α-Amylase (EC 3.2.1.1) represents the best known amylolytic enzyme. It catalyzes the hydrolysis of α-1,4-glucosidic bonds in starch and related α-glucans. In general, the α-amylase is an enzyme with a broad substrate preference and product specificity. In the sequence-based classification system of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α-amylase family, forms clan GH-H together with families GH70 and GH77 that, however, contain no α-amylase. Within the family GH13, the α-amylase specificity is currently present in several subfamilies, such as GH13_1, 5, 6, 7, 15, 24, 27, 28, 36, 37, and, possibly in a few more that are not yet defined. The α-amylases classified in family GH13 employ a reaction mechanism giving retention of configuration, share 4–7 conserved sequence regions (CSRs) and catalytic machinery, and adopt the (β/α)8-barrel catalytic domain. Although the family GH57 α-amylases also employ the retaining reaction mechanism, they possess their own five CSRs and catalytic machinery, and adopt a (β/α)7-barrel fold. These family GH57 attributes are likely to be characteristic of α-amylases from the family GH119, too. With regard to family GH126, confirmation of the unambiguous presence of the α-amylase specificity may need more biochemical investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases.

α-Amylase: an enzyme specificity found in various families of glycoside hydrolases.

Psychrophiles thriving permanently at near-zero temperatures synthesize cold-active enzymes to sustain their cell cycle. Genome sequences, proteomic, and transcriptomic studies suggest various adaptive features to maintain adequate translation and proper protein folding under cold conditions. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Several open questions in the field are also highlighted.

/pdf/psychrophilic-enzymes-from-folding-to-function-and-3da2k7hd8m.pdf

Psychrophilic enzymes: from folding to function and biotechnology.

One of the critical variables that determine the rate of any reaction is temperature. For biological systems, the effects of temperature are convoluted with myriad (and often opposing) contributions from enzyme catalysis, protein stability, and temperature-dependent regulation, for example. We have coined the phrase “macromolecular rate theory (MMRT)” to describe the temperature dependence of enzyme-catalyzed rates independent of stability or regulatory processes. Central to MMRT is the observation that enzyme-catalyzed reactions occur with significant values of ΔCp‡ that are in general negative. That is, the heat capacity (Cp) for the enzyme–substrate complex is generally larger than the Cp for the enzyme-transition state complex. Consistent with a classical description of enzyme catalysis, a negative value for ΔCp‡ is the result of the enzyme binding relatively weakly to the substrate and very tightly to the transition state. This observation of negative ΔCp‡ has important implications for the temperatu...

/pdf/on-the-temperature-dependence-of-enzyme-catalyzed-rates-11gp2aqk32.pdf

On the Temperature Dependence of Enzyme-Catalyzed Rates

Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25 oC makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications.

/pdf/discovery-molecular-mechanisms-and-industrial-applications-40im449bq9.pdf

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes.

Cold-adapted microorganisms inhabiting permanently low-temperature environments were initially just a biological curiosity but have emerged as rich sources of numerous valuable tools for application in a broad spectrum of innovative technologies. To overcome the multiple challenges inherent to life in their cold habitats, these microorganisms have developed a diverse array of highly sophisticated synergistic adaptations at all levels within their cells: from cell envelope and enzyme adaptation, to cryoprotectant and chaperone production, and novel metabolic capabilities. Basic research has provided valuable insights into how these microorganisms can thrive in their challenging habitat conditions and into the mechanisms of action of the various adaptive features employed, and such insights have served as a foundation for the knowledge-based development of numerous novel biotechnological tools. In this review, we describe the current knowledge of the adaptation strategies of cold-adapted microorganisms and the biotechnological perspectives and commercial tools emerging from this knowledge. Adaptive features and, where possible, applications, in relation to membrane fatty acids, membrane pigments, the cell wall peptidoglycan layer, the lipopolysaccharide component of the outer cell membrane, compatible solutes, antifreeze and ice-nucleating proteins, extracellular polymeric substances, biosurfactants, chaperones, storage materials such as polyhydroxyalkanoates and cyanophycins and metabolic adjustments are presented and discussed.

/pdf/psychrophilic-lifestyles-mechanisms-of-adaptation-and-3915kxj5fa.pdf

Psychrophilic lifestyles: mechanisms of adaptation and biotechnological tools

Stepwise Adaptations to Low Temperature as Revealed by Multiple Mutants of Psychrophilic α-Amylase from Antarctic Bacterium

Cuproxidases are a subset of the blue multicopper oxidases that catalyze the oxidation of toxic Cu(I) ions into less harmful Cu(II) in the bacterial periplasm. Cuproxidases from psychrophilic, mesophilic, and thermophilic bacteria display the canonical features of temperature adaptation, such as increases in structural stability and apparent optimal temperature for activity with environmental temperature as well as increases in the binding affinity for catalytic and substrate copper ions. In contrast, the oxidative activities at 25 °C for both the psychrophilic and thermophilic enzymes are similar, suggesting that the nearly temperature-independent electron transfer rate does not require peculiar adjustments. Furthermore, the structural flexibilities of both the psychrophilic and thermophilic enzymes are also similar, indicating that the firm and precise bindings of the four catalytic copper ions are essential for the oxidase function. These results show that the requirements for enzymatic electron transfer, in the absence of the selective pressure of temperature on electron transfer rates, produce a specific adaptive pattern, which is distinct from that observed in enzymes possessing a well-defined active site and relying on conformational changes such as for the induced fit mechanism.

Activity–stability relationships revisited in blue oxidases catalyzing electron transfer at extreme temperatures

Psychrophilic enzymes: cool responses to chilly problems

It is frequently overlooked that the majority (>80%) of the Earth’s biosphere is cold and permanently exposed to temperatures below 5 °C (Rodrigues & Tiedje, 2008). Such low mean temperatures mainly arise from the fact that ~70% of the Earth’s surface is covered by oceans that have a constant temperature of 4°C below 1000 m depth, irrespective of the latitude. The polar regions account for another 15%, to which the glacier and alpine regions must be added, as well as the permafrost representing more than 20% of terrestrial soils. All these low temperature biotopes have been successfully colonized by cold-adapted microorganisms, termed psychrophiles (Margesin et al., 2008). These organisms do not merely endure such low and extremely inhospitable conditions but are irreversibly adapted to these environments as most psychrophiles are unable to grow at mild (or mesophilic) temperatures. Extreme psychrophiles have been traditionally sampled from Antarctic and Arctic sites, assuming that low temperatures persisting over a geological time-scale have promoted deep and efficient adaptations to freezing conditions. In addition to ice caps and sea ice, polar regions also possess unusual microbiotopes such as porous rocks in Antarctic dry valleys hosting microbial communities surviving at -60 °C (Cary et al., 2010), the liquid brine veins between sea ice crystals harboring metabolically-active microorganisms at -20 °C (Deming, 2002) or permafrost cryopegs, i.e. salty water pockets that have remained liquid at -10 °C for about 100 000 years (Gilichinsky et al., 2005). Psychrophiles and their biomolecules also possess an interesting biotechnological potential, which has already found several applications (Margesin & Feller, 2010). Cold exerts severe physicochemical constraints on living organisms including increased water viscosity, decreased molecular diffusion rates, reduced biochemical reaction rates, perturbation of weak interactions driving molecular recognition and interaction, strengthening of hydrogen bonds that, for instance, stabilize inhibitory nucleic acid structures, increased solubility of gases and stability of toxic metabolites as well as reduced fluidity of cellular membranes (D'Amico et al., 2006; Gerday & Glansdorff, 2007; Margesin et al., 2008; Rodrigues & Tiedje, 2008). Previous biochemical studies have revealed various adaptations at the molecular level such as the synthesis of cold-active enzymes by psychrophiles or the incorporation of membrane lipids promoting homeoviscosity in cold conditions. It was shown that the high level of specific activity at low temperatures of coldadapted enzymes is a key adaptation to compensate for the exponential decrease in

/pdf/life-in-the-cold-proteomics-of-the-antarctic-bacterium-l52mr2uf7z.pdf

Life in the Cold: Proteomics of the Antarctic Bacterium Pseudoalteromonas haloplanktis

The functional and structural adaptations to temperature have been addressed in homologous chloridedependent α-amylases from a psychrophilic Antarctic bacterium, the ectothermic fruit fly, the homeothermic pig and from a thermophilic actinomycete. This series covers nearly all temperatures encountered by living organisms. We report a striking continuum in the functional properties of these enzymes coupled to their structural stability and related to the thermal regime of the source organism. In particular, thermal stability recorded by intrinsic fluorescence, circular dichroism and differential scanning calorimetry appears to be a compromise between the requirement for a stable native state and the proper structural dynamics to sustain the function at the environmental/physiological temperatures. The thermodependence of activity, the kinetic parameters, the activations parameters and fluorescence quenching support these activity-stability relationships in the investigated α-amylases.

Adaptations structurales et fonctionnelles aux températures extrêmes au sein de la famille des alpha-amylases chlorure-dépendantes

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