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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.


Representative scheme of the most typical modifications in cold-adapted enzymes. Psychrophilic and mesophilic alkaline phosphatases are compared to represent changes in the number of insertions and loop extensions, whereas psychrophilic and mesophilic α-amylases are used for visualizing changes in amino acid sequence related to the modification of several properties, listed below each type of amino acid changes. Modified from Helland et al. (2009) and Cipolla et al. (2011).
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Figure 8: Representative scheme of the most typical modifications in cold-adapted enzymes. Psychrophilic and mesophilic alkaline phosphatases are compared to represent changes in the number of insertions and loop extensions, whereas psychrophilic and mesophilic α-amylases are used for visualizing changes in amino acid sequence related to the modification of several properties, listed below each type of amino acid changes. Modified from Helland et al. (2009) and Cipolla et al. (2011).

Mentions: For example, in the case of M. marina triose phosphate isomerase, a single substitution of an alanine located within a loop that contacts the phosphate moiety of its substrate by a serine that is conserved in mesophilic enzymes is sufficient to increase the thermal stability and decrease the catalytic activity at low temperatures (Alvarez et al., 1998). The same is applicable in some cases for tuning mesophilic enzymes in order to sustain catalytic activities in the cold, as exemplified by the rationally designed single-point mutation I137M of Bacillus subtilis LipJ (Goomber et al., 2016b). Most frequently, evolutionary changes are related to multiple changes that lead to a more accessible and/or a more flexible active site due to substitution of bulky residues, insertions and deletions (Russell et al., 1998; Kim et al., 1999; Schrøder Leiros et al., 2000; Toyota et al., 2002; Aghajari et al., 2003; Van Petegem et al., 2003; Tsuruta et al., 2005, 2008; Leiros et al., 2007; Riise et al., 2007; Jung et al., 2008; Merlino et al., 2010; Jaremko et al., 2011; Malecki et al., 2013; Zheng et al., 2016), which in some cases are accompanied by the introduction of discrete amino acid substitutions in the active site that thermodynamically favor protein-ligand interactions at low temperatures, thus decreasing Km (Lonhienne et al., 2001). Finally, the most extensive changes involve large portions throughout the protein structure and are related to optimization of the surface electrostatic potential to allow better interactions with the solvent and changes in ion-pair interactions (Bell et al., 2002; de Backer et al., 2002; Leiros et al., 2003; Bae and Phillips, 2004; Kumar and Nussinov, 2004; Arnórsdóttir et al., 2005; Helland et al., 2006; De Vos et al., 2007; Fedøy et al., 2007; Wang et al., 2007; Michaux et al., 2008; Pedersen et al., 2009; Alterio et al., 2010; Arimori et al., 2013; Bujacz et al., 2015), reduction of the number of hydrogen bonds (Matsuura et al., 2002; Bae and Phillips, 2004; Altermark et al., 2008; Michaux et al., 2008; De Santi et al., 2016), changes in loop extension, amino acid content, and flexibility (Bauvois et al., 2008; Helland et al., 2009; Zhang et al., 2011; Fu et al., 2013; Miao et al., 2016; Zheng et al., 2016), introduction or loss of disulfide bonds to modulate local stability (Violot et al., 2005; Helland et al., 2006; Wang et al., 2007), differential flexibility of domains in multidomain enzymes (Watanabe et al., 2005; Bauvois et al., 2008; Angelaccio et al., 2014), and enhanced protein solvation due to increased exposure of hydrophobic residues to the solvent (Aghajari et al., 1998; Russell et al., 1998; Maes et al., 1999; Bell et al., 2002; Van Petegem et al., 2003; Zhao Y. et al., 2012; Zheng et al., 2016). A summary of the most usual modifications responsible for cold-adaptation are shown in Figure 8. It is worth noting that not all of these mechanisms are required to explain the cold-adaptation of a given enzyme (De Maayer et al., 2014), although several proteins exhibit more than one of these mechanisms occurring in parallel (Coquelle et al., 2007), which suggest that comparative analysis within protein families might be better suited to solve the sequence-structure factors that explain the evolutionary adaptations of an enzyme of interest. Although it is rare to find proteins showing other mechanisms of cold adaptation, more extensive changes in protein topology (Tsuruta et al., 2005) or modifications of the oligomerization state that allows to increase the flexibility of solvent-exposed hydrophobic regions while simultaneously stabilizing the native fold of the enzyme (Skalova et al., 2005; Zanphorlin et al., 2016) have been also observed. However, these should be considered as evolutionary alternatives rather than as general mechanisms for enhanced flexibility in cold environments.


Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes
Representative scheme of the most typical modifications in cold-adapted enzymes. Psychrophilic and mesophilic alkaline phosphatases are compared to represent changes in the number of insertions and loop extensions, whereas psychrophilic and mesophilic α-amylases are used for visualizing changes in amino acid sequence related to the modification of several properties, listed below each type of amino acid changes. Modified from Helland et al. (2009) and Cipolla et al. (2011).
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC5016527&req=5

Figure 8: Representative scheme of the most typical modifications in cold-adapted enzymes. Psychrophilic and mesophilic alkaline phosphatases are compared to represent changes in the number of insertions and loop extensions, whereas psychrophilic and mesophilic α-amylases are used for visualizing changes in amino acid sequence related to the modification of several properties, listed below each type of amino acid changes. Modified from Helland et al. (2009) and Cipolla et al. (2011).
Mentions: For example, in the case of M. marina triose phosphate isomerase, a single substitution of an alanine located within a loop that contacts the phosphate moiety of its substrate by a serine that is conserved in mesophilic enzymes is sufficient to increase the thermal stability and decrease the catalytic activity at low temperatures (Alvarez et al., 1998). The same is applicable in some cases for tuning mesophilic enzymes in order to sustain catalytic activities in the cold, as exemplified by the rationally designed single-point mutation I137M of Bacillus subtilis LipJ (Goomber et al., 2016b). Most frequently, evolutionary changes are related to multiple changes that lead to a more accessible and/or a more flexible active site due to substitution of bulky residues, insertions and deletions (Russell et al., 1998; Kim et al., 1999; Schrøder Leiros et al., 2000; Toyota et al., 2002; Aghajari et al., 2003; Van Petegem et al., 2003; Tsuruta et al., 2005, 2008; Leiros et al., 2007; Riise et al., 2007; Jung et al., 2008; Merlino et al., 2010; Jaremko et al., 2011; Malecki et al., 2013; Zheng et al., 2016), which in some cases are accompanied by the introduction of discrete amino acid substitutions in the active site that thermodynamically favor protein-ligand interactions at low temperatures, thus decreasing Km (Lonhienne et al., 2001). Finally, the most extensive changes involve large portions throughout the protein structure and are related to optimization of the surface electrostatic potential to allow better interactions with the solvent and changes in ion-pair interactions (Bell et al., 2002; de Backer et al., 2002; Leiros et al., 2003; Bae and Phillips, 2004; Kumar and Nussinov, 2004; Arnórsdóttir et al., 2005; Helland et al., 2006; De Vos et al., 2007; Fedøy et al., 2007; Wang et al., 2007; Michaux et al., 2008; Pedersen et al., 2009; Alterio et al., 2010; Arimori et al., 2013; Bujacz et al., 2015), reduction of the number of hydrogen bonds (Matsuura et al., 2002; Bae and Phillips, 2004; Altermark et al., 2008; Michaux et al., 2008; De Santi et al., 2016), changes in loop extension, amino acid content, and flexibility (Bauvois et al., 2008; Helland et al., 2009; Zhang et al., 2011; Fu et al., 2013; Miao et al., 2016; Zheng et al., 2016), introduction or loss of disulfide bonds to modulate local stability (Violot et al., 2005; Helland et al., 2006; Wang et al., 2007), differential flexibility of domains in multidomain enzymes (Watanabe et al., 2005; Bauvois et al., 2008; Angelaccio et al., 2014), and enhanced protein solvation due to increased exposure of hydrophobic residues to the solvent (Aghajari et al., 1998; Russell et al., 1998; Maes et al., 1999; Bell et al., 2002; Van Petegem et al., 2003; Zhao Y. et al., 2012; Zheng et al., 2016). A summary of the most usual modifications responsible for cold-adaptation are shown in Figure 8. It is worth noting that not all of these mechanisms are required to explain the cold-adaptation of a given enzyme (De Maayer et al., 2014), although several proteins exhibit more than one of these mechanisms occurring in parallel (Coquelle et al., 2007), which suggest that comparative analysis within protein families might be better suited to solve the sequence-structure factors that explain the evolutionary adaptations of an enzyme of interest. Although it is rare to find proteins showing other mechanisms of cold adaptation, more extensive changes in protein topology (Tsuruta et al., 2005) or modifications of the oligomerization state that allows to increase the flexibility of solvent-exposed hydrophobic regions while simultaneously stabilizing the native fold of the enzyme (Skalova et al., 2005; Zanphorlin et al., 2016) have been also observed. However, these should be considered as evolutionary alternatives rather than as general mechanisms for enhanced flexibility in cold environments.

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.