Alteration of lipase chain length specificity in the hydrolysis of esters by random mutagenesis.
The feasibility of altering the chain length specificity of industrially important Rhizomucor miehei lipase was investigated by randomly mutating Phe94 in the protein groove which is responsible for accommodating the acyl chain of the substrate. The recombinant lipase was initially expressed in E. coli. 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 preliminary screen, one mutant, Phe94Gly, was selected and purified to homogeneity 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 against resorufin ester but displayed 3-4 times higher activity with short chain substrates such as butyric acid esters. The observed alteration to the enzyme specificity was rationalised using the available 3D structure of the lipase.
Gaskin
D J
DJ
Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK.
Romojaro
A
A
Turner
N A
NA
Jenkins
J
J
Vulfson
E N
EN
D019943:Amino Acid Substitution; D001665:Binding Sites; D020148:Butyric Acid / Q000737:chemistry / Q000378:metabolism; D002847:Chromatography, Agarose / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D004952:Esters / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016296:Mutagenesis; D010649:Phenylalanine; D010843:Pichia / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020103:Rhizomucor / Q000201:enzymology; D013379:Substrate Specificity
D004952:Esters; D011994:Recombinant Proteins; D020148:Butyric Acid; D010649:Phenylalanine; D008049:Lipase