Enzymatic depolymerization of alginate by two novel thermostable alginate lyases from Rhodothermus marinus

Alginate (alginic acid) is a linear polysaccharide, wherein (1 -> 4)-linked beta-D-mannuronic acid and its C5 epimer, alpha-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1 -> 4) glycosidic linkages between the monomers by a beta-elimination mechanism, to yield unsaturated 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid (Delta) at the non-reducing end of resulting oligosaccharides (alpha-L-erythro configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers. In this study, two alginate lyase genes, designated alyRm3 and alyRm4, from the marine thermophilic bacterium Rhodothermus marinus (strain MAT378), were cloned and expressed in Escherichia coli. The recombinant enzymes were characterized, and their substrate specificity and product structures determined. AlyRm3 (PL39) and AlyRm4 (PL17) are among the most thermophilic and thermostable alginate lyases described to date with temperature optimum of activity at similar to 75 and 81 degrees C, respectively. The pH optimum of activity of AlyRm3 is similar to 5.5 and AlyRm4 at pH 6.5. Detailed NMR analysis of the incubation products demonstrated that AlyRm3 is an endolytic lyase, while AlyRm4 is an exolytic lyase, cleaving monomers from the non-reducing end of oligo/poly-alginates.

Automated summary

Alginate lyases are enzymes that catalyze the depolymerization of alginate, a linear polysaccharide. In this study, two alginate lyase genes were cloned and expressed from a marine thermophilic bacterium. The recombinant enzymes showed high thermostability and activity at elevated temperatures. The NMR analysis revealed that one of the enzymes is an endolytic lyase, while the other is an exolytic lyase.