Penicillin G acylase (PGA) is one of the most important enzymes for the production of semisynthetic p-lactam antibiotics and their key intermediates. To enhance its expression, the PGA gene from Bacillus megaterium was amplified by PCR and subcloned into an expression vector under the control of the P43 promoter. The resulting construct was transferred into Bacillus subtilis WB600 and the transformant producing the most PGA was selected and designated SI-BAS205. In contrast to the parent cells, which have to be induced by phenylacetic acid and cultured at 28 and 25 degreesC successively to produce PGA, the recombinant cells needed neither induction nor thermoregulation during fermentation at 37 degreesC. PGA was secreted and reached an expression level of 40 U/mL under optimized conditions. The enzyme was separated by centrifugation and purified by Al2O3 adsorption and phenyl-Sepharose CL-4B hydrophobic chromatography with a yield of 85%. The purified enzyme had a specific activity of 45 U/mg protein.