A unique method that applied a multilayer-immobilization strategy was developed to prepare nanofibrous enzymes for biosynthesis. LiCl co-electrospun with polyurethane nanofibers enabled strong physical adsorption of bovine serum albumin (BSA), forming the first layer of protein on the nanofibers; lipase AK was subsequently crosslinked to BSA as an outer layer of enzyme. The content of LiCl in nanofibers was found to be a sensitive factor affecting the activity and stability of the immobilized lipase. For bio-diesel synthesis from soybean oil and methanol in isooctane, the reaction rate catalyzed by nanofibrious lipase carrying 5 wt% LiCl was 6.6-fold higher than fibers without LiCl, with a conversion of 91% was achieved within 2 h. LiCl also induced much improved enzyme stability. The nanofibrous lipase with 5% LiCl could be repeatedly used for 42 cycles without apparent activity loss, while the immobilized lipase without LiCl lost over 90% activity within 13 reuse cycles. (C) 2011 Elsevier Ltd. All rights reserved.