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Applied and Environmental Microbiology, January 2002, p. 211-218, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.211-218.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Cloning, Sequence Analysis, and Expression in Escherichia coli of the Gene Encoding an {alpha}-Amino Acid Ester Hydrolase from Acetobacter turbidans

Jolanda J. Polderman-Tijmes,1 Peter A. Jekel,1 Erik J. de Vries,1 Annet E. J. van Merode,1 René Floris,{dagger},1 Jan-Metske van der Laan,2 Theo Sonke,3 and Dick B. Janssen1*

Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, NL-9747 AG Groningen,1 DSM Food Specialties, 2600 MA, Delft,2 DSM Research, 6160 MD Geleen, The Netherlands3

Received 9 August 2001/ Accepted 2 November 2001

The {alpha}-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing ß-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified {alpha}-amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an A. turbidans genomic library. The gene, designated aehA, encodes a polypeptide with a molecular weight of 72,000. Comparison of the determined N-terminal sequence and the deduced amino acid sequence indicated the presence of an N-terminal leader sequence of 40 amino acids. The aehA gene was subcloned in the pET9 expression plasmid and expressed in Escherichia coli. The recombinant protein was purified and found to be dimeric with subunits of 70 kDa. A sequence similarity search revealed 26% identity with a glutaryl 7-ACA acylase precursor from Bacillus laterosporus, but no homology was found with other known penicillin or cephalosporin acylases. There was some similarity to serine proteases, including the conservation of the active site motif, GXSYXG. Together with database searches, this suggested that the {alpha}-amino acid ester hydrolase is a ß-lactam antibiotic acylase that belongs to a class of hydrolases that is different from the Ntn hydrolase superfamily to which the well-characterized penicillin acylase from E. coli belongs. The {alpha}-amino acid ester hydrolase of A. turbidans represents a subclass of this new class of ß-lactam antibiotic acylases.

* Corresponding author. Mailing address: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, NL-9747 AG Groningen, The Netherlands. Phone: 31-50-3634209. Fax: 31-5-3634165. E-mail: D.B.Janssen{at}chem.rug.nl.

{dagger} Present address: NIZO, 6710 BA, Ede, The Netherlands.

Applied and Environmental Microbiology, January 2002, p. 211-218, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.211-218.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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