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Alteration of substrate specificity of aspartase by directed evolution
- Source :
-
Biomolecular Engineering . Jun2005, Vol. 22 Issue 1-3, p95-101. 7p. - Publication Year :
- 2005
-
Abstract
- 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 K m value and V max value for the l-aspartic acid of the mutated enzyme were 28.3mM and 0.26U/mg, respectively. The K m value and V max value for the l-aspartic acid-α-amide of the mutated enzyme were 1450mM and 0.47U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme. [Copyright &y& Elsevier]
- Subjects :
- *AMMONIA
*LYASES
*ENZYMES
*ASPARTIC acid
Subjects
Details
- Language :
- English
- ISSN :
- 13890344
- Volume :
- 22
- Issue :
- 1-3
- Database :
- Academic Search Index
- Journal :
- Biomolecular Engineering
- Publication Type :
- Academic Journal
- Accession number :
- 17699208
- Full Text :
- https://doi.org/10.1016/j.bioeng.2004.12.002