Zymomonas mobilis IFO 13756 (ATCC 29191) produces three kinds of sucrose-hydrolyzing enzymes, E1, E2, and E3. Extracellular enzymes E2 and E3 bound to the cell surface were released from cells by suspension in 20 mM potassium phosphate buffer (pH 7.0) and incubation at 30 degrees C for 10 min with gentle shaking. After centrifugation of the cell suspension, E3 was isolated from the supernatant as crystals in a 52-fold purification. The enzyme consisted of a monomer subunit having a molecular mass of 58 kDa and its isoelectric point was pH 3.2. The N-terminal amino acid sequence was MFNFNASRWTRAQAMKVNKFDL. The enzyme catalyzed the hydrolysis of sucrose, and was identified as an invertase that had a strict substrate specificity for sucrose. The optimum pH and temperature were pH 5.5 and 50 degrees C, respectively. Thiol reagents inhibited the enzyme activity markedly.