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Applied and Environmental Microbiology, June 2004, p. 3321-3328, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3321-3328.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Purification, Characterization, and Sequencing of an Extracellular Cold-Active Aminopeptidase Produced by Marine Psychrophile Colwellia psychrerythraea Strain 34H

Adrienne L. Huston,1* Barbara Methe,2 and Jody W. Deming1

University of Washington School of Oceanography, Seattle, Washington 98195,1 The Institute for Genomic Research, Rockville, Maryland 208502

Received 9 October 2003/ Accepted 26 February 2004

The limited database on cold-active extracellular proteases from marine bacteria was expanded by successful purification and initial biochemical and structural characterization of a family M1 aminopeptidase (designated ColAP) produced by the marine psychrophile Colwellia psychrerythraea strain 34H. The 71-kDa enzyme displayed a low optimum temperature (19°C) and narrow pH range (pH 6 to 8.5) for activity and greater thermolability than other extracellular proteases. Sequencing of the gene encoding ColAP revealed a predicted amino acid sequence with the highest levels of identity (45 to 55%) to M1 aminopeptidases from mesophilic members of the {gamma} subclass of the Proteobacteria and the next highest levels of identity (35 to 36%) to leukotriene A4 hydrolases from mammalian sources. Compared to mesophilic homologs, ColAP had structural differences thought to increase the flexibility for activity in the cold; for example, it had fewer proline residues, fewer ion pairs, and a lower hydrophobic residue content. In addition to intrinsic properties that determine enzyme activity and stability, we also investigated effects of extracellular polymeric substances (EPS) from spent culture medium of strain 34H on ColAP activity at an environmentally relevant temperature (0°C) and at 45°C (the maximum temperature for activity). In both cases, ColAP stability increased significantly in the presence of EPS, indicating the importance of considering environmentally relevant extrinsic factors when enzyme structure and function are investigated.

* Corresponding author. Present address: Laboratory of Biochemistry, University of Liege B6a, Sart-Tilman, B-4000 Liege, Belgium. Phone: 0 11 32 (0)4 237 01 33. Fax: 0 11 32 (0)4 366 33 64. E-mail: ahuston{at}u.washington.edu.

Applied and Environmental Microbiology, June 2004, p. 3321-3328, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3321-3328.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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