*2.2. Molecular Cloning and Sequence Analysis*

Deoxyribonucleic acid sequencing showed that *gal* consisted of an open reading frame of 3174 bp, encoding 1058 amino acid residues. The theoretical Mw and pI of the enzyme was 118,543 Da and 4.96, respectively. According to the BLAST results, *gal* had a highest identity of 99% to a putative β-galactosidase from *Alteromonas addita* (WP\_062085674.1). At the time of writing, two characterized β-galactosidase genes from *Pseudoalteromonas* sp. 22b (AAR92204.1) and *Pseudoalteromonas haloplanktis* (CAA10470.1) exhibited the highest sequence identity (65%) to *gal*. Based on sequence comparisons, Gal was classified into glycoside hydrolase family 2. Amino acid sequence comparison of Gal and other characterized GH2 β-galactosidases are shown in Figure 3.

**Figure 3.** Sequence alignment of Gal and other GH2 β-galactosidase from different microorganisms. Identical residues are shaded in black and conserved residues are shaded in gray. The putative nucleophilic and catalytic amino acids are marked by a red asterisk and the regions relative to the formation of a tetramer are marked by blue boxes. Protein accession numbers and species are as follows: AAR92204.1, *Pseudoalteromonas* sp. 22b; CAA10470.1, *Pseudoalteromonas haloplanktis*; ABN72582.1, *E. coli* K-12; CAD29775.1, *Arthrobacter* sp. C2-2.
