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The additive stabilizing effect of E518I/S662R/Q706P may becaused by the relatively distant location on the enzyme structureof these residues without direct interaction among these threesites[10,37]; therefore, the individual contribution of each mutation was reflected in the combined mutant. Similar reports onB.deramificanspullulanase also indicated that the stabilizing effect ofsingle-site mutation can be combined. The mutant (D437H/D503Y)ofB. deramificanspullulanase displayed a higher optimal temperature and half-life than that of the WT enzyme and single-sitemutants[3].
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