Cloning and sequence analysis of the heat-stable acrylamidase from a newly isolated thermophilic bacterium, Geobacillus thermoglucosidasius AUT-01.
A thermophilic bacterium capable of degrading acrylamide, AUT-01, was isolated from soil collected from a hot spring area in Montana, USA. The thermophilic strain grew with 0.2 % glucose as the sole carbon source and 1.4 mM acrylamide as the sole nitrogen source. The isolate AUT-01 was identified as Geobacillus thermoglucosidasius based on 16S rDNA sequence. An enzyme from the strain capable of transforming acrylamide to acrylic acid was purified by a series of chromatographic columns. The molecular weight of the enzyme was estimated to be 38 kDa by SDS-PAGE. The enzyme activity had pH and temperature optima of 6.2 and 70 ºC, respectively. The influence of different metals and amino acids on the ability of the purified protein to transform acrylamide to acrylic acid was evaluated. The gene from G. thermoglucosidasius encoding the acrylamidase was cloned, sequenced, and compared to aliphatic amidases from other bacterial strains. The G. thermoglucosidasius gene, amiE, encoded a 38 kDa, monomeric, heat-stable amidase that catalysed the cleavage of carbon-nitrogen bonds in acrylamide. Comparison of the amino acid sequence to other bacterial amidases revealed 99 and 82 % similarity to the amino acid sequences of Bacillus stearothermophilus and Pseudomonas aeruginosa, respectively.
Department of Bacteriology and Department of Food Science, University of Wisconsin-Madison, 1550 Linden Dr., Microbial Sciences Building, Madison, WI 53706, USA. mcha@uwalumni.com
Minseok
M
Cha
Glenn H
GH
Chambliss
D020106:Acrylamide / Q000378:metabolism; D000179:Acrylates / Q000378:metabolism; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D056506:Geobacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D045482:Hot Springs / Q000382:microbiology*; D006358:Hot Temperature; D008969:Molecular Sequence Data
D000179:Acrylates; D001426:Bacterial Proteins; D020106:Acrylamide; D000581:Amidohydrolases; C036658:acrylic acid