| Summary: | Recently, there has been increasing use of agro-byproducts in microbial fermentation to produce a variety of value-added products. In this study, among various kinds of agro-byproducts, pomelo albedo powder (PAP) was found to be the most effective carbon source for the production of sucrose hydrolyzing enzyme by <i>Bacillus licheniformis</i> TKU004. The optimal medium for sucrolytic enzyme production contained 2% PAP, 0.75% NH<sub>4</sub>NO<sub>3</sub>, 0.05% MgSO<sub>4</sub>, and 0.05% NaH<sub>2</sub>PO<sub>4</sub> and the optimal culture conditions were pH 6.7, 35 °C, 150 rpm, and 24 h. Accordingly, the highest sucrolytic activity was 1.87 U/mL, 4.79-fold higher than that from standard conditions using sucrose as the carbon source. The purified sucrolytic enzyme (sleTKU004) is a 53 kDa monomeric protein and belongs to the glycoside hydrolase family 68. The optimum temperature and pH of sleTKU004 were 50 °C, and pH = 6, respectively. SleTKU004 could hydrolyze sucrose, raffinose, and stachyose by attacking the glycoside linkage between glucose and fructose molecules of the sucrose unit. The <i>K<sub>m</sub></i> and <i>V<sub>max</sub></i> of sleTKU004 were 1.16 M and 5.99 µmol/min, respectively. Finally, sleTKU004 showed strong sucrose tolerance and presented the highest hydrolytic activity at the sucrose concentration of 1.2 M–1.5 M.
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