Characterization and comparison of acrylamidases isolated from two different acrylamide-utilizing bacteria, Ralstonia eutropha AUM-01 and Geobacillus thermoglucosidasius AUT-01

Acrylamide, a neurotoxic molecule, spontaneously produced in many types of foods and used in a variety of industrial applications, was found to be degraded by microorganisms present in soil and air. Two mesophilic bacteria capable of utilizing acrylamide were isolated, AUM-01 from soil collected from leaf litter on Picnic Point on the UW-Madison campus and AUM-02 by air contamination in the laboratory. One thermophilic strain, AUT-01, was isolated from soil collected from a hot spring area in Montana. Thirteen thermophiles were purchased from the Bacillus Genetic Stock Center at the Ohio State University, and three of them were found to degrade acrylamide at 65°C. In minimal medium with acrylamide as the sole carbon and nitrogen source, batch cultures of AUM-01 and AUM-02 completely degraded 14.0 mM acrylamide in 7 and 18 hours, respectively. The four thermophilic strains grew with 0.2% glucose as the sole carbon source and 1.4 mM acrylamide as the sole nitrogen source. AUT-01 converted acrylamide to acrylic acid within 10 hours. The three other thermophiles required more than 30 hours to convert acrylamide to acrylic acid completely. Degradation was verified by HPLC analysis of acrylamide and acrylic acid concentrations in the test cultures. The acrylamide-utilizing mesophiles, AUM-01 and AUM-02, were identified as Ralstonia eutropha and Pseudomonas aeruginosa, respectively, based on morphological observations, the BiOLOG GN2 MicroPlate(TM) identification system for Gram-negative bacteria, and addition physiological tests. Isolate AUT-01 was identified as Geobacillus thermoglucosidasius based on 16S rDNA sequence, and characterized by the API 50 CH kit. Enzymes capable of transforming acrylamide to acrylic acid were purified by a series of column chromatography from two of the bacterial strains, R. eutropha AUM-01 and G. thermoglucosidasius AUT-01. The molecular weights of the enzymes from both bacteria were estimated to be 38 kDa by SDS-PAGE. The enzyme activities had pH and temperature optima of 6.3 and 55°C, and 6.2 and 70°C, respectively. The influence of different metals and amino acids on the ability of the purified proteins to transform acrylamide to acrylic acid was evaluated. The gene encoding the acrylamide-degrading protein from G. thermoglucosidasius AUT-01 was cloned and sequenced. Comparison of the sequence of the cloned gene to that of known genes demonstrated that this acrylamide-degrading enzyme is an aliphatic amidase. Enzyme preparations from both mesophiles and thermophiles were tested for their ability to degrade acrylamide in coffee.