A gene for a streptavidin-lipase fusion protein was constructed by cloning a PCR-modified Pseudomonas fluorescens B52 lipase gene into a streptavidin chimeric protein expression vector, pStp4. The plasmids, pSTLP1 or pSTLP2, were used to transform Escherichia coli NM522 or DH5 alpha. Plasmid pSTLP2, which contains a lacl(q) gene upstream from the promoter, gave better expression of the bifunctional fusion protein when the cells were induced. Correct insertion of the fused gene was confirmed by DNA sequencing of the linker region and the lipase gene, by identification of a protein band of the correct molecular size (66 kDa) on SDS-PAGE, and by Western blot analysis rising antistreptavidin antibody. Streptavidin-lipase fusion protein was purified and immobilized in a single step from recombinant cell lysates by bioselective adsorption on 6-(biotinamido)hexanamidopropyl-controlled pore glass. The hydrolytic activity of the resulting immobilized enzyme exhibited a pH optimum between 7 and 8 and a preference for short-chain fatty acids, Comparison of the hydrolytic activity of immobilized B52 lipase with that of commercial SAM-2 lipase from P. fluorescens suggests that about 30% of the activity was retained upon immobilization. Transesterification of tricaprylin with oleic acid in hexane by immobilized lipase indicated that 11% of the reaction products were derived from transesterification.