Biochemical and molecular biological characterization of a lipase producedby the fungus Rhizopus delemar

The results of a comprehensive biochemical and molecular biological investi gation of the lipase produced by the mycelial fungus Rhizopus delemar are d escribed. This enzyme cleaves and synthesizes primary esters and related bo nds, 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 bi ocatalysis. Use of glycerol as main carbon source rather than glucose or li pid supported mycelial growth and lipase production. The enzyme was purifie d to homogeneity and characterized. Pure lipase was crystallized and its th ree-dimensional structure determined. The enzyme was found to adopt a confi guration similar to those of other members of its homologous family. The st ructural data also indicated that lipases possess greater configurational m obility than had been previously appreciated. A complementary DNA clone was isolated that contained the full length lipase gene. The nucleic acid sequ ence of this cDNA indicated that it was initially synthesized as a preproen zyme, and allowed determination of the complete predicted amino acid sequen ce of the lipase, and its comparison to the sequences of related enzymes. T runcated forms of the cloned cDNA were produced that encoded either mature or prepro-lipase. These DNAs were introduced into a rightly regulated E. co li expression system, overcoming the toxicity of the enzyme while also allo wing overproduction of lipase. Molecular modelling was employed to guide th e rational mutagenesis of the enzyme, identifying sites within the substrat e 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 lipolyt ic enzymes.