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Lehmann, M. & Wyss, M. Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution. Curr. Opin. Biotechnol. 12, 371-375

F Hoffmann-La Roche Ltd., Vitamins and Fine Chemicals Division, Department VFB, Building 203, CH-4070 Basel, Switzerland.
Current Opinion in Biotechnology (Impact Factor: 7.12). 09/2001; 12(4):371-5. DOI: 10.1016/S0958-1669(00)00229-9
Source: PubMed

ABSTRACT

With the advent of directed evolution techniques, protein engineering has received a fresh impetus. Engineering proteins for thermostability is a particularly exciting and challenging field, as it is crucial for broadening the industrial use of recombinant proteins. In addition to directed evolution, a variety of partially successful rational concepts for engineering thermostability have been developed in the past. Recent results suggest that amino acid sequence comparisons of mesophilic proteins alone can be used efficiently to engineer thermostable proteins. The potential benefits of the underlying, semirational 'consensus concept' are compared with those of rational design and directed evolution approaches.

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    • "Given the usefulness of thermostable enzymes in molecular biological laboratory methods, it is not surprising that they have been proposed as powerful tools for industrial catalysis as well (Zamost et al., 1991;Vieille and Zeikus, 2001;Atomi et al., 2011). It is also becoming increasingly possible to improve the thermostability of mesophilic enzymes, either through protein engineering or techniques such as enzyme immobilization (Lehmann and Wyss, 2001;Harris et al., 2010;Steiner and Schwab, 2012;Singh et al., 2013), so that thermally-based bioprocessing can be considered. High-temperature bioprocessing has a number of advantages, including reduced risk of contamination as compared to mesophilic hosts such as E. coli and S. cerevisiae, lowered chances of phage infection, improved solubility of substrates such as lignocellulosic biomass, continuous recovery of volatile chemical products directly from fermentation broth, and reduced cooling costs due to the greater temperature differential between the fermenter and the ambient air, which is the ultimate heat acceptor (Frock and Kelly, 2012;Keller et al., 2014). "
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    ABSTRACT: Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus, and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye toward potential technological advantages for high temperature industrial biotechnology.
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    • "By understanding the importance of these enzymes, designing of a thermostable haloalkane dehalogenase would be very much important. The current in silico approaches in the protein engineering and design to enhance the thermostability in enzymes have been ■■■ employed as a potential method in many events as explained by many researchers (Lehmann and Wyss, 2001; Wijma and Janssen, 2013; Damborsky and Brezovsky, 2014; Basu and Sen, 2013). A typical sequence/structure based design to enhance thermostability assumes that the conserved amino acids observed in related sequences are singles which mainly contributes favoring stability. "
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    ABSTRACT: Thermostability of enzymes is a major prerequisite for use in industrial enzymology. There are as such no simple general principles for achieving thermostability in case of enzymes as many factors are required to fulfil for different enzymes. The present study describes computational methods to design thermostable haloalkane dehalogenase enzyme using the crystal structure available Protein Data Bank (PDB ID: 1EDE). In in silico design strategy rule-based approaches such as disulfide bond geometry, new hydrophobic pocket design, new salt bridge construction and multiple mutations (combination of the above approaches) were introduced to the original enzyme. After each design strategy the functional effect was confirmed in terms of enzyme substrate binding by molecular docking using Autodock vina tool. Best design strategy was evaluated by comparative molecular dynamics simulation applying simulated annealing method at 8 ns using GROMACS tool. The surface hydrophobicity which is the key factor for thermostability in haloalkane dehalogenase was obtained from the simulation result. Upon optimizing the parameters, thermostability of mutant enzyme under consideration was also confirmed by the 5 ns molecular dynamics simulation at 400, 500 and 600 K.
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    • "Protein modification is a covalent reaction between the functional groups on the surface of enzyme molecule and chemicals, e.g., polymers in order to change the chemical feature of enzyme surface, which can lead to greater stability of the pretreated enzyme [4] [5] [6] [7] [8] [9]. Protein stabilization techniques, such as protein engineering [10] [11] [12] [13], reaction medium engineering [7] [14] [15] and protein immobilization are often applied methods in order to stabilize enzyme catalytic activity, i.e., to increase its functional time [6], though immobilized enzymes often have lesser activity than the native ones [16] [17]. Protein or enzyme immobilization means an attachment of protein to the surface or onto the inner cavities of greater structures with adsorption or covalent linkage [4] [5]. "
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