A novel cold-active β-D-galactosidase from the Paracoccus sp. 32d--gene cloning, purification and characterization

Abstract

Background: β-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.

Results: 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 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.

Conclusions: A 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.

Figure 1

Phylogenetic tree based on a neighbor-joining analysis of the 16S rDNA gene of strain 32d and closely related Paracoccus species Gene sequences from the following organisms were used (the numbers in parentheses are the GenBank accession numbers): P. alcaliphilus JCM7364 (D32238), P. alkenifer (Y13827), P. aminophilus JCM7686 (D32239), P. aminovorans JCM7685 (D32240), P. carotinifaciens (AB006899), P. denitrificans ATCC17741T (Y16927), P. haeundaesis BC74171 (AY189743), P. halotolerans YIM90738 (DQ923133), P. homiensis DD-R11 (DQ342239), P. kocurii JCM7684 (D32241), P. kondratievae (AF250332), P. marcusii (Y12703), P. marinus KLL-B9 (AB185959), P. methylutens (AF250334), P. pantotrophus ATCC35512T (Y16933), P. seriniphilus MBT-A4 (AJ428275), P. solventivorans (Y13826), P. thiocyanatus THI011 (D32242), P. versutus ATCC25364 (Y16932), P. yeeii G1212 (AY014173), and P. zeaxanthinifaciens ATCC21588 (AF461158). Bootstrap 1000.

Figure 2

Topographic presentation of Pfam domains for selected LacZ β-D-galactosidases and Paracoccus sp. 32d β-D-galactosidase BgaL The domains presented were suggested by the Pfam database http://www.sanger.ac.uk/software/Pfam and are indicated by different colors. The numbers in parentheses are the GenBank accession numbers.

Figure 3

Alignment of the amino acids sequences of β-D-galactosidase (A) acid-base active sites, and (B) the consensus nucleophilic region of the selected LacZ enzymes and Paracoccus sp. 32d β-D-galactosidase The numbers in parentheses are GenBank accession numbers. Proposed active site glutamic acid residues are indicated by the black arrows. Para.32d - Paracoccus sp. 32d BgaL (ACY69080), Pseudo.22b - Pseudoalteromonas sp. 22b LacZ (AAR92204), Pseudo.hal - Pseudoalteromonas haloplanktis TAE79 LacZ (AJI31635), Arthr. 20B - Arthrobacter sp. 20B LacZ (ACI41243), Athr. SB - Arthrobacter sp. SB LacZ (AAQ19029), Arthr.C2-2 - Arthrobacter sp. C2-2 LacZ (CAD29775), Art.psych - Arthrobacter psychrolactophilus strain F2 LacZ (ABN72582) and E.coliLacZ - E. coli LacZ (ABN72582).

Figure 4

SDS-PAGE (12% polyacrylamide gel) protein profiles of fractions collected after successive purification steps carried out on recombinant Paracoccus sp. 32d β-D-galactosidase from E. coli strain LMG194 Lane M - protein molecular weight marker; lane CE - cell extract; lane F - pooled fraction after Fractogel EMD DEAE chromatography; lane R - pooled fraction after Resource Q chromatography.

Figure 5

The effect of temperature on the recombinant Paracoccus sp. 32d β-D-galactosidase activity.

Figure 6

The effect of temperature on the recombinant Paracoccus sp. 32d β-D-galactosidase stability.

Figure 7

The effect of pH on the recombinant Paracoccus sp. 32d β-D-galactosidase activity.

Figure 8

The effect of pH on the recombinant Paracoccus sp. 32d β-D-galactosidase stability.

Figure 9

Hydrolysis of milk lactose by 1U of Paracoccus sp. 32d β-D-galactosidase as a function of time-course.