Abstract
The Azotobacter vinelandii enzyme AlgE1 is a member of a family of secreted mannuronan C-5-epimerases. These enzymes convert beta-D-mannuronic acid residues (M) to alpha-L-guluronic acid residues (G) at the polymer level in the industrially important polysaccharide alginate, leading to altered physical and immunological properties of the polymer. The reaction product of AlgE1 was found to be a mixture of blocks of continuous G residues (G-blocks) and blocks containing alternating M and G residues (MG-blocks). The enzyme is dependent on Ca2+ for activity, and only Sr2+ of those tested was able to replace Ca2+. Zn2+ blocked the activity even at low concentrations. algE1 has been divided into two parts based on the modular type of structure previously reported to be a characteristic of the secreted epimerases, and each part has been expressed in Escherichia coli. These experiments showed that 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. However, the two halves of AlgE1 seem to cooperate in such a way that they contribute approximately equally to the overall epimerization reaction.
Highlights
Alginate is a biopolymer synthesized by brown algae and by some bacteria belonging to the genera Azotobacter and Pseudomonas [1,2,3,4]
The G residues are introduced at the polymer level by the enzyme mannuronan C-5-epimerase
AlgE1-1 was made such that its N terminus is the same as that of AlgE1, while the C terminus contains the 5 first amino acids of the second A-module followed by 7 vector-encoded amino acids
Summary
Growth of Bacteria—E. coli JM109 [13] was used in this study. For site-specific mutagenesis, the strain BMH71-18 mutS (Promega) was used. For enzyme purifications the cells containing plasmid were grown in a medium containing 30 g/liter tryptone, 15 g/liter yeast extract, and 5 g/liter NaCl. Standard Laboratory Methods—Standard recombinant DNA procedures and protein gel electrophoresis were performed according to Sambrook et al [14]. The epimerizations of alginate for NMR spectroscopy were performed using 7.5 mg of unlabeled alginate in a volume of 6 ml of the same buffer, the concentrations of CaCl2 are stated for each experiment. Proteins were eluted by a continuous NaCl gradient (0 –1 M) in the same buffer, and fractions containing epimerase activity were adjusted to 1 M (NH4)2SO4 and applied to a hydrophobic interaction column (Pharmacia HiTrap Phenyl-Sepharose High Performance) equilibrated with 20 mM MOPS (pH 6.9), 5 mM CaCl2, and 1 M (NH4)2SO4. Fractions displaying epimerase activity were gel filtrated through a Pharmacia Sephacryl S200 column equilibrated with 20 mM MOPS (pH 6.9), 150 mM NaCl, 1 mM CaCl2
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