Alteration of substrate specificity of aspartase by directed evolution

Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1), which catalyzes the reversible deamination Of L-aspartic acid to yield fumaric acid and ammonia, is highly selective towards L-aspartic acid. We screened for enzyme variants with altered substrate specificity by a directed evolution method. Random mutagenesis was performed on all Escherichia coli aspartase gene (aspA) by error-prone PCR to construct a mutant library. The mutant library was introduced to E. coli and the transformants were screened for production of fumaric acid-mono amide front L-aspartic acid-alpha-amide. Through the screening, one mutant, MA2100, catalyzing deamination Of L-aspartic acid-alpha-amide was achieved. Gene analysis of the MA2100 mutant indicated that the mutated enzyme had a K327N mutation. The characteristics of the mutated enzyme were examined. The optimum pH values for the L-aspartic acid and L-aspartic acid-alpha-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The K-m value and V-max value for the L-aspartic acid of the mutated enzyme were 28.3 mM and 0.26 U/mg, respectively. The K-m value and V-max value for the L-aspartic acid-alpha-amide of the mutated enzyme were 1450 mM and 0.47 U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme. (C) 2005 Elsevier B.V. All rights reserved.