The yields in the synthesis of antibiotics catalyzed by Penicillin G acylase under very mild experimental conditions by using esters of side chains as acylating agents depend on the ratio between synthetase and hydrolase activities of penicillin G acylase. The presence of low concentrations of methanol in the reaction medium exerts complex and interesting changes in the catalytic properties of immobilized penicillin G acylase. When phenylglycine methyl ester is used as acylating agent, the presence of methanol has a double effect. It increased the ratio synthesis/hydrolysis of antibiotic, but decreased the ratio between synthesis of antibiotic and direct hydrolysis of the acyl donor ester. In this way, synthetic yields are increased when using an excess of acylating substrate (e.g., from 63% and 73%) because the key point in this reaction is to decrease the hydrolysis of the already synthesized antibiotic. When using an excess of antibiotics nucleus, the hydrolysis of antibiotic is strongly inhibited and then the ratio synthesis of antibiotic/hydrolysis of the acyl donor is the key point and the yields suffered a decrease (e.g., from 90 to 60%). On the contrary, when using mandelic acid methyl ester as acylating agent, methanol increases both ratios (hydrolysis/synthesis of the antibiotic and synthesis of antibiotic/hydrolysis of the acyl donor); thus, the yields increased by almost a twofold factor (e.g., from 38 to 70%). Finally, the contradictory effects observed in the presence of methanol can be modulated by using different enzyme derivatives. In fact, the effect of methanol on the Penicillin G acylase structure could be cleared out by increasing the stability of the enzyme derivative or by "fixing" the enzyme conformation in the presence of the organic solvent during the immobilization process.