Application of in vivo selective methods to investigate novel activity in the enolase superfamily

• Main Author: Royer, Sylvain F. R. M.
• Published: University of Edinburgh 2005
• Subjects: 572.7
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.661443

High-throughput screening is now widely used to isolate specific or improved activities of various enzymes. However, most of the current screening methods are based on in vitro procedures. The aim of this project is the development of an in vivo directed evolution approach to study a novel racemase enzyme. Although in vivo selection in itself is not new, this project emphasises a new approach to the high throughput in vivo concept, selecting an enzyme activity apparrently unintended by nature. The selection, which can screen a library of 10⁶-10⁸ mutants per assay, is complementary to in vitro screening, and applies new genetic tools for strain construction. Since the racemase under investigation is active on commercially important compounds, this method may also be suitable to identify and improve new industrial biocatalysts. The focus of the study is an N-acylamino acid racemase from Amycolatopsis which displays relaxed substrate specificity upon N-acetylated amino acids but no activity towards free amino acids. As such, this enzyme is potentially valuable to use with well established enantioselective acylases for the dynamic kinetic resolution of L- or D-amino acids. However, the racemase displays properties which limit its usefulness as a biocatalyst, such as a low substrate turnover, and inhibition of activity at substrate concentrations exceeding 50mM. The racemase also appears to have evolved serendipitously from a different activity of the enolase superfamily. The in vivo selection developed in this work is based on the construction of specific auxotrophic mutants, which require expression of this racemase activity for growth. This offers a way to explore this very interesting and novel N-acylamino acid racemase activity of an enzyme in the enolase superfamily. The screening of libraries of variants derived from the wild type enzyme was applied, to unveil mutations which improve activity, and demonstrate the value of the in vivo approach. This in vivo selection could therefore potentially help to understand the evolution of new activities in the enolase superfamily and develop a new class of biocatalyst to expand the utility of acylase resolutions by industry.