Abstract
Alginate lyases have been widely used for the preparation of bioactive alginate oligosaccharides. An alginate lyase AlgL-CD was rationally designed by introducing alkaline amino acid residues near active center to increase activity. One of its mutants E226K presented much higher activity than wild-type AlgL-CD. Substrate affinity of E226K increased 10 folds as the Km values indicated. The spectra of intrinsic emission fluorescence and circular dichroism of E226K suggested the whole enzyme turned to be more flexible. The 8-anilino-1-naphthalenesulfonate (ANS)-binding assay showed that the hydrophobic active center of E226K was more available to ligand. Molecular dynamic analysis of the enzyme-substrate complex showed that lid loops of the active center in E226K turned to be more opened up, which might contribute to the increase of substrate-binding affinity. Meanwhile, the catalytic residue of E226K was closer to the hydrogen donor C5 atom of the substrate to increase catalysis rate. The final degradation products of alginate by E226K were determined to be identical with that of AlgL-CD. This study provides guidance for improving enzymatic preparation efficiency of bioactive alginate oligosaccharides.
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