Mannuronan C-5 epimerases suited for tailoring of specific alginate structures obtained by high-throughput screening of an epimerase mutant library.

The polysaccharide alginate is produced by brown algae and some bacteria and is composed of the two monomers, ß-D-mannuronic acid (M) and α-L-guluronic acid (G). The distribution and composition of M/G are important for the chemical-physical properties of alginate and result from the activity of a family of mannuronan C-5 epimerases that converts M to G in the initially synthesized polyM. Traditionally, G-rich alginates are commercially most interesting due to gelling and viscosifying properties. From a library of mutant epimerases we have isolated enzymes that introduce a high level of G-blocks in polyM more efficiently than the wild-type enzymes from Azotobacter vinelandii when employed for in vitro epimerization reactions. This was achieved by developing a high-throughput screening method to discriminate between different alginate structures. Furthermore, genetic and biochemical analyses of the mutant enzymes have revealed structural features that are important for the differences in epimerization pattern found for the various epimerases.

Characterization of three new Azotobacter vinelandii alginate lyases, one of which is involved in cyst germination.

Alginates are polysaccharides composed of 1-4-linked beta-D-mannuronic acid and alpha-L-guluronic acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a beta-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca(2+). All three enzymes preferably cleave the bond between guluronic acid and mannuronic acid, resulting in a guluronic acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.