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　K-m　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.