Characterization of two new alginate lyases from Pseudomonas mendocina E03

Alginate lyases, which degrade alginate into oligosaccharides, have broad applications in biorefinery, biomedical, and industrial fields. The Polysaccharide Lyase Family 7 (PL7) is particularly notable for its alginate lyase activity. In this study, two novel alginate lyases, PmAlg7A and PmAlg7B, from Pseudomonas mendocina E03 were cloned, heterologously expressed, and characterized. PmAlg7B exhibited limited activity toward alginate (0.10 U/mg-protein), while PmAlg7A demonstrated higher activity with a specific activity of 0.76 U/mg-protein. PmAlg7A was identified as an MG-specific alginate lyase, producing oligosaccharides with degrees of polymerization (Dp) ranging from 2 to 5. The enzyme exhibited optimal activity at a temperature of 30 °C and a pH of 8.0, with a K_m of 7.94 ± 0.92 mg/ml and a k_cat of 1.23 ± 0.06 s^-1. Structural comparisons and amino acid sequence alignments indicated a potential role for residue 55 in loop B in modulating the activity of PmAlg7B, which was supported by mutagenesis experiments and molecular dynamics simulations. These findings enhance our understanding of the critical role of loop B in regulating substrate binding in PL7 alginate lyases.