Cold Adaptation of Proteins. Purification, Characterization, and Sequence of the Heat-Labile Subtilisin from the Antarctic Psychrophile Bacillus Ta41

The gene of subtilisin S41, an alkaline protease secreted by the psychrophile Bacillus TA41, encodes for a preproenzyme of 419 amino acids residues. The nucleotide sequence and NH2- and COOH-terminal amino acid sequencing of the purified enzyme indicate that the mature subtilisin S41 is composed of 309 residues with a predicted M(r) = 31,224. Subtilisin S41 shares most of its properties with mesophilic subtilisins (structure of the precursor, 52% amino acid sequence identity, alkaline pH optimum, broad specificity, Ca2+ binding) but is characterized by a higher specific activity on macromolecular substrate, by a shift of the optimum of activity toward low temperatures, and by a low thermal stability. The enzyme also differs by an acidic pI (5.3) and the presence of one disulfide bond. It is proposed that the psychrophilic enzyme possesses a more flexible molecular structure when compared to mesophilic and thermophilic subtilases in order to compensate for the reduction of reaction rates at low temperatures. The model of subtilisin S41 indeed reveals several features able to induce a more flexible, heat-labile conformation: the occurrence of four extended surface loops, a very hydrophilic surface through 11 extra Asp residues, and the lack of several salt bridges and aromatic-aromatic interactions. The low affinity of the Ca1 calcium binding site (Kd(app) = 10(-6) M), resulting possibly from one chelating side chain substitution and the stacking of Gly residues, also reflect a less compact conformation. The difference of free energy of stabilization between subtilisin S41 and a mesophilic subtilisin suggests that the balance of exo- and endothermically formed weak bonds is critical for the enzyme flexibility

Nucleotide and Derived Amino Acid Sequence of the Subtilisin from the Antarctic Psychrotroph Bacillus Ta39

The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph Bacillus TA39 was determined. The primary structure of the subtilisin precursor is composed of 420 amino acids giving rise to a mature enzyme of 309 amino acids. Asp-145, His-185 and Ser-361 are the proposed catalytic residues of the active site

Parvalbumin in the Cardiac Muscle of Normal and Haemoglobin-Myoglobin-Free Antarctic Fish

Three parvalbumin isotypes were isolated from the white muscle of haemoglobin-myoglobin-free antarctic fish Channichthys rhinoceratus. Antibodies against the parvalbumin mixture were raised in rabbits and used for discovery, quantitation and isolation by affinity chromatography of parvalbumin in the cardiac muscle of three antarctic fish species: Channichthys rhinoceratus, Champsocephalus gunnari and Notothenia neglecta. The cardiac muscle of these species contains parvalbumin in concentration close to 1 mumol per kg wet weight

Sequence of the Subtilisin-Encoding Gene from an Antarctic Psychrotroph Bacillus Ta41

peer reviewedThe nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph, Bacillus TA41, was determined. The primary structure of the subtilisin precursor corresponds to a preproenzyme of 419 amino acids. Asp144, His181 and Ser359 are the proposed catalytic residues of the protease active site

Temperature dependence of growth, enzyme-secretion and activity of psychrophilic Antartic bacteria.

peer reviewedFive 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 degrees C. However, production of lipase from Moraxella, alpha-amylase from Alteromonas haloplanctis, beta-lactamase from Psychrobacter immobilis and protease from Bacillus is maximal at temperatures close to that of their environment (-2 to 4 degrees 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 degrees C, i.e. about 20 degrees C lower than that of their mesophilic counterparts

Enzymes from psychrophilic organisms

Psychrophilic organisms such as micro-organisms and other ectothermic species living in polar, deep- sea or any constantly low temperature environments, produce enzymes adapted to function at low temperature. These enzymes are characterized by a high catalytic efficiency at low and moderate temperatures but are rather thermolabile. Due to their high specific activity and their rapid inactivation at temperatures as low as 30 degrees C, they offer, along with the producing micro-organisms, a great potential in biotechnology. The molecular basis of the adaptation of cold cu-amylase, subtilisin, triose phosphate isomerase from Antarctic bacteria and of trypsin from fish living in North Atlantic and in Antarctic sea waters have been studied. The comparison of the 3D structures obtained either by protein modelling or by X-ray crystallography (North Atlantic trypsin) with those of their mesophilic counterparts indicates that the molecular changes tend to increase the flexibility of the structure by a weakening of the intramolecular interactions and by an increase of the interactions with the solvent. For each enzyme, the most appropriate strategy enabling it to accommodate the substrate at a low energy cost is selected. There is a price to pay in terms of thermosensibility because the selective pressure is essentially oriented towards the harmonization of the specific activity with ambient thermal conditions. However, as demonstrated by site-directed mutagenesis experiments carried out on the Antarctic subtilisin, the possibility remains to stabilize the structure of these enzymes without affecting their high catalytic efficiency

Expression of Psychrophilic Genes in Mesophilic Hosts: Assessment of the Folding State of a Recombinant α-Amylase

α-Amylase from the antarctic psychrophile Alteromonas haloplanktis is synthesized at 0 ± 2°C by the wild strain. This heat-labile α-amylase folds correctly when overexpressed in Escherichia coli, providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and E. coli growth rate is reached at 18°C

Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples.

In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carboxymethyl cellulose as a substrate. Moreover, the study of pH and the thermal dependence of the hydrolytic activity shows that RBcel1 was active from pH 6 to pH 9 and remained significantly active when temperature decreased (18% of activity at 10 degrees C). It is interesting that RBcel1 was able to synthetize non-reticulated cellulose using cellobiose as a substrate. Moreover, by a combination of bioinformatics and enzyme analysis, the physiological relevance of the RBcel1 protein and its mesophilic homologous Pst_2494 protein from P. stutzeri, A1501, was established as the key enzymes involved in the production of cellulose by bacteria. In addition, RBcel1 and Pst_2494 are the two primary enzymes belonging to the GH5 family involved in this process.The ISME Journal advance online publication, 21 May 2009; doi:10.1038/ismej.2009.48