The arylsulf atase gene (astA) from Pseudoalteromonas carrageenovora genome was subcloned into pHCEIA vector, in which the hyper constitutive expression (HCE) promoter from the D-amino acid aminotransferase (DAAT) gene of Geobacillus toebii was employed. When the constructed pHCE-AST was introduced into E. coli, the transformant showed the hydrolyzing activity for 4-methylumbelliferyl-sulfate and p-nitrophenyl-sulfate. When the cell was cultured on fermentor containing MaxyBroth-HD medium with 1% glycerol, the enzyme activity reached 12.8unit/mL. On MaxyBroth-HD medium with 2% glycerol, the cell showed 2.7-fold higher arylsulfatase expression than that with 1% glycerol. The fed-batch cultivation employing MaxyBroth-HD medium and additional feeding of glycerol gave about 143 unit/mL of arylsulfatase at 20 h, which corresponds to 4-fold higher enzyme activity than that of 2% glycerol batch culture. Most of arylsulfatase activity in fed-batch culture was produced in the extracellular medium, whereas the activity in the batch cultures was localized in the periplasmic cell space.