Thermostability

Last updated
Crystal structure of b-glucosidase from Thermotoga neapolitana (PDB: 5IDI). Thermostable protein, active at 80degC and with unfolding temperature of 101degC. ThBgl1A.tif
Crystal structure of β-glucosidase from Thermotoga neapolitana (PDB: 5IDI). Thermostable protein, active at 80°C and with unfolding temperature of 101°C.

In materials science and molecular biology, thermostability is the ability of a substance to resist irreversible change in its chemical or physical structure, often by resisting decomposition or polymerization, at a high relative temperature.

Contents

Thermostable materials may be used industrially as fire retardants. A thermostable plastic , an uncommon and unconventional term, is likely to refer to a thermosetting plastic that cannot be reshaped when heated, than to a thermoplastic that can be remelted and recast.

Thermostability is also a property of some proteins. To be a thermostable protein means to be resistant to changes in protein structure due to applied heat.

Thermostable proteins

As heat is added, this disrupts the intramolecular bonds found in tertiary structure of proteins, causing the protein to unfold and become inactive. Process of Denaturation.svg
As heat is added, this disrupts the intramolecular bonds found in tertiary structure of proteins, causing the protein to unfold and become inactive.

Most life-forms on Earth live at temperatures of less than 50 °C, commonly from 15 to 50 °C. Within these organisms are macromolecules (proteins and nucleic acids) which form the three-dimensional structures essential to their enzymatic activity. [2] Above the native temperature of the organism, thermal energy may cause the unfolding and denaturation, as the heat can disrupt the intramolecular bonds in the tertiary and quaternary structure. This unfolding will result in loss in enzymatic activity, which is understandably deleterious to continuing life-functions. An example of such is the denaturing of proteins in albumen from a clear, nearly colourless liquid to an opaque white, insoluble gel.

Proteins capable of withstanding such high temperatures compared to proteins that cannot, are generally from microorganisms that are hyperthermophiles. Such organisms can withstand above 50 °C temperatures as they usually live within environments of 85 °C and above. [3] Certain thermophilic life-forms exist which can withstand temperatures above this, and have corresponding adaptations to preserve protein function at these temperatures. [4] These can include altered bulk properties of the cell to stabilize all proteins, [5] and specific changes to individual proteins. Comparing homologous proteins present in these thermophiles and other organisms reveal some differences in the protein structure. One notable difference is the presence of extra hydrogen bonds in the thermophile's proteins—meaning that the protein structure is more resistant to unfolding. Similarly, thermostable proteins are rich in salt bridges or/and extra disulfide bridges stabilizing the structure. [6] [7] Other factors of protein thermostability are compactness of protein structure, [8] oligomerization, [9] and strength interaction between subunits.

Uses and applications

Polymerase chain reactions

Thermostable enzymes such as Taq polymerase and Pfu DNA polymerase are used in polymerase chain reactions (PCR) where temperatures of 94 °C or over are used to melt DNA strands in the denaturation step of PCR. [10] This resistance to high temperature allows for DNA polymerase to elongate DNA with a desired sequence of interest with the presence of dNTPs.

Feed additives

Enzymes are often added to animal feed to improve the health and growth of farmed animals, particularly chickens and pigs. The feed is typically treated with high pressure steam to kill bacteria such as Salmonella. Therefore the added enzymes (e.g. phytase and xylanase) must be able to withstand this thermal challenge without being irreversibly inactivated. [11]

Protein purification

Knowledge of an enzyme's resistance to high temperatures is especially beneficial in protein purification. In the procedure of heat denaturation, one can subject a mixture of proteins to high temperatures, which will result in the denaturation of proteins that are not thermostable, and the isolation of the protein that is thermodynamically stable. One notable example of this is found in the purification of alkaline phosphatase from the hyperthermophile Pyrococcus abyssi . This enzyme is known for being heat stable at temperatures greater than 95 °C, and therefore can be partially purified by heating when heterologously expressed in E. coli. [12] The increase in temperature causes the E. coli proteins to precipitate, while the P. abyssi alkaline phosphatase remains stably in solution.

Glycoside hydrolases

Another important group of thermostable enzymes are glycoside hydrolases. These enzymes are responsible of the degradation of the major fraction of biomass, the polysaccharides present in starch and lignocellulose. Thus, glycoside hydrolases are gaining great interest in biorefining applications in the future bioeconomy. [13] Some examples are the production of monosaccharides for food applications as well as use as carbon source for microbial conversion in fuels (ethanol) and chemical intermediates, production of oligosaccharides for prebiotic applications and production of surfactants alkyl glycoside type. All of these processes often involve thermal treatments to facilitate the polysaccharide hydrolysis, hence give thermostable variants of glycoside hydrolases an important role in this context.

Approaches to improve thermostability of proteins

Protein engineering can be used to enhance the thermostability of proteins. A number of site-directed and random mutagenesis techniques, [14] [15] in addition to directed evolution, [16] have been used to increase the thermostability of target proteins. Comparative methods have been used to increase the stability of mesophilic proteins based on comparison to thermophilic homologs. [17] [18] [19] [20] Additionally, analysis of the protein unfolding by molecular dynamics can be used to understand the process of unfolding and then design stabilizing mutations. [21] Rational protein engineering for increasing protein thermostability includes mutations which truncate loops, increase salt bridges [22] or hydrogen bonds, introduced disulfide bonds. [23] In addition, ligand binding can increase the stability of the protein, particularly when purified. [24] There are various different forces that allow for the thermostability of a particular protein. These forces include hydrophobic interactions, electrostatic interactions, and the presence of disulfide bonds. The overall amount of hydrophobicity present in a particular protein is responsible for its thermostability. Another type of force that is responsible for thermostability of a protein is the electrostatic interactions between molecules. These interactions include salt bridges and hydrogen bonds. Salt bridges are unaffected by high temperatures, therefore, are necessary for protein and enzyme stability. A third force used to increase thermostability in proteins and enzymes is the presence of disulfide bonds. They present covalent cross-linkages between the polypeptide chains. These bonds are the strongest because they're covalent bonds, making them stronger than intermolecular forces. [25] Glycosylation is another way to improve the thermostability of proteins. Stereoelectronic effects in stabilizing interactions between carbohydrate and protein can lead to the thermostabilization of the glycosylated protein. [26] Cyclizing enzymes by covalently linking the N-terminus to the C-terminus has been applied to increase the thermostability of many enzymes. Intein cyclization and SpyTag/SpyCatcher cyclization have often been employed. [27] [28]

Thermostable toxins

Certain poisonous fungi contain thermostable toxins, such as amatoxin found in the death cap and autumn skullcap mushrooms and patulin from molds. Therefore, applying heat to these will not remove the toxicity and is of particular concern for food safety. [29]

See also

Thermophiles

Related Research Articles

<span class="mw-page-title-main">Denaturation (biochemistry)</span> Loss of structure in proteins and nucleic acids due to external stress

In biochemistry, denaturation is a process in which proteins or nucleic acids lose the quaternary structure, tertiary structure, and secondary structure which is present in their native state, by application of some external stress or compound such as a strong acid or base, a concentrated inorganic salt, an organic solvent, agitation and radiation or heat. If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death. Protein denaturation is also a consequence of cell death. Denatured proteins can exhibit a wide range of characteristics, from conformational change and loss of solubility to aggregation due to the exposure of hydrophobic groups. The loss of solubility as a result of denaturation is called coagulation. Denatured proteins lose their 3D structure and therefore cannot function.

<span class="mw-page-title-main">Thermophile</span> Organism that thrives at relatively high temperatures

A thermophile is an organism—a type of extremophile—that thrives at relatively high temperatures, between 41 and 122 °C. Many thermophiles are archaea, though some of them are bacteria and fungi. Thermophilic eubacteria are suggested to have been among the earliest bacteria.

<span class="mw-page-title-main">Protein folding</span> Change of a linear protein chain to a 3D structure

Protein folding is the physical process by which a protein, after synthesis by a ribosome as a linear chain of amino acids, changes from an unstable random coil into a more ordered three-dimensional structure. This structure permits the protein to become biologically functional.

<span class="mw-page-title-main">Lysozyme</span> Antimicrobial enzyme produced by animals

Lysozyme is an antimicrobial enzyme produced by animals that forms part of the innate immune system. It is a glycoside hydrolase that catalyzes the following process:

<i>Thermus aquaticus</i> Species of bacterium

Thermus aquaticus is a species of bacteria that can tolerate high temperatures, one of several thermophilic bacteria that belong to the Deinococcota phylum. It is the source of the heat-resistant enzyme Taq DNA polymerase, one of the most important enzymes in molecular biology because of its use in the polymerase chain reaction (PCR) DNA amplification technique.

A hyperthermophile is an organism that thrives in extremely hot environments—from 60 °C (140 °F) upwards. An optimal temperature for the existence of hyperthermophiles is often above 80 °C (176 °F). Hyperthermophiles are often within the domain Archaea, although some bacteria are also able to tolerate extreme temperatures. Some of these bacteria are able to live at temperatures greater than 100 °C, deep in the ocean where high pressures increase the boiling point of water. Many hyperthermophiles are also able to withstand other environmental extremes, such as high acidity or high radiation levels. Hyperthermophiles are a subset of extremophiles. Their existence may support the possibility of extraterrestrial life, showing that life can thrive in environmental extremes.

<span class="mw-page-title-main">Protein structure</span> Three-dimensional arrangement of atoms in an amino acid-chain molecule

Protein structure is the three-dimensional arrangement of atoms in an amino acid-chain molecule. Proteins are polymers – specifically polypeptides – formed from sequences of amino acids, which are the monomers of the polymer. A single amino acid monomer may also be called a residue, which indicates a repeating unit of a polymer. Proteins form by amino acids undergoing condensation reactions, in which the amino acids lose one water molecule per reaction in order to attach to one another with a peptide bond. By convention, a chain under 30 amino acids is often identified as a peptide, rather than a protein. To be able to perform their biological function, proteins fold into one or more specific spatial conformations driven by a number of non-covalent interactions, such as hydrogen bonding, ionic interactions, Van der Waals forces, and hydrophobic packing. To understand the functions of proteins at a molecular level, it is often necessary to determine their three-dimensional structure. This is the topic of the scientific field of structural biology, which employs techniques such as X-ray crystallography, NMR spectroscopy, cryo-electron microscopy (cryo-EM) and dual polarisation interferometry, to determine the structure of proteins.

<i>Taq</i> polymerase Thermostable form of DNA polymerase I used in polymerase chain reaction

Taq polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism Thermus aquaticus, from which it was originally isolated by Chien et al. in 1976. Its name is often abbreviated to Taq or Taq pol. It is frequently used in the polymerase chain reaction (PCR), a method for greatly amplifying the quantity of short segments of DNA.

<i>Pyrococcus furiosus</i> Species of archaeon

Pyrococcus furiosus is a heterotrophic, strictly anaerobic, extremophilic, model species of archaea. It is classified as a hyperthermophile because it thrives best under extremely high temperatures, and is notable for having an optimum growth temperature of 100 °C. P. furiosus belongs to the Pyrococcus genus, most commonly found in extreme environmental conditions of hydrothermal vents. It is one of the few prokaryotic organisms that has enzymes containing tungsten, an element rarely found in biological molecules.

Protein metabolism denotes the various biochemical processes responsible for the synthesis of proteins and amino acids (anabolism), and the breakdown of proteins by catabolism.

<span class="mw-page-title-main">Thermolysin</span>

Thermolysin is a thermostable neutral metalloproteinase enzyme produced by the Gram-positive bacteria Bacillus thermoproteolyticus. It requires one zinc ion for enzyme activity and four calcium ions for structural stability. Thermolysin specifically catalyzes the hydrolysis of peptide bonds containing hydrophobic amino acids. However thermolysin is also widely used for peptide bond formation through the reverse reaction of hydrolysis. Thermolysin is the most stable member of a family of metalloproteinases produced by various Bacillus species. These enzymes are also termed 'neutral' proteinases or thermolysin -like proteinases (TLPs).

The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization which have been developed by molecular biologists to improve PCR performance and minimize failure.

<i>Pyrococcus</i> Genus of archaea

Pyrococcus is a genus of Thermococcaceaen archaean.

In taxonomy, Staphylothermus is a genus of the Desulfurococcaceae.[1]

In molecular biology, glycoside hydrolase family 52 is a family of glycoside hydrolases.

In molecular biology, glycoside hydrolase family 57 is a family of glycoside hydrolases.

Thermococcus kodakarensis is a species of thermophilic archaea. The type strain T. kodakarensis KOD1 is one of the best-studied members of the genus.

A thermal shift assay (TSA) measures changes in the thermal denaturation temperature and hence stability of a protein under varying conditions such as variations in drug concentration, buffer pH or ionic strength, redox potential, or sequence mutation. The most common method for measuring protein thermal shifts is differential scanning fluorimetry (DSF) or thermofluor, which utilizes specialized fluorogenic dyes.

Pyrococcus abyssi is a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent in the North Fiji Basin at 2,000 metres (6,600 ft). It is anaerobic, sulfur-metabolizing, gram-negative, coccus-shaped and highly motile. Its optimum growth temperature is 96 °C (205 °F). Its type strain is GE5. Pyrococcus abyssi has been used as a model organism in studies of DNA polymerase. This species can also grow at high cell densities in bioreactors.

Saccharolobus solfataricus is a species of thermophilic archaeon. It was transferred from the genus Sulfolobus to the new genus Saccharolobus with the description of Saccharolobus caldissimus in 2018.

References

  1. ↑ Kulkarni TS, Khan S, Villagomez R, Mahmood T, Lindahl S, Logan DT, et al. (May 2017). "Crystal structure of β-glucosidase 1A from Thermotoga neapolitana and comparison of active site mutants for hydrolysis of flavonoid glucosides". Proteins. 85 (5): 872–884. doi:10.1002/prot.25256. PMID   28142197. S2CID   27832389.
  2. ↑ Kandhari N, Sinha S (June 26, 2017). "Complex network analysis of thermostable mutants of Bacillus subtilis Lipase A". Applied Network Science. 2 (1): 18. doi:10.1007/s41109-017-0039-y. PMC   6214246 . PMID   30443573.
  3. ↑ Danson MJ, Hough DW, Russell RJ, Taylor GL, Pearl L (August 1996). "Enzyme thermostability and thermoactivity". Protein Engineering. 9 (8): 629–630. doi: 10.1093/protein/9.8.629 . PMID   8875639.
  4. ↑ Takami H, Takaki Y, Chee GJ, Nishi S, Shimamura S, Suzuki H, et al. (2004). "Thermoadaptation trait revealed by the genome sequence of thermophilic Geobacillus kaustophilus". Nucleic Acids Research. 32 (21): 6292–6303. doi:10.1093/nar/gkh970. PMC   535678 . PMID   15576355.
  5. ↑ Neves C, da Costa MS, Santos H (December 2005). "Compatible solutes of the hyperthermophile Palaeococcus ferrophilus: osmoadaptation and thermoadaptation in the order thermococcales". Applied and Environmental Microbiology. 71 (12): 8091–8098. Bibcode:2005ApEnM..71.8091N. doi:10.1128/AEM.71.12.8091-8098.2005. PMC   1317470 . PMID   16332790.
  6. ↑ Das R, Gerstein M (May 2000). "The stability of thermophilic proteins: a study based on comprehensive genome comparison". Functional & Integrative Genomics. 1 (1): 76–88. doi:10.1007/s101420000003. PMID   11793224. S2CID   2717885.
  7. ↑ Matsumura M, Becktel WJ, Levitt M, Matthews BW (September 1989). "Stabilization of phage T4 lysozyme by engineered disulfide bonds". Proceedings of the National Academy of Sciences of the United States of America. 86 (17): 6562–6566. Bibcode:1989PNAS...86.6562M. doi: 10.1073/pnas.86.17.6562 . PMC   297884 . PMID   2671995.
  8. ↑ Thompson MJ, Eisenberg D (July 1999). "Transproteomic evidence of a loop-deletion mechanism for enhancing protein thermostability". Journal of Molecular Biology. 290 (2): 595–604. doi:10.1006/jmbi.1999.2889. PMID   10390356.
  9. ↑ Tanaka Y, Tsumoto K, Yasutake Y, Umetsu M, Yao M, Fukada H, et al. (July 2004). "How oligomerization contributes to the thermostability of an archaeon protein. Protein L-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii". The Journal of Biological Chemistry. 279 (31): 32957–32967. doi: 10.1074/jbc.M404405200 . PMID   15169774.
  10. ↑ Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. (January 1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science. 239 (4839): 487–491. Bibcode:1988Sci...239..487S. doi:10.1126/science.239.4839.487. PMID   2448875.
  11. ↑ Corrêa TL, de Araújo EF (September 2020). "Fungal phytases: from genes to applications". Brazilian Journal of Microbiology. 51 (3): 1009–1020. doi:10.1007/s42770-020-00289-y. PMC   7455620 . PMID   32410091.
  12. ↑ Zappa S, Rolland JL, Flament D, Gueguen Y, Boudrant J, Dietrich J (October 2001). "Characterization of a highly thermostable alkaline phosphatase from the euryarchaeon Pyrococcus abyssi". Applied and Environmental Microbiology. 67 (10): 4504–4511. Bibcode:2001ApEnM..67.4504Z. doi:10.1128/AEM.67.10.4504-4511.2001. PMC   93196 . PMID   11571149.
  13. ↑ Linares-Pasten JA, Andersson M, N Karlsson E (2014). "Thermostable glycoside hydrolases in biorefinery technologies". Current Biotechnology. 3 (1): 26–44. doi:10.2174/22115501113026660041.
  14. ↑ Sarkar CA, Dodevski I, Kenig M, Dudli S, Mohr A, Hermans E, Plückthun A (September 2008). "Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity". Proceedings of the National Academy of Sciences of the United States of America. 105 (39): 14808–14813. Bibcode:2008PNAS..10514808S. doi: 10.1073/pnas.0803103105 . PMC   2567449 . PMID   18812512.
  15. ↑ Asial I, Cheng YX, Engman H, Dollhopf M, Wu B, Nordlund P, Cornvik T (2013). "Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell". Nature Communications. 4: 2901. Bibcode:2013NatCo...4.2901A. doi: 10.1038/ncomms3901 . PMID   24352381.
  16. ↑ Hoseki J, Yano T, Koyama Y, Kuramitsu S, Kagamiyama H (November 1999). "Directed evolution of thermostable kanamycin-resistance gene: a convenient selection marker for Thermus thermophilus". Journal of Biochemistry. 126 (5): 951–956. doi:10.1093/oxfordjournals.jbchem.a022539. PMID   10544290.
  17. ↑ Sayed A, Ghazy MA, Ferreira AJ, Setubal JC, Chambergo FS, Ouf A, et al. (January 2014). "A novel mercuric reductase from the unique deep brine environment of Atlantis II in the Red Sea". The Journal of Biological Chemistry. 289 (3): 1675–1687. doi: 10.1074/jbc.M113.493429 . PMC   3894346 . PMID   24280218.
  18. ↑ Perl D, Mueller U, Heinemann U, Schmid FX (May 2000). "Two exposed amino acid residues confer thermostability on a cold shock protein". Nature Structural Biology. 7 (5): 380–383. doi:10.1038/75151. PMID   10802734. S2CID   21850845.
  19. ↑ Lehmann M, Pasamontes L, Lassen SF, Wyss M (December 2000). "The consensus concept for thermostability engineering of proteins". Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1543 (2): 408–415. doi:10.1016/s0167-4838(00)00238-7. PMID   11150616.
  20. ↑ Sauer DB, Karpowich NK, Song JM, Wang DN (October 2015). "Rapid Bioinformatic Identification of Thermostabilizing Mutations". Biophysical Journal. 109 (7): 1420–1428. Bibcode:2015BpJ...109.1420S. doi:10.1016/j.bpj.2015.07.026. PMC   4601007 . PMID   26445442.
  21. ↑ Liu HL, Wang WC (January 2003). "Protein engineering to improve the thermostability of glucoamylase from Aspergillus awamori based on molecular dynamics simulations". Protein Engineering. 16 (1): 19–25. doi:10.1093/proeng/gzg007. PMID   12646689.
  22. ↑ Lee CW, Wang HJ, Hwang JK, Tseng CP (2014). "Protein thermal stability enhancement by designing salt bridges: a combined computational and experimental study". PLOS ONE. 9 (11): e112751. Bibcode:2014PLoSO...9k2751L. doi: 10.1371/journal.pone.0112751 . PMC   4231051 . PMID   25393107.
  23. ↑ Mansfeld J, Vriend G, Dijkstra BW, Veltman OR, Van den Burg B, Venema G, et al. (April 1997). "Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond". The Journal of Biological Chemistry. 272 (17): 11152–11156. doi: 10.1074/jbc.272.17.11152 . PMID   9111013.
  24. ↑ Mancusso R, Karpowich NK, Czyzewski BK, Wang DN (December 2011). "Simple screening method for improving membrane protein thermostability". Methods. 55 (4): 324–329. doi:10.1016/j.ymeth.2011.07.008. PMC   3220791 . PMID   21840396.
  25. ↑ Tigerström A (2005). "Thermostability of Proteins". BIOS. 76 (1): 22–27. doi:10.1893/0005-3155(2005)076[0022:TBFTOP]2.0.CO;2. JSTOR   4608725. S2CID   85654007.
  26. ↑ Ardejani MS, Noodleman L, Powers ET, Kelly JW (May 2021). "Stereoelectronic effects in stabilizing protein-N-glycan interactions revealed by experiment and machine learning". Nature Chemistry. 13 (5): 480–487. Bibcode:2021NatCh..13..480A. doi:10.1038/s41557-021-00646-w. PMC   8102341 . PMID   33723379.
  27. ↑ Iwai H, Plückthun A (October 1999). "Circular beta-lactamase: stability enhancement by cyclizing the backbone". FEBS Letters. 459 (2): 166–172. doi: 10.1016/s0014-5793(99)01220-x . PMID   10518012. S2CID   85415249.
  28. ↑ Keeble AH, Howarth M (July 2020). "Power to the protein: enhancing and combining activities using the Spy toolbox". Chemical Science. 11 (28): 7281–7291. doi:10.1039/d0sc01878c. PMC   7844731 . PMID   33552459.
  29. ↑ Aleccia JN (4 November 2011). "FDA: Moldy applesauce repackaged by school lunch supplier". NBC News. Retrieved 15 April 2015.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.