Structure--function studies of the recombinant protein bioemulsifier AlnA.
Acinetobacter radioresistens KA53 produces an extracellular bioemulsifier, referred to as alasan. The surface active component of alasan is a 35.77 kDa protein AlnA. Although AlnA and the Escherichia coli outer membrane protein A (OmpA) have a high amino acid sequence homology, E. coli OmpA has no emulsifying activity. Comparison of the amino acid sequences of AlnA and E. coli OmpA revealed four hydrophobic regions in AlnA that were absent in E. coli OmpA. Deletions and substitutions (with the homologous OmpA sequence) were constructed in each of the four hydrophobic regions of AlnA by site-directed polymerase chain reaction (PCR) mutagenesis, using the overlap PCR method. Analysis of the emulsifying activities of the mutated AlnA molecules demonstrated that all four hydrophobic regions were necessary for emulsifying activity. However, most of the inactive mutated proteins still adhered avidly to hexadecane. These findings indicate that in addition to binding to hydrocarbons, the protein emulsifier must form a specific structure on the surface of the hydrocarbon that prevents coalescence of oil droplets. This is the first structure-function study of a protein bioemulsifer.
Toren
Amir
A
Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel 69978. eros@post.tau.ac.il
Segal
Gil
G
Ron
Eliora Z
EZ
Rosenberg
Eugene
E
D000150:Acinetobacter / Q000737:chemistry* / Q000378:metabolism; D000473:Alkanes / Q000737:chemistry; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D005079:Excipients / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011135:Polysaccharides, Bacterial / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins; D013499:Surface Properties
D000473:Alkanes; C443986:AlnA protein, Acinetobacter radioresistens; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D005079:Excipients; D011135:Polysaccharides, Bacterial; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C443985:alasan protein, Acinetobacter radioresistens; C078167:OMPA outer membrane proteins; C007932:n-hexadecane