Alteration of substrate specificity of aspartase by directed evolution

• Published in: Biomolecular engineering Vol. 22; no. 1; pp. 95 - 101
• Main Authors: Asano, Yasuhisa, Kira, Ikuo, Yokozeki, Kenzo
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2005
• Subjects: Alteration of substrate specificity; Aspartase; Aspartate Ammonia-Lyase - chemistry; Aspartate Ammonia-Lyase - genetics; Directed evolution; Directed Molecular Evolution - methods; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Substrate Specificity - genetics; Aspartase; Directed evolution; Alteration of substrate specificity
• ISSN: 1389-0344; 1878-559X
• DOI: 10.1016/j.bioeng.2004.12.002

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 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-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.