Cloning, Expression, and Characterization of a New PL25 Family Ulvan Lyase from Marine Bacterium Alteromonas sp. A321

Abstract

Ulvan lyases can degrade ulvan to oligosaccharides with potent biological activity. A new ulvan lyase gene, <i>ALT3695</i>, was identified in <i>Alteromonas</i> sp. A321. Soluble expression of <i>ALT3695</i> was achieved in <i>Escherichia coli</i> BL21 (DE3). The 1314-bp gene encoded a protein with 437 amino acid residues. The amino acid sequence of ALT3695 exhibited low sequence identity with polysaccharide lyase family 25 (PL25) ulvan lyases from <i>Pseudoalteromonas</i> sp. PLSV (64.14% identity), <i>Alteromonas</i> sp. LOR (62.68% identity), and <i>Nonlabens ulvanivorans</i> PLR (57.37% identity). Recombinant ALT3695 was purified and the apparent molecular weight was about 53 kDa, which is different from that of other polysaccharide-degrading enzymes identified in <i>Alteromonas</i> sp. A321. ALT3695 exhibited maximal activity in 50 mM Tris-HCl buffer at pH 8.0 and 50 °C. ALT3695 was relatively thermostable, as 90% activity was observed after incubation at 40 °C for 3 h. The <i>K_m</i> and <i>V_max</i> values of ALT3695 towards ulvan were 0.43 mg·mL^-1 and 0.11 μmol·min^-1·mL^-1, respectively. ESI-MS analysis showed that <i>enzymatic products</i> were mainly disaccharides and tetrasaccharides. This study reports a new PL25 family ulvan lyase, ALT3695, with properties that suggest its great potential for the preparation of ulvan oligosaccharides.