CLONING AND EXPRESSION OF A STREPTAVIDIN-LIPASE FUSION GENE IN ESCHERICHIA-COLI AND CHARACTERIZATION OF THE IMMOBILIZED FUSION PROTEIN

A gene for a streptavidin-lipase fusion protein was constructed by clo ning a PCR-modified Pseudomonas fluorescens B52 lipase gene into a str eptavidin chimeric protein expression vector, pStp4. The plasmids, pST LP1 or pSTLP2, were used to transform Escherichia coli NM522 or DH5 al pha. Plasmid pSTLP2, which contains a lacl(q) gene upstream from the p romoter, gave better expression of the bifunctional fusion protein whe n the cells were induced. Correct insertion of the fused gene was conf irmed by DNA sequencing of the linker region and the lipase gene, by i dentification of a protein band of the correct molecular size (66 kDa) on SDS-PAGE, and by Western blot analysis rising antistreptavidin ant ibody. Streptavidin-lipase fusion protein was purified and immobilized in a single step from recombinant cell lysates by bioselective adsorp tion on 6-(biotinamido)hexanamidopropyl-controlled pore glass. The hyd rolytic activity of the resulting immobilized enzyme exhibited a pH op timum between 7 and 8 and a preference for short-chain fatty acids, Co mparison of the hydrolytic activity of immobilized B52 lipase with tha t of commercial SAM-2 lipase from P. fluorescens suggests that about 3 0% of the activity was retained upon immobilization. Transesterificati on of tricaprylin with oleic acid in hexane by immobilized lipase indi cated that 11% of the reaction products were derived from transesterif ication. (C) 1998 Elsevier Science Inc.