Abstract
SummaryA high‐throughput method (≥ 106 of clones can be analysed on a single agar plate) for the selection of ester‐hydrolysing enzymes was developed based on the uridine auxotrophy of Escherichia coli strain DH10B ΔpyrFEC and the acylated derivatives 2′,3′,5′‐O‐tri‐acetyluridine and 2′,3′,5′‐O‐tri‐hexanoyluridine as the sole source of uridine. The proposed approach permits the selection of hydrolases belonging to different families and active towards different substrates. Moreover, the ester group of the substrate used for the selection, at least partly, determined the specificity of the selected enzymes.
Highlights
Discovery of novel enzymes and their engineering is the basis of the tremendously fast-developing industrial biotechnology
Since only a few of such systems have been developed for the selection of ester hydrolases, the hydrolysis reaction, which liberates glycerol as a carbon source, has been applied for the generation of active esterase mutants (Bornscheuer et al, 1998)
We present a method of functional screening of hydrolases based on uridine auxothrophy and the appropriate uridine derivatives that serve as substrates for the ester-hydrolysing enzymes
Summary
A high-throughput method (≥ 106 of clones can be analysed on a single agar plate) for the selection of ester-hydrolysing enzymes was developed based on the uridine auxotrophy of Escherichia coli strain DH10B DpyrFEC and the acylated derivatives 20,30,50O-tri-acetyluridine and 20,30,50-O-tri-hexanoyluridine as the sole source of uridine. The proposed approach permits the selection of hydrolases belonging to different families and active towards different substrates. The ester group of the substrate used for the selection, at least partly, determined the specificity of the selected enzymes
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