| A novel strain of Paenibacillus apiarius, SOS 1CA3.1 was isolated from an Antarctic
environmental sample from the Bratina Island area. Paenibacillus apiarius SOS 1CA3.1 was
selected because of its expressed β-galactosidase activity and protease activity. Once isolated, the
16S rRNA gene was amplified, and sequenced and a phylogenetic tree was constructed. The gene
of a Family 35 β-galactosidase was identified through genome sequencing and annotation.
Primers were designed for the gene to be amplified and cloned into various cloning systems. The
gene was successfully amplified, cloned, and expressed in the pBAD TOPO TA system and
sequenced to confirm whole gene insertion with no errors. Expression and purification
conditions were optimized. Once purified the Family 35 β-galactosidase was characterized to
determine conditions for optimal activity. Characterization included determining optimal
conditions for activity, substrate specificity, inhibitory effects of various substrates, and kinetic
parameters of enzyme activity. It was found that the enzyme had an optimal temperature of
activity of 57°C, was active on both ONPG and PNPG, retained activity in high salt
concentrations, is highly dependent on Mg2+
, and was not inhibited by lactose or its byproducts.
The enzyme was not active on lactose and future work will involve optimizing methods to
change activity on lactose.
Keywords: [Antarctica], [16S rRNA], [Enzyme], [β-galactosidase], [Characterization] |