Study of the acyl transfer activity of a recombinant amidase overproduced in an Escherichia coli strain. Application for short-chain hydroxamic acid and acid hydrazide synthesis

Abstract

The study of acyl transfer activity of a wide spectrum amidase from Rhodococcus sp. R312, overproduced in an Escherichia coli strain, revealed that the ‘bi-bi-ping-pong’ type reaction was efficient with only four very-short chain (C 2–C 3) aliphatic amides as substrates. The optimum working pH was 7.0 for all neutral amides. Very short-chain aliphatic carboxylic acids were 10–1000-fold less efficient and the corresponding optimum working pH values depended on the acid used. Very polar molecules, such as water, hydroxylamine and hydrazine, were good acyl acceptors. An [acyl donor]/[acyl acceptor] ratio lower than 0.3-0.5 had to be maintained to avoid enzyme inhibition by excess acyl donor. The different acyl-enzyme complexes generally exhibited high affinity for hydroxylamine or hydrazine (except the propionyl-enzyme complex), so that the residual hydrolysis activities were almost totally inhibited at appropriate acyl acceptor concentrations. Molar conversion yields were higher with hydrazine as acyl acceptor (e.g., 97% with acetamide as acyl donor) because of the higher V max values, but in all cases, interesting quantities of short-chain hydroxamic acids (2.9-6.5 g l −1) and acid hydrazides (6.4–7.8 g l −1) could be quickly obtained (10–60 min) with small amounts of enzyme (0.04-0.20 g l −1).