Effect of pegylation on the activities and stabilities of horseradish peroxidase (HRP) and N-carbamoyl-L-amino acid amidohydrolase (L-N-carbamoylase) was investigated. Pegylation was favored under alkaline conditions with high PEG/enzyme molar ratios. The lysine residues in L-N-carbamoylase were found to be more readily accessible for pegylation than those in HRP, leading to the formation of multi-pegylated enzymes. Pegylation and addition of mPEG as an additive improved HRP activity by 64% and 42%, respectively, via the formation of protective layers against the free radical-induced inactivation. The activity of pegylated L-N-carbamoylase, however, declined due to the additional mass transfer resistance introduced by the PEG layers. The extent of reduction in L-N-carbamoylase activity increased with the level of pegylation. In terms of thermostability, both the addition of mPEG as an additive and pegylation with PEG(5000) led to a decline in thermostability. Nevertheless, pegylation of HRP with PEG(2000) resulted in a PEG-HRP conjugate exhibiting a thermostability two-fold that of the control at 70 degrees C, probably due to the capability of the relatively hydrophilic PEG domains on the conjugate in avoiding enzyme aggregation. After a 4-day incubation at room temperature, pegylated HRP retained significantly higher activity, 89%, than the control, 12%. (C) 2015 Published by Elsevier Ltd.