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

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Current Opinion in Biotechnology (Impact Factor: 7.12). 09/2001; 12(4):371-5. DOI: 10.1016/S0958-1669(00)00229-9
Source: PubMed


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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Article: 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

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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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    ABSTRACT: The drug carrier function of single protein nanoparticles, i.e., each individual protein molecule covered by a very thin, porous and few nanometer thick polymer layer, has been investigated. This layer around protein molecule is very thin, about 3-5 nm thick and highly porous, thus it does not reduce seriously the enzymatic function of protein molecule. The spatial structure of encapsulated protein molecule, which is essential in its function, can stabilize by thispolymer layer. Bovine serum albumin was used as protein drug molecule and it was encapsulated with acrylamide-bisarylamide random copolymer. The polymerization, starting from the modified sites of the surface of bovine serum albumin molecules was initiated by TEMED (tetramethylethylenediamine). These single albumin nanoparticles were painted with fluorescein isothiocyanate. This material was then injected into the inferior vena cava of rats. The treated rats were decapitated after 1 to 10 minutes and its brain was investigated by fluorescent microscopy. It was proved that bovine serum albumin molecules as drugs encapsulated in polymer nano-layer with a reduced size (about 10 nm) can pass through the blood brain barrier. The results suggest that this method is capable of transformation of biomacromolecules to access the brain tissue via the blood.
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    • "A serious shortcoming of such a random approach is that it can only be applied to enzymes for which high-throughput expression and activity screens are available. Other methods to stabilize enzymes are consensus design (Lehmann and Wyss, 2001; Bommarius et al., 2006), rational protein engineering (Eijsink et al., 2004), the creation of chimeric enzymes (Romero et al., 2013) and computational design (Korkegian et al., 2005; Gribenko et al., 2009; Joo et al., 2011). The number of stabilizing mutations that are introduced is usually rather low and currently none of these methods work well enough to reliably achieve a large stability increase of a target enzyme. "
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    ABSTRACT: The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications. Here, we demonstrate that computational design of a library with chemically diverse stabilizing mutations allows the engineering of drastically stabilized and fully functional variants of the mesostable enzyme limonene epoxide hydrolase. First, point mutations were selected if they significantly improved the predicted free energy of protein folding. Disulfide bonds were designed using sampling of backbone conformational space, which tripled the number of experimentally stabilizing disulfide bridges. Next, orthogonal in silico screening steps were used to remove chemically unreasonable mutations and mutations that are predicted to increase protein flexibility. The resulting library of 64 variants was experimentally screened, which revealed 21 (pairs of) stabilizing mutations located both in relatively rigid and in flexible areas of the enzyme. Finally, combining 10-12 of these confirmed mutations resulted in multi-site mutants with an increase in apparent melting temperature from 50 to 85°C, enhanced catalytic activity, preserved regioselectivity and a >250-fold longer half-life. The developed Framework for Rapid Enzyme Stabilization by Computational libraries (FRESCO) requires far less screening than conventional directed evolution.
    Protein Engineering Design and Selection 01/2014; 27(2). DOI:10.1093/protein/gzt061 · 2.54 Impact Factor
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