Studies on the biotransformation of acrylonitrile to acrylic acid using Rhodococcus Ruber Aksh-84

Abstract

A versatile potent nitrile-degrading bacterium was isolated through enrichment culturing of petroleum contaminated water and sludge samples from Essar Oil Refinery Ltd, Vadinar, Gujarat. The strain was characterized for its physicochemical, biochemical and growth characteristics. It was identified as Rhodococcus ruber AKSH-84 and its 16S rDNA sequence was submitted to the EMBL sequence database under the accession number FM995614. This organism was determined to be a potential biocatalyst in that it contains a single enzyme system with strong nitrile-converting activity comprising nitrilase. The development of a suitable assay for measuring the activity of the enzymes of interest was explored. A pH sensitive indicator-based assay was found to be suitable only for colorimetrically identifying highly concentrated enzymes with acid-forming activity. Using this colorimetric method, 11 isolates exhibiting nitrilase activity were screened out of 108 isolates. This assay was found to be applicable to select the positive screens, but the assay could not be used to measure the activity of Rhodococcus ruber AKSH-84. A simple, rapid and most suitable RP-HPLC method was developed for reliable and quantitative measurement of nitrile hydrolysis and is applicable to monitoring activities of whole-cell and cell-free extracts. Initial analysis of acrylic acid, acrylamide and acrylonitrile, was performed by HPLC to measure linearly the average retention area, amount and absorbance of the compounds up to 10 mM concentrations. The conversion of the substrate acrylonitrile was further analyzed with respect to time and growth of the organism. The maximum production of enzyme took place within 26 h of incubation and the enzyme is constitutive in nature. Initially conversion of acrylonitrile to acrylic acid by the nitrilase was slow and could be measured in 1 h. LC-Mass spectral analysis was used to confirm, at a structural level, relatively less volatile reactant compounds including acrylamide and acrylic acid.