Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a b-elimination mechanism. A putative alginate lyase (Smlt1473) from S. maltophilia was heterologously expressed in E. coli, purified in a one-step fashion via affinity chromatography, and activity as well as specificity determined for a range of polysaccharides. Interestingly, Smlt1473 catalyzed the degradation of not only alginate, but poly-b-D-glucuronic acid and hyaluronic acid as well. Furthermore, the pH optimum for enzymatic activity is substrate-dependent, with optimal hyaluronic acid degradation at pH 5, poly-b-D-glucuronic acid degradation at pH 7, and alginate degradation at pH 9. Additionally, single point mutations of His221 andArg312, two putative substrate binding residues located in the active site cleft, resulted in increased activity and specificity towards poly-�-D-mannuronic acid and poly-b-D-glucuronic acid, respectively. Collectively, these results imply that Smlt1473 is a multifunctional PL that exhibits broad substrate specificity, but utilizes pH as a mechanism to achieve selectivity. Furthermore, mutation of substrate binding residues resulted in significant changes in substrate specificity, indicating the potential use of Smlt1473 as a platform to engineer lyases which generate products of a desired size and composition for various industrial applications.

Polysaccharide lysae from <i>S. maltophilia</i> with unique, pH-regulated substrate specificity

248th National Meeting (2014)

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Polysaccharide lysae from S. maltophilia with unique, pH-regulated substrate specificity