Hydrolysis of amylopectin by a debranching enzyme, isoamylase, was investigated under pressurized carbon dioxide in a reactor of one liter for various agitation rates and pH values. The experimental results indicated that agitation significantly inactivated the enzyme with pressurized carbon dioxide. The pH shift due to higher solubility of carbon dioxide in the system also decreased the rate. With proper tuning of agitation rate and pH, the enzyme hydrolysis reaction under supercritical carbon dioxide demonstrated better result than that under atmospheric conditions at higher substrate concentration.