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

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.


Graphical representation of the distribution of the optimal temperatures of cold-active enzymes. The optimal temperature reported for enzymes from Table 1 is represented in a frequency plot noticing that temperatures are distributed between 5 and 90°C and the majority of the enzymes have a Topt between 20 and 45°C.
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Figure 3: Graphical representation of the distribution of the optimal temperatures of cold-active enzymes. The optimal temperature reported for enzymes from Table 1 is represented in a frequency plot noticing that temperatures are distributed between 5 and 90°C and the majority of the enzymes have a Topt between 20 and 45°C.

Mentions: Half of the cold-adapted genes were cloned in plasmids from the pET system for their expression. Only five of the genes were cloned in pCold vectors, whose advantages are described later in this review. Fusion constructs were also used for cloning 10 genes, eight in pGEX-6P-1, which allow the fusion expression of proteins to GST, and two in pMAL-c, which express proteins fusion to MBP. Other vectors are detailed in Table 1. Concerning enzyme purification, for more than half of the enzymes from Table 1 the purification process was aided by fusion to a His tag. The majority of the enzymes were overproduced in the cytoplasm in a soluble form (72%). Only 15% were secreted and 8% were insoluble. Only two enzymes were purified from the periplasm and one was expressed in the outer membrane through fusion with an autotransporter domain (Petrovskaya et al., 2015; Table 1). Almost all enzymes were characterized, providing data from their optimal temperature (Topt), optimal pH (pHopt) and kinetic parameters like kcat and Km. The distribution of the optimal temperatures of the enzymes is displayed in Figure 3, and shows that Topt are distributed between 5 and 90°C, with 80% of the enzymes having a Topt between 20 and 45°C.


Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes
Graphical representation of the distribution of the optimal temperatures of cold-active enzymes. The optimal temperature reported for enzymes from Table 1 is represented in a frequency plot noticing that temperatures are distributed between 5 and 90°C and the majority of the enzymes have a Topt between 20 and 45°C.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016527&req=5

Figure 3: Graphical representation of the distribution of the optimal temperatures of cold-active enzymes. The optimal temperature reported for enzymes from Table 1 is represented in a frequency plot noticing that temperatures are distributed between 5 and 90°C and the majority of the enzymes have a Topt between 20 and 45°C.
Mentions: Half of the cold-adapted genes were cloned in plasmids from the pET system for their expression. Only five of the genes were cloned in pCold vectors, whose advantages are described later in this review. Fusion constructs were also used for cloning 10 genes, eight in pGEX-6P-1, which allow the fusion expression of proteins to GST, and two in pMAL-c, which express proteins fusion to MBP. Other vectors are detailed in Table 1. Concerning enzyme purification, for more than half of the enzymes from Table 1 the purification process was aided by fusion to a His tag. The majority of the enzymes were overproduced in the cytoplasm in a soluble form (72%). Only 15% were secreted and 8% were insoluble. Only two enzymes were purified from the periplasm and one was expressed in the outer membrane through fusion with an autotransporter domain (Petrovskaya et al., 2015; Table 1). Almost all enzymes were characterized, providing data from their optimal temperature (Topt), optimal pH (pHopt) and kinetic parameters like kcat and Km. The distribution of the optimal temperatures of the enzymes is displayed in Figure 3, and shows that Topt are distributed between 5 and 90°C, with 80% of the enzymes having a Topt between 20 and 45°C.

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.