </sub>values of 2.94 and 1.17 mM and <it>k</it><sub>cat </sub>values 43.23 and 71.81 s<sup>-1</sup>, 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.</p> <p>Conclusions</p> <p>A novel <it>bgaL </it>gene was isolated from <it>Paracoccus </it>sp. 32d encoded a novel cold-active β-<smcaps>D</smcaps>-galactosidase. An <it>E. coli </it>expression system has enabled efficient production of soluble form of BgaL <it>Paracoccus </it>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-<smcaps>D</smcaps>-galactosidases belonging to Glycoside Hydrolase Family 2. Finally, the enzymatic properties of <it>Paracoccus </it>sp. 32d β-<smcaps>D</smcaps>-galactosidase shows its potential for being applied to development of a new industrial biocatalyst for efficient lactose hydrolysis in milk.</p>
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