Protein engineering: Opportunities and challenges

Laboratory of Bioprocess Engineering, Helsinki University of Technology, P.O. Box 6100, 02015 HUT, Espoo, Finland.
Applied Microbiology and Biotechnology (Impact Factor: 3.34). 07/2007; 75(6):1225-32. DOI: 10.1007/s00253-007-0964-2
Source: PubMed


The extraordinary properties of natural proteins demonstrate that life-like protein engineering is both achievable and valuable. Rapid progress and impressive results have been made towards this goal using rational design and random techniques or a combination of both. However, we still do not have a general theory on how to specify a structure that is suited to a target function nor can we specify a sequence that folds to a target structure. There is also overreliance on the Darwinian blind search to obtain practical results. In the long run, random methods cannot replace insight in constructing life-like proteins. For the near future, however, in enzyme development, we need to rely on a combination of both.

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Available from: Matti Leisola, Oct 10, 2015
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    • "The liberated hemicellulose itself is also a source of potentially useful carbohydrates and other compounds (Saha 2003; Xiong et al. 2005). While the thermostabilization of mesophilic or mildly thermophilic enzymes showing high activity can lead to useful enzyme versions (Leisola and Turunen 2007; Wang et al. 2012), the vast pool of enzyme variants in different ecological locations is still an attractive source for finding new enzymes with industrially promising performance. For example, family GH10 contains highly extremophilic xylanases (Winterhalter and Liebl 1995; Kumar and Satyanarayana 2013). "
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    ABSTRACT: GH10 xylanase from Thermoascus aurantiacus strain SL16W (TasXyn10A) showed high stability and activity up to 70–75 °C. The enzyme’s half-lives were 101 h, 65 h, 63 min and 6 min at 60, 70, 75 and 80 °C, respectively. The melting point (T m), as measured by DSC, was 78.5 °C, which is in line with a strong activity decrease at 75–80 °C. The biomass-dissolving ionic liquid 1-ethyl-3-methylimidazolium acetate ([emim]OAc) in 30 % concentration had a small effect on the stability of TasXyn10A; T m decreased by only 5 °C. It was also observed that [emim]OAc inhibited much less GH10 xylanase (TasXyn10A) than the studied GH11 xylanases. The K m of TasXyn10A increased 3.5-fold in 15 % [emim]OAc with xylan as the substrate, whereas the approximate level of V max was not altered. The inhibition of enzyme activity by [emim]OAc was lesser at higher substrate concentrations. Therefore, high solid concentrations in industrial conditions may mitigate the inhibition of enzyme activity by ionic liquids. Molecular docking experiments indicated that the [emim] cation has major binding sites near the catalytic residues but in lower amounts in GH10 than in GH11 xylanases. Therefore, [emim] cation likely competes with the substrate when binding to the active site. The docking results indicated why the effect is lower in GH10.
    Extremophiles 07/2014; 18(6). DOI:10.1007/s00792-014-0679-0 · 2.31 Impact Factor
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    • "Thermostabilizing mutations protect xylanases also in high pressure [22]. In general, directed evolution and rational design methods have been used in various forms to engineer new enzymes, including xylanases [23] [24]. Ionic liquids (ILs) have been successfully used as dissolving agents in the pretreatment of lignocellulose for improved hydrolysis by enzymes. "
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    ABSTRACT: In the present study, an extremophilic GH11 xylanase was stabilized by an engineered N-terminal disulphide bridge. The effect of the stabilization was then tested against high temperatures and in the presence of a biomass-dissolving ionic liquid, 1-ethyl-3-methylimidazolium acetate ([emim]OAc). The N-terminal disulfide bridge increased the half-life of a GH11 xylanase (XYNB) from the hyperthermophilic bacterium Dictyoglomus thermophilum by 10-fold at 100°C. The apparent temperature optimum increased only by ∼5°C, which is less than the corresponding increase in mesophilic (∼15°C) and moderately thermophilic (∼10°C) xylanases. The performance of the enzyme was increased significantly at 100-110°C. The increasing concentration of [emim]OAc almost linearly increased the inactivation level of the enzyme activity and 25% [emim]OAc inactivated the enzyme almost fully. On the contrary, the apparent temperature optimum did not decrease to a similar extent, and the degree of denaturation of the enzyme was also much lower according to the residual activity assays. Also, 5% [emim]OAc largely counteracted the benefit obtained by the stabilizing disulfide bridge in the temperature-dependent activity assays, but not in the stability assays. Km was increased in the presence of [emim]OAc, indicating that [emim]OAc interfered the substrate-enzyme interactions. These results indicate that the effect of [emim]OAc is targeted more to the functioning of the enzyme than the basic stability of the hyperthermophilic GH11 xylanase.
    12/2013; 53(6-7):414-9. DOI:10.1016/j.enzmictec.2013.09.004
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    • "Members of glycoside hydrolase fam- ily10 (GH10) have (␣/␤) 8 barrel fold compared to GH11 have a higher molecular mass and a low pI (Collins et al., 2005). Directed evolution is a technique that can overcome the limitations of natural enzymes used as biocatalysts, for this technique does not rely on a detailed understanding of the relationship between enzyme structure and function, but on the simple powerful Darwinian principles of mutation and selection (Stemmer, 1994; Leisola and Turunen, 2007). An important step in a directed evolution experiment is to explore sequence space through random mutagenesis, and currently error-prone PCR based on the inaccurate amplification of genes is frequently used due to its high efficiency in exploring sequence space (Wong et al., 2004; Lin et al., 2009; Stephens et al., 2009). "
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    ABSTRACT: Endo-β-1, 4-xylanase was cloned from Geobacillus stearothermophilus 1A05583 by PCR. Enzymes with improved catalytic efficiency were obtained using error-prone PCR and a 96-well plate high-throughout screening system. Two variants 1-B8 and 2-H6 were screened from the mutant library containing 9,000 colonies, which, when compared with the wild-type enzyme increased the catalytic efficiency (kcat/Km) by 25% and 89%, respectively, acting on beechwood xylan. By sequencing 1-B8 and 2-H6, an identical mutation point H179Y was detected and found to overlap in the active site cleft. Following the introduction of the remaining 19 amino acids into position 179 by site-saturation mutagenesis, the catalytic efficiency of H179F was found to be 3.46-fold that of the wild-type. When Whistidine was substituted by tryptophan, arginine, methionine or proline, the enzyme lost activity. Therefore, the position 179 site may play an important role in regulating the catalytic efficiency.
    Journal of Biotechnology 10/2013; 168(4). DOI:10.1016/j.jbiotec.2013.09.014 · 2.87 Impact Factor
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