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

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ABSTRACT

Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25°C 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.

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Localized mutations are responsible for the temperature adaptations of lactate dehydrogenases in notothenioid fishes. (A) Three-dimensional structure of the tetramer of lactate dehydrogenase from the Antarctic fish C. gunnari, showing the position of the mutations responsible for the changes between orthologs of these enzymes in thermal stability (measured as residual activity upon incubation at 50°C) and catalytic activity at low temperatures. The localization of these mutations compared to the consensus sequence are indicated as blue, magenta and red spheres for proteins with low, mild and high thermal stability, respectively. Most of them are located in structural elements (labeled in A) surrounding the active site. (B) The effect of mutations in the different positions indicated in A lead to changes in the catalytic rate of these enzymes in the cold, due to increased flexibility of regions neighboring the active-site, such that enzymes from notothenioids with lower body temperatures exhibit higher catalytic activities, as represented by the lineal regression shown in red (y = −4.6 × [s] + 231 [s−1]). Modified from Fields and Somero (1998).
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Figure 7: Localized mutations are responsible for the temperature adaptations of lactate dehydrogenases in notothenioid fishes. (A) Three-dimensional structure of the tetramer of lactate dehydrogenase from the Antarctic fish C. gunnari, showing the position of the mutations responsible for the changes between orthologs of these enzymes in thermal stability (measured as residual activity upon incubation at 50°C) and catalytic activity at low temperatures. The localization of these mutations compared to the consensus sequence are indicated as blue, magenta and red spheres for proteins with low, mild and high thermal stability, respectively. Most of them are located in structural elements (labeled in A) surrounding the active site. (B) The effect of mutations in the different positions indicated in A lead to changes in the catalytic rate of these enzymes in the cold, due to increased flexibility of regions neighboring the active-site, such that enzymes from notothenioids with lower body temperatures exhibit higher catalytic activities, as represented by the lineal regression shown in red (y = −4.6 × [s] + 231 [s−1]). Modified from Fields and Somero (1998).

Mentions: Some of the first and most detailed evidences of this apparent increase in conformational flexibility came from the study of A4 lactate dehydrogenases (A4-LDH) from nine Antarctic and three South American notothenioid teleosts, which inhabited niches with temperatures ranging from −1.8 to 10°C (Fields and Somero, 1998). Enzyme activity assays revealed that the catalytic rate of A4-LDH from teleosts inhabiting the coldest environments were higher at 0°C than their homologs, with kcat decreasing linearly as a function of average body temperature. More importantly, deduction of their amino acid sequences from RT-PCR and DNA sequencing showed that most of the minimal residue substitutions between A4-LDH that led to these catalytic differences were not distributed randomly, but located in two regions in the vicinity of the active site (helix αH and an extended loop connecting an helix with catalytic residues) whose conformational changes are rate-limiting steps for catalysis (Figure 7). Their results suggested that the observed substitutions increased the flexibility of these regions, leading to more rapid conformational changes and thus increasing kcat (Fields and Somero, 1998).


Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes
Localized mutations are responsible for the temperature adaptations of lactate dehydrogenases in notothenioid fishes. (A) Three-dimensional structure of the tetramer of lactate dehydrogenase from the Antarctic fish C. gunnari, showing the position of the mutations responsible for the changes between orthologs of these enzymes in thermal stability (measured as residual activity upon incubation at 50°C) and catalytic activity at low temperatures. The localization of these mutations compared to the consensus sequence are indicated as blue, magenta and red spheres for proteins with low, mild and high thermal stability, respectively. Most of them are located in structural elements (labeled in A) surrounding the active site. (B) The effect of mutations in the different positions indicated in A lead to changes in the catalytic rate of these enzymes in the cold, due to increased flexibility of regions neighboring the active-site, such that enzymes from notothenioids with lower body temperatures exhibit higher catalytic activities, as represented by the lineal regression shown in red (y = −4.6 × [s] + 231 [s−1]). Modified from Fields and Somero (1998).
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Related In: Results  -  Collection

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Figure 7: Localized mutations are responsible for the temperature adaptations of lactate dehydrogenases in notothenioid fishes. (A) Three-dimensional structure of the tetramer of lactate dehydrogenase from the Antarctic fish C. gunnari, showing the position of the mutations responsible for the changes between orthologs of these enzymes in thermal stability (measured as residual activity upon incubation at 50°C) and catalytic activity at low temperatures. The localization of these mutations compared to the consensus sequence are indicated as blue, magenta and red spheres for proteins with low, mild and high thermal stability, respectively. Most of them are located in structural elements (labeled in A) surrounding the active site. (B) The effect of mutations in the different positions indicated in A lead to changes in the catalytic rate of these enzymes in the cold, due to increased flexibility of regions neighboring the active-site, such that enzymes from notothenioids with lower body temperatures exhibit higher catalytic activities, as represented by the lineal regression shown in red (y = −4.6 × [s] + 231 [s−1]). Modified from Fields and Somero (1998).
Mentions: Some of the first and most detailed evidences of this apparent increase in conformational flexibility came from the study of A4 lactate dehydrogenases (A4-LDH) from nine Antarctic and three South American notothenioid teleosts, which inhabited niches with temperatures ranging from −1.8 to 10°C (Fields and Somero, 1998). Enzyme activity assays revealed that the catalytic rate of A4-LDH from teleosts inhabiting the coldest environments were higher at 0°C than their homologs, with kcat decreasing linearly as a function of average body temperature. More importantly, deduction of their amino acid sequences from RT-PCR and DNA sequencing showed that most of the minimal residue substitutions between A4-LDH that led to these catalytic differences were not distributed randomly, but located in two regions in the vicinity of the active site (helix αH and an extended loop connecting an helix with catalytic residues) whose conformational changes are rate-limiting steps for catalysis (Figure 7). Their results suggested that the observed substitutions increased the flexibility of these regions, leading to more rapid conformational changes and thus increasing kcat (Fields and Somero, 1998).

View Article: PubMed Central - PubMed

ABSTRACT

Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25°C 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.

No MeSH data available.


Related in: MedlinePlus