Mannuronan C-5 Epimerases Suited for Tailoring of Specific Alginate Structures Obtained by High-Throughput Screening of an Epimerase Mutant Library

Abstract

The polysaccharide alginate is produced by brown algae and some bacteria and is composed of the two monomers β-D-mannuronic acid (M) and α-L-guluronic acid (G). The distribution and composition of M/G is 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 synthesised 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 analysis of the mutant enzymes have revealed structural features that are important for the differences in epimerisation pattern found for the various epimerases.

Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii

“…Swapping the R-modules between AlgE4 and AlgE6 shows that they also have a strong influence on the epimerization pattern of the epimerase, which was also observed in a mutant epimerase (20). Improving the alginate binding ability of AlgE6A by introducing the AlgE4R module, creating the AlgE64 epimerase mutant, leads to increased G-block formation.…”

“…Both groups of amino acids can bind alginate through hydrogen bonds and probably stabilizing the proteins by avoiding electrostatic repulsion of the basic residues that might otherwise cause labile protein structures. A recent study shows that the presence of a negative charge at the substrate binding groove had a significant effect on the ability of an epimerase to move in a well defined manner (20). This points out that if the binding groove only had basic residues, the alginate polymer would not be able to dissociate from the enzyme surface and/or the epimerase could not move along the alginate polymer chain.…”

“…The cell culture was incubated on ice for 5 min. Expression was induced by addition of isopropyl 1-thio-␤-D-galactopyranoside (final concentration 1 mM), and then the culture was incubated at 15°C for 16 -20 h. pAT116 and pAT117 was transformed to E. coli strains XL10-gold and RV308, respectively, and recombinant protein production was performed as described previously (20,22). The cells were harvested by centrifugation and resuspended in 20 mM HEPES, pH 6.9, 800 mM NaCl, 5 mM CaCl 2 , and 0.1% Triton X-100.…”

“…Therefore, it was suggested that the R-module helps to orient the alginate in the active site and to keep AlgE4 tightly bound to the polymer during epimerization. The role of the R-modules in G-block-producing epimerases is not clear; however, recent results obtained with a library of epimerases with mutated A-modules have indicated that R-modules can influence the epimerization pattern (20). These mutant enzymes were constructed with the R-module from AlgE4 to stimulate the activity.…”

“…These mutant enzymes were constructed with the R-module from AlgE4 to stimulate the activity. Aiming at generating even more potent G-block-forming epimerases, the A-modules of the high G-block-forming epimerase mutants were combined with the three R-modules from AlgE6 (20). Surprisingly, this resulted in mutant epimerases with very similar G-block-forming abilities as AlgE6.…”