Aliphatic amidase of Rhodococcus rhodochrous PA-34: Purification, characterization and application in synthesis of acrylic acid

Abstract

An intracellular aliphatic amide degrading inducible amidase produced by Rhodococcus rhodochrous PA-34 was characterized and acrylic acid synthesis from acrylamide was carried out using whole cell amidase. A bioprocess was developed at 50 ml fed batch reaction using 400 mM acrylamide feeding at an interval of 30 min resulted in the production of 4 g acrylic acid with volumetric and catalytic productivity of 80 g/l and 19 g/g/h respectively. The amidase of this organism had molecular weight of 40 kDa and was purified to 8.5 fold with 8% yield. This enzyme was active within the temperature range of 30 to 60 °C, with optimum temperature 45 °C and pH 7.5. The Vmax, Km, and kcat of purified amidase were calculated as 250 U/mg protein, 4.5 mM, and 166 sec-1 for acrylamide. The enzyme showed tolerance to metal chelating agent (EDTA) and was strongly inhibited by heavy metal ions Hg2+, Ag2+, Cu2+ and Co2+. R. rhodochrous PA-34 amidase preferentially hydrolyzed small aliphatic toxic amide such as acrylamide. Thus, the amidase of R. rhodochrous PA-34 is promising biocatalyst for the synthesis of industrially important acids and biodegradation of toxic amides.

Development and application of a transcriptional sensor for detection of heterologous acrylic acid production in E. coli

“…Acrylic acid can be made via enzymatic deamination of acrylamide as demonstrated using aliphatic amidases [18, 19]. We coupled this relatively simple pathway to AA sensor output by expressing the G. pallidus RAPc8 amidase [19] in E. coli and measuring sensor fluorescence by FACS after incubation with acrylamide.…”

Characterization Of A Bacterium – Producer Of Amidase

“…The multi-ton synthesis of acrylamide (AA) over many years, launched at several production sites around the world, is regarded as the most outstanding example of the cellular process for this group of bacteria, as well as the possibility of being used as biocatalysts for the production of other useful chemicals, in particular, acrylic acid. Acrylic acid is a raw material for the production of polymers and copolymers for various purposes, widely used in all spheres of the national economy [7][8][9][10].…”