5.1.3.37 D152G mutation eliminates almost all of both the lyase and epimerase activities 652156 5.1.3.37 D173E 54% residual activity 734159 5.1.3.37 D178E complete loss of activity 734159 5.1.3.37 D178N complete loss of activity 734159 5.1.3.37 D317A about 5% of wild-type activity 734293 5.1.3.37 D320A complete loss of activity -, 734293 5.1.3.37 D368N about 5% of wild-type activity -, 734293 5.1.3.37 D452A about 70% of wild-type activity 734293 5.1.3.37 F122Y 65% residual activity 734159 5.1.3.37 H154F complete loss of activity 734159 5.1.3.37 H154R complete loss of activity 734159 5.1.3.37 H319A complete loss of activity -, 734293 5.1.3.37 H339A about 50% of wild-type activity -, 734293 5.1.3.37 K117 R 24% residual activity 734159 5.1.3.37 K117A 16% residual activity 734159 5.1.3.37 K255A 8% residual activity 734159 5.1.3.37 K255R 51% residual activity 734159 5.1.3.37 K338A about 90% of wild-type activity -, 734293 5.1.3.37 additional information a truncated form of isoform AlgE1 (AlgE1-1) is converted to a combined epimerase and lyase by replacing the 5'-798 base pairs in the algE1-1 gene with the corresponding A-module-encoding DNA sequence from algE7 652156 5.1.3.37 additional information a truncated form of isoform AlgE1 (AlgE1-1) is converted to a combined epimerase and lyase by replacing the 5'-798 base pairs in the algE1-1 gene with the corresponding A-module-encoding DNA sequence from bifunctional isoform algE7 652156 5.1.3.37 additional information construction of a variety of truncated forms of isoform AlgE1. An A module alone is sufficient for epimerization and module A1 catalyzes the formation of contiguous stretches of G residues in the polymer, while module A2 introduces single G residues. The epimerization reaction is Ca2+ dependent, and both the A and R modules bind this cation. The R modules appear to reduce the Ca2+ concentration needed for full activity and also stimulate the reaction rate when positioned both N- and C-terminally 734060 5.1.3.37 additional information construction of mutant enzymes that introduce a high level of G-blocks in poly(beta-(1->4)-D-mannuronate) more efficiently than the wild-type enzymes from Azotobacter vinelandii when employed for in vitro epimerization reactions. Shuffling of the genes encoding isoforms AlgE1 to AlgE6 leads to two epimerases that are more efficient in introducing G-blocks in poly(beta-(1->4)-D-mannuronate) than the naturally occurring enzymes, and one of these acts kinetically different than the G-block former AlgE6 733461 5.1.3.37 additional information division of algE1 into two parts based on the modular type of structure, and expression of each part in Escherichia coli. AlgE1 contains two catalytic domains, AlgE1-1, which introduces both G-blocks and MG-blocks, and AlgE1-2, which only introduces MG-blocks. AlgE1-1 has a much lower specific activity than both AlgE1-2 and AlgE1 734128 5.1.3.37 additional information exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6 748180 5.1.3.37 additional information mannuronan C5-epimerases (AlgE1-AlgE7) produced by Azotobacter vinelandii are able to convert beta-D-mannuronate to its epimer alpha-L-guluronate in alginates. The introduction of new G-blocks into the polymer by in vitro epimerization is a strategy to improve the mechanical properties of alginates as biomaterial. Epimerization is hampered when the substrate is modified or in the gelled state. Reducing the size of the epimerases enables the epimerization of otherwise inaccessible regions in the alginate polymer. Even though the reduction of the size affects the productive binding of epimerases to the substrate, and hence their activity, the smaller epimerases can more freely diffuse into calcium-alginate hydrogel and epimerize it 747457 5.1.3.37 additional information mannuronan C5-epimerases (AlgE1-AlgE7) produced by Azotobacter vinelandii are able to convert beta-D-mannuronate to its epimer alpha-L-guluronate in alginates. The introduction of new G-blocks into the polymer by in vitro epimerization is a strategy to improve the mechanical properties of alginates as biomaterial. Epimerization is hampered when the substrate is modified or in the gelled state. Reducing the size of the epimerases enables the epimerization of otherwise inaccessible regions in the alginate polymer. Even though the reduction of the size affects the productive binding of epimerases to the substrate, and hence their activity, the smaller epimerases can more freely diffuse into calcium-alginate hydrogel and epimerize it. Construction of thehybrid enzyme AlgE6A with A-module, and the hybrid enzyme AlgE64 constituted by the Amodule from AlgE6 and the R-module from AlgE4, modular structure, overview. The A-module is the minimal size for an active epimerase, even though the active site is located in proximity of the N-terminus. Reducing the size of AlgE6 influences the epimerization of modified alginates in solution 747457 5.1.3.37 additional information mannuronan C5-epimerases (AlgE1-AlgE7) produced by Azotobacter vinelandii are able to convert beta-D-mannuronate to its epimer alpha-L-guluronate in alginates. The introduction of new G-blocks into the polymer by in vitro epimerization is a strategy to improve the mechanical properties of alginates as biomaterial. Epimerization is hampered when the substrate is modified or in the gelled state. Reducing the size of the epimerases enables the epimerization of otherwise inaccessible regions in the alginate polymer. Even though the reduction of the size affects the productive binding of epimerases to the substrate, and hence their activity, the smaller epimerases can more freely diffuse into calcium-alginate hydrogel and epimerize it. Enzyme modular structure, overview 747457 5.1.3.37 P153A 10% residual activity 734159 5.1.3.37 P153A/D173E 4% residual activity 734159 5.1.3.37 Q156A 10% residual activity 734159 5.1.3.37 Q225A 9% residual activity 734159 5.1.3.37 Q225E 4% residual activity 734159 5.1.3.37 Q225N 6% residual activity 734159 5.1.3.37 R249A 46% residual activity 734159 5.1.3.37 R321K about 75% of wild-type activity 734293 5.1.3.37 R345A about 10% of wild-type activity 734293 5.1.3.37 R345K about 40% of wild-type activity 734293 5.1.3.37 R345Q about125% of wild-type activity 734293 5.1.3.37 R353E complete loss of activity 734293 5.1.3.37 R369A about 25% of wild-type activity 734293 5.1.3.37 R415C complete loss of activity 734293 5.1.3.37 S344A about 55% of wild-type activity 734293 5.1.3.37 Y149F complete loss of activity 734159 5.1.3.37 Y149H complete loss of activity 734159 5.1.3.37 Y291F about 85% of wild-type activity 734293 5.1.3.37 Y294A about 25% of wild-type activity 734293 5.1.3.37 Y294F about 65% of wild-type activity 734293 5.1.3.37 Y296A about 65% of wild-type activity 734293 5.1.3.37 Y314F about 5% of wild-type activity 734293 5.1.3.37 Y392A about 65% of wild-type activity 734293 5.1.3.37 Y392F about 70% of wild-type activity 734293