Improving the Catalytic Properties of Xylanase from Alteromones Macleadii H35 Through Sequence Analysis

Endo-1,4-β-xylanase is a key xylanolytic enzyme, and our study aimed to enhance the catalytic properties of Alteromones Macleadii xylanase (Xyn ZT-2) through sequence-guided design approach. Analysis of the amino acid sequence revealed highly conserved residues near the active site, with few differences. Introducing various mutations allowed us to modify the enzyme's catalytic performance. Particularly, the A152G mutation led to a 9.8-fold increase in activity and a 23.2-fold increase in catalytic efficiency. Moreover, A152G exhibited an optimal temperature of 65 °C, 20 °C higher than that of Xyn ZT-2, while the T287S mutant showed a 4.9-fold increase in half-life. These results underscore the role of amino acid evolution in shaping xylanase catalysis. Through targeted sequence analysis and a focused mutation library, we effectively improved catalytic performance, providing a straightforward approach for enhancing enzyme efficiency.