Trypsin was coated onto electrospun polystyrene-poly(styrene-co-maleic anhydride) (PS-PSMA) nanofibers with varied concentrations of maleic anhydride group, which enables an easy conjugation of enzymes onto PS-PSMA nanofibers. The concentration of maleic anhydride group, determined by the amount of PSMA in the mixture of PS and PSMA for electrospinning, correlated well with the enzyme loading and activity of covalently attached trypsin. Trypsin-coated nanofibers, prepared via covalent attachment of seed enzyme molecules and follow-up enzyme cross-linking, resulted in highly stable nanobiocatalytic systems, irrespective of added PSMA amounts. The variation of PSMA amounts enables the facile control of various properties of trypsin-coated nanofibers, such as enzyme loading, activity, structure, and mass transfer limitation. Especially, the mass transfer limitation of substrate through the enzyme coating matrix correlated well with the conjugation site concentration, and this feature would be useful in optimizing the enzyme coatings for the biocatalytic conversion of large-size substrates, such as protein digestion as an example. Facile property control of highly stable enzyme coatings would provide a powerful tool for more successful applications of enzyme coatings in various fields. (c) 2010 Elsevier Inc. All rights reserved.