Characterization of a Novel Polysaccharide Lyase Family 5 Alginate Lyase with PolyM Substrate Specificity.

Abstract

Alginate lyases (ALyases) have been widely applied in enzymatically degrading alginate for the preparation of alginate oligosaccharides (AOS), which possess a range of excellent physiological benefits including immunoregulatory, antivirus, and antidiabetic properties. Among the characterized ALyases, the number of ALyases with strict substrate specificity which possess potential in directed preparation of AOS is quite small. ALyases of polysaccharides lyase (PL) 5 family have been reported to perform poly-β-D-mannuronic acid (Poly-M) substrate specificity. However, there have been fewer studies with a comprehensive characterization and comparison of PL 5 family ALyases. In this study, a putative PL 5 family ALyase PMD was cloned from Pseudomonas mendocina and expressed in Escherichia coli. The novel ALyase presented maximum activity at 30 °C and pH 7.0. PMD displayed pH stability properties under the range of pH 5 to pH 9, which retained more than 80% relative activity, even when incubated for 48 h. Product analysis indicated that PMD might be an endolytic ALyase with strict Poly M substrate specificity and yield disaccharide and trisaccharide as main products. In addition, residues K58, R66, Y248, and R344 were proposed to be the potential key residues for catalysis via site-directed mutation. Detailed characterization of PMD and comprehensive comparisons could supply some different information about properties of PL 5 ALyases which might be helpful for its application in the directed production of AOS.