Thermostabilization of Bacillus circulans xylanase: Computational optimization of unstable residues based on thermal fluctuation analysis

Joo, Jeong Chan; Pack, Seung Pil; Kim, Yong Hwan; Yoo, Young Je

doi:10.1016/j.jbiotec.2010.10.002

Cited by 108 publications

(56 citation statements)

References 37 publications

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“…This can be well rationalized, for example, by reducing van der Waals strain as was found for TEM-1 b-lactamase, 34 substitution of hydrophobic residues on the surface, increase of secondary structure propensity or removal of flexible side-chains. 13 Often both thermodynamic and kinetic stability of an enzyme are improved by in vitro evolution simultaneously. It happened to the LipA in the previous study, where loop tightening aided by hydrogen bonding, 27 electrostatic repulsion and limited conformational freedom around proline were indicated as factors stabilizing the enzyme.…”

Section: Protein Denaturation Studies Using CDmentioning

confidence: 99%

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Augustyniak

Brzezinska

Pijning³

et al. 2012

Protein Science

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Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65°C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.

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Section: Protein Denaturation Studies Using CDmentioning

confidence: 99%

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Augustyniak

Brzezinska

Pijning³

et al. 2012

Protein Science

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“…Larger errors were detected for mutations that introduced changes in the electrostatics of buried residues or large structure fluctuation: mutations to glycine, involving bulky and/or well packed residues, etc.. Due to the impossibility of scoring the entire sequence (mutation) space, several strategies have been developed to focus the search in smaller regions. These include: i) the identification of flexible backbone sites which can be rigidified [129,130] introducing salt bridges [131] and/or disulphide bonds [132]; ii) the optimization of surface charge-charge interactions [133][134][135]; iii) the optimization of core packing [136]; iv) the removal of unsatisfied buried polar groups [137]; v) the localization of critical residues in the active site entry tunnels, especially for cosolute tolerance, with MD [138] or other algorithms like our in-house software PELE [113].…”

Section: Protein Stability Improvementmentioning

confidence: 99%

“…By full we obviously do not refer to a complete study of the sequence space (all residues and all possible mutations), but to an exhaustive random mutagenesis combination on a large subset of selected mutants. For 100 residues, for example, we have a sequence space of ~10 130 , which would take several lifetimes to be evaluated even if using current supercomputers. If we restrict the exploration to single, double and triple mutants, we have now ~10…”

Section: Catalytic Rate Constant Enhancementmentioning

confidence: 99%

Molecular Modeling in Enzyme Design, Toward In Silico Guided Directed Evolution

Monza

Acebes

Lucas

et al. 2017

Directed Enzyme Evolution: Advances and Applications

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Directed evolution creates diversity in subsequent rounds of mutagenesis in the quest of increased protein stability, substrate binding and catalysis. Although this technique does not require any structural/mechanistic knowledge of the system, the frequency of improved mutations is usually low. For this reason, computational tools are increasingly used to focus the search in sequence space, enhancing the efficiency of laboratory evolution. In particular, molecular modeling methods provide a unique tool to grasp the sequence/structure/function relationship The final publication is available at Springer via https://link.springer.com/chapter/10.1007/978-3-319-50413-1_10/fulltext.html of the protein to evolve, with the only condition that a structural model is provided. With this book chapter, we tried to guide the reader through the state of art of molecular modeling, discussing their strengths, limitations and directions. In addition, we suggest a possible future template for in silico directed evolution where we underline two main points: a hierarchical computational protocol combining several different techniques, and a synergic effort between simulations and experimental validation. IntroductionBiotechnology needs catalysts that can work under harsh conditions, catalyze a broad range of substrates, generate maximum amount of product, and tolerate changes in the environment. Enzymes, which are biodegradable and reusable catalysts [1], in addition to remarkable reaction rates, can work in environmentally friendly pH and temperature ranges, and display control over stereochemistry and regioselectivity which makes them ideal for many applications [2,3]. When thinking about enzymes, people normally associate them to expressions such as "perfect catalysts" or "outstanding reaction rate". In fact, there are examples of enzymes that catalyze reactions at extremely high rates such as triose phosphate isomerase, superoxide dismutase or carbonic anhydrase [4]. These are often limited only by the rate of ligand diffusion into the active site (diffusion-controlled rate). Nevertheless, an extensive analysis by Bar-Even et al., of nearly 2000 enzymes, showed that the median maximal turnover rate value over all measured enzymes is about 10 s −1 nowhere close to the values of 10 5 or 10 6 normally associated with catalysts [5,6].So, it would appear that natural enzymes are "just good enough" for the function they must perform in a given organism [7]. One might conclude that if they had evolved to their optimum performance then trying to improve them (from a kinetic point of view) would be attempting the impossible. On the contrary, as seen by the distribution of reaction rates, k cat , most enzymes function at a lower rate than the diffusion-limit and thus, there is space to further increase their kinetic properties to meet industrial needs. Additionally, we need enzymes capable of catalyzing reactions for which no known enzymes exist, to work with different substrates and for particular conditions that are industrially...

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“…The factors affecting thermostability are important for understanding the functions of proteins and their use in various industries (Ruller et al, 2008). The advantages of employing enzymes with high optimum temperatures in biotechnological processes or biocatalytic conversions industrial include the lower risk of microbial contamination by common mesophiles, the improvement of substrate solubility, increased reaction rates and decreased viscosity (Joo et al, 2011;Haki and Rakshit, 2003). The tolerance of the enzymes to high temperatures for long periods may be associated with their conformational structures, composition and/or amino acid sequences, and the origins of the enzymes (Gomes et al, 2007;Techapun et al, 2003).…”

Section: Thermostable Xylanase and Its Propertiesmentioning

confidence: 99%

Thermostable xylanase from thermophilic fungi: Biochemical properties and industrial applications

Torre¹,

Kadowaki²

2017

Afr. J. Microbiol. Res.

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Filamentous fungi have been investigated as producer of xylanases with relevant characteristics for application in different industrial sectors, such as bakery, beverage, biofuel, textile, animal feed, pharmaceutical, pulp and paper. Thus, this review will focus on biochemical properties and industrial use of thermostable xylanases produced by different filamentous fungi, as well as mechanisms of adaptation of thermophilic organisms to tolerate in high-temperature environments. These enzymatic properties of thermal and pH stability are crucial, especially in processes such as the manufacture of animal feed, pulp and paper industry. Reports on cha nges in enzyme structure, such as site -directed mutagenesis, insertion or substitution of amino acids, addition of disulfide bonds in the alpha helix or beta-sheet structure for improving the thermal stability will also be reported. However, strains of Thermomyces lanuginosus has been described as good producers of thermostable xylanases, as well as promising enzymes, because it does not require any change in structure to increase the tolerance to high temperatures.

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Thermostabilization of Bacillus circulans xylanase: Computational optimization of unstable residues based on thermal fluctuation analysis

Cited by 108 publications

References 37 publications

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Molecular Modeling in Enzyme Design, Toward In Silico Guided Directed Evolution

Thermostable xylanase from thermophilic fungi: Biochemical properties and industrial applications

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