ALTERATION OF CHAIN-LENGTH SELECTIVITY OF A RHIZOPUS-DELEMAR LIPASE THROUGH SITE-DIRECTED MUTAGENESIS

The coding sequences of the Rhizopus delemar lipase and prolipase were altered by oligonucleotide-directed mutagenesis to introduce amino ac id substitutions. The resulting mutant enzymes, synthesized by the bac terial host Escherichia coil BL21 (DE3), were tested for their ability to hydrolyze the triglycerides triolein (TO), tricaprylin (TC) and tr ibutyrin (TB). Mutagenesis and lipase gene expression were carried out using plasmid vectors derived from previously described recombinant p lasmids [Joerger and Haas (1993) Lipids 28, 81-88] by introduction of the origin of replication of bacteriophage f1. Substitution of threoni ne 83 (thr83), a residue thought to be involved in oxyanion binding, b y alanine essentially eliminated lipolytic activity toward all substra tes examined (TB, TO and TC). Replacement of thr83 with serine caused from two- to sevenfold reductions in the activity toward these substra tes. Introduction of tryptophan (trp) at position 89, where such a res idue is found in closely related fungal lipases, reduced the specific activity toward the three triglyceride substrates, For the mutagenesis of residues in the predicted acyl chain binding groove, mutagenic pri mers were designed to cause the replacement of a specific codon within the prolipase gene with codons for all other amino acids. Phenylalani ne 95 (phe95), phe112, valine 206 (val206) and val209, were targeted. A phenotypic screen was successfully employed to identify cells produc ing prolipase with altered preference for olive oil, TC or TB. In assa ys involving equimolar mixtures of the three triglycerides, a prolipas e with a phe95 --> aspartate mutation showed an almost twofold increas e in the relative activity toward TC. Substitution of trp for phe112 c aused an almost threefold decrease in the relative preference for TC, but elevated relative TB hydrolysis. Replacement of val209 with trp re sulted in an enzyme with a two- and fourfold enhanced preference for T C and TB, respectively.