ABSTRACT
The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high molecular weight complex of a polysaccharide and three proteins (AlnA, AlnB and AlnC). AlnA has previously been shown to be an OmpA-like protein that is largely responsible for the emulsifying activity of alasan. To further elucidate the nature of alasan, the gene coding for AlnB was cloned, sequenced and overexpressed in Escherichia coli. The overall 561 bp sequence of the hypothetical AlnB showed strong homology, including all conserved regions and residues known to be essential for enzymatic activity, to the ubiquitous family of thiol-specific antioxidant enzymes known as peroxiredoxins. Transgenic E. coli overexpressing AlnB exhibited increased resistance to cumene hydroperoxide both in liquid culture and on agar medium. Recombinant AlnB had no emulsifying activity but stabilized oil-in-water emulsion generated by AlnA.
Subject(s)
Acinetobacter/chemistry , Bacterial Proteins/chemistry , Peroxidases/chemistry , Polysaccharides, Bacterial/chemistry , Cloning, Molecular , Emulsifying Agents/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Excipients/chemistry , Excipients/isolation & purification , Excipients/metabolism , Peroxidases/metabolism , Peroxiredoxins , Recombinant Proteins/biosynthesisABSTRACT
AlnA is the protein responsible for the emulsifying and solubilizing activity of the Acinetobacter radioresistens KA53 bioemulsifier alasan. AlnA was produced in Escherichia coli, purified to homogeneity and then used to measure the enhanced solubility of 12 polyaromatic hydrocarbons (PAHs). The amount of PAH solubilized was directly proportional to AlnA concentration. The ratio of PAH solubilized by 40 micro g/ml AlnA compared to that soluble in the aqueous buffer varied greatly, from 4 (fluorene) to 81 (hexylbenzylcyclosilane). Calculations of moles PAH solubilized per mole AlnA yielded values from 4.3 (hexylphenylbenzene) to 55.8 (1,10-phenanthrolene). There was no obvious relationship between the amount of PAH solubilized and its molecular weight or intrinsic solubility. Native gel electrophoresis indicated that AlnA formed hexamers in the presence of PAHs. With molar ratios of fluorene to AlnA of 0.75 or less, only the monomer was observed, whereas at ratios of 7.5 or higher, only the hexamer was detected. At an intermediate molar ratio of 2.6, both monomer and hexamer appeared. The data indicate that PAHs are initially solubilized by binding to the monomeric form of AlnA, and as the amount bound increases above one molecule PAH per AlnA, the protein aggregates to form a specific oligomer of 5-8 monomers which allows for the binding and solubilization of more PAH.
