Abstrakt

Beta-D-galactosidase (EC 3.2.1.23) catalyze the hydrolysis of terminal non-reducing beta-D-galactose residues in beta-D-galactosides. Cold-active beta-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 beta-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. A 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 beta-D-galactosidase was isolated. The in silico analysis of the enzyme amino acid sequence revealed that the beta-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 decidet 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 degrees C and pH 7.5. Moreover, BgaL was able to hydrolyze both lactose and o-nitrophenyl-betaD-galactopyranoside at 10 degrees 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 degrees C. A novel bgaL gene was isolated from Paracoccus sp. 32d encoded a novel cold-active beta-D-galactosidase. An E. coli expression system has enabled efficient production of soluble form of BgaL Paracoccus sp. 32d.

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