A heat-labile fl-lactamase has been purified from culture supernatants of Psychrobacter irnmobilis AS grown at 4°C and the corresponding chromosomal ainpC gene has been cloned and sequenced. All structural and kinetic properties clearly relate this enzyme to class C P-lactamases. The kinetic parameters of P irnrnobilis p-lactamase for the hydrolysis of some /!-lactam antibiotics are in the same range as the values recorded for the highly specialized cephalosporinases from pathogenic mesophilic bacteria. By contrast, the enzyme displays a low apparent optimum temperature of activity and a reduced thermal stability. Structural factors responsible for the latter property were analysed from the three-dimensional structure built by homology modelling. The deletion of proline residues in loops, the low number of arginine-mediated H-bonds and aromatic-aromatic interactions, the lower global hydrophobicity and the improved solvent interactions through additional surface acidic residues appear to be the main determinants of the enzyme flexibility.Keywords: psychrophile ; extremophile ; Antarctic ; P-lactamase ; cephalosporinase.Psychrophilic microorganisms represent a major class of the microbial world if one considers the vast extent of permanently cold environments on Earth (deep-sea waters, polar and alpine regions). In spite of the diversity and abundance of these extremophiles, the numerous physiological and biochemical adaptations to the life at temperatures close to 0°C remain poorly documented [I, 21. The dominating character of cold-adapted enzymes is probably their enhanced turnover number (kcat) and catalytic efficiency (kc.,&C,,,) : improving these kinetic parameters compensates for the reduction of reaction rates at low teniperatures and adequate metabolic fluxes are therefore maintained 131. According to the current hypothesis [4], optimization of the catalytic parameters can originate from the highly flexible structure of these proteins which provides enhanced abilities to undergo conformational changes during catalysis at low temperatures. The usually observed thermal lability is therefore regarded as a consequence of the folded structure flexibility. The molecular origin of this flexibility has been analysed from the primary structure of some psychrophilic bacterial enzymes. All studies suggest a potentially low number of weak interactions stabilizing the folded conformation [ S , 61. We report here the kinetic characterization, the nucleotide sequence and a structural analysis of the P-lactamase secreted by the antarctic psychrophile Psyclzrobacter irnrnobilis A5, a gram-negative bacterial strain collected in an environment ranging in temperature from -20 to +2"C. A brief account of some properties of another P-lactamase from a closely related strain has been given recently 171.

Five psychrophilic Antarctic bacteria have been selected for their capacity to secrete exoenzymes into culture medium. These strains are able to grow from 0 to about 25 ° C. However, production of lipase from Moraxella, a-amylase from Alteromonas haloplanctis, /3-1actamase from Psychrobacter immobilis and protease from Bacillus is maximal at temperatures close to that of their environment @2 to 4 ° C) and is strongly inhibited at higher temperatures. This thermal effect involves alterations in the secretory pathway in the upper range of temperatures, losses due to the enzyme thermal lability and in some cases to reduction in cell development. The apparent optimal activity temperature of these enzymes is between 30 and 40 ° C, i.e. about 20°C lower than that of their mesophilic counterparts.

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