Biochemical and molecular biological characterization of a lipase produced by the fungusRhizopus delemar

Abstract

The results of a comprehensive biochemical and molecular biological investigation of the lipase produced by the mycelial fungus Rhizopus delemar are described. This enzyme cleaves and synthesizes primary esters and related bonds, exhibits 1,3-positional selectivity in its actions on glycerides, and is a member of a family of enzymes that have been widely used in applied biocatalysis. Use of glycerol as main carbon source rather than glucose or lipid supported mycelial growth and lipase production. The enzyme was purified to homogeneity and characterized. Pure lipase was crystallized and its three-dimensional structure determined. The enzyme was found to adopt a configuration similar to those of other members of its homologous family. The structural data also indicated that lipases possess greater configurational mobility than had been previously appreciated. A complementary DNA clone was isolated that contained the full length lipase gene. The nucleic acid sequence of this cDNA indicated that it was initially synthesized as a preproenzyme, and allowed determination of the complete predicted amino acid sequence of the lipase, and its comparison to the sequences of related enzymes. Truncated forms of the cloned cDNA were produced that encoded either mature or prepro-lipase. These DNAs were introduced into a tightly regulated E. coli expression system, overcoming the toxicity of the enzyme while also allowing overproduction of lipase. Molecular modelling was employed to guide the rational mutagenesis of the enzyme, identifying sites within the substrate binding region that regulated substrate selectivity. Mutant lipases were generated with altered substrate specificities, creating novel enzymes and beginning the definition of structure-function relationships in the lipolytic enzymes.