In this study, a recombinant strain Bacillus subtilis WB600 (pMA5) was used to produce Alcaligenes faecalis penicillin G acylase (PGA). The PGA activity and overall PGA productivity were respectively 378 and 7.13 U/l in a batch culture using a complex medium containing 25 g/l of soluble starch. Fed-batch cultures indicated that glucose had a negative effect on the production of PGA. Starch is a good carbon source, but it could not be directly used as a feed due to high viscosity after heat sterilization. A feed was prepared by limited hydrolysis of starch with a small amount of alpha-amylase, which greatly improved the fluidity with little formation of glucose. The production of PGA increased to 546 U/l by feeding of the partially hydrolyzed starch. When a mixture containing the partially hydrolyzed starch and Tryptone was added by using a pH-stat strategy, the PGA activity reached 1960 U/l, over five-fold of that obtained in the batch culture, and the overall productivity reached 19.6 U/l h, which was more than two-fold of that in the batch culture. (c) 2006 Elsevier Inc. All rights reserved.