Temperature adaptations in psychrophilic, mesophilic and thermophilic chloride-dependent alpha-amylases

Cipolla, Alexandre; Delbrassine, François; Lage, Jean-Luc Da; Feller, Georges

doi:10.1016/j.biochi.2012.05.013

Cited by 71 publications

(61 citation statements)

References 55 publications

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“…that they cannot become less stable without losing the native and active conformation (Feller and Gerday ). Importantly, cold adaptation does not seem to shift the temperature of maximal stability, but reduces thermodynamic stability at all temperatures (Cipolla et al ) (Fig. B).…”

Section: Temperature and Mutational Robustnessmentioning

confidence: 90%

Temperature‐dependent mutational robustness can explain faster molecular evolution at warm temperatures, affecting speciation rate and global patterns of species diversity

et al. 2015

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Distribution of species across the Earth shows strong latitudinal and altitudinal gradients with the number of species decreasing with declining temperatures. While these patterns have been recognized for well over a century, the mechanisms generating and maintaining them have remained elusive. Here, we propose a mechanistic explanation for temperature‐dependent rates of molecular evolution that can influence speciation rates and global biodiversity gradients. Our hypothesis is based on the effects of temperature and temperature‐adaptation on stability of proteins and other catalytic biomolecules. First, due to the nature of physical forces between biomolecules and water, stability of biomolecules is maximal around + 20°C and decreases as temperature either decreases or increases. Second, organisms that have adapted to cold temperatures have evolved especially flexible (but unstable) proteins to facilitate catalytic reactions in cold, where molecular movements slow down. Both these effects should result in mutations being on average more detrimental at cold temperatures (i.e. lower mutational robustness in cold). At high temperatures, destabilizing water–biomolecule interactions, and the need to maintain structures that withstand heat denaturation, should decrease mutational robustness similarly. Decreased mutational robustness at extreme temperatures will slow down molecular evolution, as a larger fraction of new mutations will be removed by selection. Lower mutational robustness may also select for reduced mutation rates, further slowing down the rate of molecular evolution. As speciation requires the evolution of epistatic incompatibilities that prevent gene flow among incipient species, slow rate of molecular evolution at extreme temperatures will directly slow down the rate at which new species arise. The proposed mechanism can thus explain why molecular evolution is faster at warm temperatures, contributing to higher speciation rate and elevated species richness in environments characterized by stable and warm temperatures.

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Section: Temperature and Mutational Robustnessmentioning

confidence: 90%

Temperature‐dependent mutational robustness can explain faster molecular evolution at warm temperatures, affecting speciation rate and global patterns of species diversity

et al. 2015

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“…Conversely, because the chemical reaction rates increase as temperatures are increased, thermophilic enzymes benefit from high temperatures to boost activity. Therefore, a strong selective pressure for high low-temperature activity should be less essential for these enzymes (48,58). For the WF146 protease, the effect of the absence of the above-mentioned stabilizing forces could be compensated by the presence of other stabilizing interactions around the active-site region.…”

Section: Discussionmentioning

confidence: 99%

“…As a result, the geometric arrangement of the catalytic triad is less likely to be affected, thereby rendering the enzyme stable and active for catalysis at high temperatures. Due to a lack of strong selective pressure for highly active thermophilic enzymes that benefit from thermally induced activity in nature (58), the low-temperature activity of the thermophilic WF146 protease can be artificially improved by incorporating structural elements of psychrophilic S41. This enhancement in low-temperature activity will then be accompanied by an increase in enzyme thermostability if the structural elements (e.g., Asn136 in S41) contribute to enzymatic activity by stabilizing key components that are involved in the catalytic cycle (e.g., the catalytic triad).…”

Section: Discussionmentioning

confidence: 99%

Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart

Bin

Dai

Chen

et al. 2015

Appl Environ Microbiol

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bThe incorporation of the structural elements of thermostable enzymes into their less stable counterparts is generally used to improve enzyme thermostability. However, the process of engineering enzymes with both high thermostability and high activity remains an important challenge. Here, we report that the thermostability and activity of a thermophilic subtilase were simultaneously improved by incorporating structural elements of a psychrophilic subtilase. There were 64 variable regions/residues (VRs) in the alignment of the thermophilic WF146 protease, mesophilic sphericase, and psychrophilic S41. The WF146 protease was subjected to systematic mutagenesis, in which each of its VRs was replaced with those from S41 and sphericase. After successive rounds of combination and screening, we constructed the variant PBL5X with eight amino acid residues from S41. The halflife of PBL5X at 85°C (57.1 min) was approximately 9-fold longer than that of the wild-type (WT) WF146 protease (6.3 min). The substitutions also led to an increase in the apparent thermal denaturation midpoint temperature (T m ) of the enzyme by 5.5°C, as determined by differential scanning calorimetry. Compared to the WT, PBL5X exhibited high caseinolytic activity (25 to 95°C) and high values of K m and k cat (25 to 80°C). Our study may provide a rational basis for developing highly stable and active enzymes, which are highly desired in industrial applications. Many microorganisms have successfully colonized extreme temperature environments ranging from Ϫ20°C to 122°C (1, 2). Enzymes from psychrophiles, mesophiles, and (hyper)thermophiles usually perform efficient catalysis at low, moderate, and high temperatures, respectively. Psychrophilic enzymes are characterized as cold active but heat labile, and these characteristics may arise from an increase in either the global or localized flexibility of enzyme structure (3, 4). Compared to their psychrophilic and mesophilic counterparts, (hyper)thermophilic enzymes generally exhibit enhanced conformational rigidity (5-7). However, some (hyper)thermophilic enzymes may combine local flexibility in their active site with high overall rigidity, thus making them more thermostable and cold active than their mesophilic counterparts (5-7).Accumulating evidence suggests that the cumulative effect of minor improvements of local interactions enhances the intrinsic stability of (hyper)thermophilic enzymes (5, 8). Identification of protein stabilization mechanisms is normally based on comparative studies of homologous enzymes that are adapted to different temperatures, on mutational analyses, on directed evolution and on computational methods (6, 9). The results of these studies have provided a rational basis for improving enzyme stability by sitedirected mutagenesis (SDM) (10). Nevertheless, the process of engineering enzymes for higher thermostability and activity remains important and difficult. One reason for this problem is that structural differences between homologous enzymes that are adapted to different tempe...

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“…The significance of the intended objectives is better appreciated if cognizance is taken of the fact that it is the hydrolysis of the bond that is the rate limiting step; this is the reason while thermophiles in particular are less active at low temperatures due to low conformational flexibility of the active site domain, unlike psychrophiles whose cold temperature environment does not inhibit its activity because its active site domain is already in a state of conformational flexibility, eliminating the need for higher temperature dependence [17,24]. Apart from swallowing starchrich diet, by-passing partial digestion in oral cavity as suggested elsewhere [16,20], a complex process of gene transplant coding for amylase with lower capacity to hydrolyze glycosidic bond or ingestion of capsules encapsulating such enzyme may also enhance the control of digestion and plasma sugar level in diabetics.…”

Section: Introductionmentioning

confidence: 99%

Untitled

2017

JAPLR

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Background: There has been an equation which unifies all common kinetic parameters to the exclusion of pseudo-first order rate constant. Much attention had been paid to rate constant for the production of reducing sugars during amylolysis catalyzed by alpha amylase. There seems not be concern for the rate (k 2[S] ) of amylolysis of glycosidic bond and making of bonds. Modification of active site for therapeutic and scientific reasons can alter the value of k 2[S] .Objectives: The objectives of this research are the formulation of an additional, simple, and verifiable mathematical models that can be used to determine the parameter, exp Method:The theoretical aspect entailed the derivation of different equations while the experimental aspect entailed the application of Bernfeld method of enzyme assay; this was used to determine the molar concentration of reducing sugar produced. ~1/2 nd of k 2 ; this shows that hydrolysis of glycosidic bond and making of new bonds is the rate limiting step. Estimation of the duration of assay needed to produce a desired amount of reducing sugar may be for feature investigation. Results: Unpaired t -test showed that

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Temperature adaptations in psychrophilic, mesophilic and thermophilic chloride-dependent alpha-amylases

Cited by 71 publications

References 55 publications

Temperature‐dependent mutational robustness can explain faster molecular evolution at warm temperatures, affecting speciation rate and global patterns of species diversity

Temperature‐dependent mutational robustness can explain faster molecular evolution at warm temperatures, affecting speciation rate and global patterns of species diversity

Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart

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