Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9602419 | Biomolecular Engineering | 2005 | 7 Pages |
Abstract
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 an 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 from l-aspartic acid-α-amide. Through the screening, one mutant, MA2100, catalyzing deamination of l-aspartic acid-α-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-α-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The Km value and Vmax value for the l-aspartic acid of the mutated enzyme were 28.3 mM and 0.26 U/mg, respectively. The Km value and Vmax value for the l-aspartic acid-α-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.
Keywords
Related Topics
Physical Sciences and Engineering
Chemical Engineering
Bioengineering
Authors
Yasuhisa Asano, Ikuo Kira, Kenzo Yokozeki,