Jean-Pierre Chessa

Cold-adapted enzymes: from fundamentals to biotechnology

Psychrophilic enzymes produced by cold-adapted microorganisms display a high catalytic efficiency and are most often, if not always, associated with high thermosensitivity. Using X-ray crystallography, these properties are beginning to become understood, and the rules governing their adaptation to cold appear to be relatively diverse. The application of these enzymes offers considerable potential to the biotechnology industry, for example, in the detergent and food industries, for the production of fine chemicals and in bioremediation processes.

Psychrophilic enzymes: a thermodynamic challenge

Psychrophilic microorganisms, hosts of permanently cold habitats, produce enzymes which are adapted to work at low temperatures. When compared to their mesophilic counterparts, these enzymes display a higher catalytic efficiency over a temperature range of roughly 0-30 degrees C and a high thermosensitivity. The molecular characteristics of cold enzymes originating from Antarctic bacteria have been approached through protein modelling and X-ray crystallography. The deduced three-dimensional structures of cold alpha-amylase, beta-lactamase, lipase and subtilisin have been compared to their mesophilic homologs. It appears that the molecular adaptation resides in a weakening of the intramolecular interactions, and in some cases in an increase of the interaction with the solvent, leading to more flexible molecular edifices capable of performing catalysis at a lower energy cost.

Crystal structures of a psychrophilic metalloprotease reveal new insights into catalysis by cold-adapted proteases

Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermostability and a higher specific activity at low and moderate temperatures. It is in general accepted that psychrophilic enzymes are more flexible to allow easy accommodation and transformation of the substrates at low energy costs. Here, we report the structures of two crystal forms of the alkaline protease from an Antarctic Pseudomonas species (PAP), solved to 2.1‐ and 1.96‐Å resolution, respectively. Comparative studies of PAP structures with mesophilic counterparts show that the overall structures are similar but that the conformation of the substrate‐free active site in PAP resembles that of the substrate‐bound region of the mesophilic homolog, with both an active‐site tyrosine and a substrate‐binding loop displaying a conformation as in the substrate‐bound form of the mesophilic proteases. Further, a region in the catalytic domain of PAP undergoes a conformational change with a loop movement as large as 13 Å, induced by the binding of an extra calcium ion. Finally, the active site is more accessible due to deletions occurring in surrounding loop regions. Proteins 2003;50:636–647.

Xylanase from the psychrophilic yeast Cryptococcus adeliae.

Abstract A xylanase belonging to family 10 is produced by Cryptococcus adeliae, an Antarctic yeast that exhibits optimal growth at low temperature. The mature glycosylated xylanase secreted by C. adeliae is composed of 338 amino acid residues and 26 ± 3 osidic residues, and shares 84% identity with its mesophilic counterpart from C. albidus. The xylanase from C. adeliae is less thermostable than its mesophilic homologue when the residual activities are compared, and this difference was confirmed by differential scanning calorimetry experiments. In the range 0°–20°C, the cold-adapted xylanase displays a lower activation energy and a higher catalytic efficiency. All these observations suggest a less compact, more flexible molecular structure. Analysis of computerized molecular models built up for both psychrophilic and mesophilic xylanases indicates that the adaptation to cold consists of discrete changes in the tridimensional structure: of 53 substitutions, 22 are presumably involved in the adaptation process. These changes lead mainly to a less compact hydrophobic packing, to the loss of one salt bridge, and to a destabilization of the macrodipoles of the helices.

Purification, physico-chemical characterization and sequence of a heat labile alkaline metalloprotease isolated from a psychrophilic Pseudomonas species.

The psychrophilic alkaline metalloprotease (PAP) produced by a Pseudomonas bacterium isolated from Antarctica has been purified and characterized. The gene encoding PAP has been cloned and sequenced and the derived amino acid sequence shows 66% identity with the mesophilic alkaline metalloprotease from Pseudomonas aeruginosa IFO 3455 (AP). Compared to the purified AP, PAP is three times more active at 20 degrees C, is very sensitive to chelating agents and is rapidly inactivated at 45 degrees C. The lower thermostability of PAP can tentatively be explained by a loss of a stabilizing Ca(2+), a decrease in the content of hydrophobic residues and a smaller aliphatic index.

Cold enzymes: a hot topic

Chemical reaction rates often show a strong temperature dependency and a decrease of 10°C from room temperature typically divides the rate by a factor oscillating between 1.5 and 4. The decrease of the rate constant k indeed obeys an equation proposed by Svante Arrhenius as early as in 1889:

Preliminary crystal structure determination of the alkaline protease from the Antarctic psychrophile pseudomonas aeruginosa

K = A{e^{ - Ea/RT}}

Purification and characterization of the heat-labile α-amylase secreted by the psychrophilic bacterium TAC 240B

(1) in which E a is the activation energy, R the gas constant (8.31 kJ mol−1) and T the temperature in Kelvin.