Alteration of lipase chain length specificity in the hydrolysis of esters by random mutagenesis

The feasibility of altering the chain length specificity of industrially im portant Rhizomucor miehei lipase was investigated by randomly mutating Phe9 4 in the protein groove which is responsible for accommodating the acyl cha in of the substrate. The recombinant lipase was initially expressed in E. c oli. Individual colonies were selected, grown, and the DNA sequence of the lipase gene determined. Fourteen of the 19 possible mutants were identified and each of these was transformed into Pichia pastoris which expresses the enzyme extracellularly. The yeast was grown and the supernatants assessed in several assays with long and short chain substrates. Based on this preli minary screen, one mutant Phe94Gly, was selected and purified to homogeneit y for further analysis. It was found that the substitution of phenylalanine 94 with glycine led to an enzyme which was about six times less active aga inst resorufin ester but displayed 3-4 times higher activity with short cha in substrates such as butyric acid esters. The observed alteration to the e nzyme specificity was rationalised using the available 3D structure of the lipase. (C) 2001 John Wiley & Sons, Inc.