A method to rationally increase protein stability based on the charge–charge interaction, with application to lipase LipK107

Zhang, Lujia; Tang, Xiaomang; Cui, Dongbing; Yao, Zhiqiang; Gao, Bei; Jiang, Shuiqin; Yin, Biaolin; Yuan, Yusheng; Wei, Dongzhi

doi:10.1002/pro.2388

Cited by 37 publications

(31 citation statements)

References 49 publications

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“…This model was then improved by introducing solvent accessibility (SA) [ 29 ], Gibbs free energy [ 30 ] and electrostatic interactions [ 31 ]. Using a suite of enzyme redesign algorithms, enzyme thermal stability system (ETSS) [ 32 ] was developed to refine the calculation of the TK-SA model and surface charge–charge interaction analysis. By replacing positively charged residues with negatively charged or neutral ones, the E ij value is decreased and the enzyme thermostability would be improved.…”

Section: Introductionmentioning

confidence: 99%

Improvement of the thermostability and catalytic efficiency of a highly active β-glucanase from Talaromyces leycettanus JCM12802 by optimizing residual charge–charge interactions

You

Zhang

et al. 2016

Biotechnol Biofuels

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Backgroundβ-Glucanase is one of the most extensively used biocatalysts in biofuel, food and animal feed industries. However, the poor thermostability and low catalytic efficiency of most reported β-glucanases limit their applications. Currently, two strategies are used to overcome these bottlenecks, i.e., mining for novel enzymes from extremophiles and engineering existing enzymes.ResultsA novel endo-β-1,3-1,4-glucanase of GH16 (Tlglu16A) from the thermophilic fungus Talaromyces leycettanus JCM12802 was produced in Pichia pastoris and characterized. For potential industrial applications, recombinant TlGlu16A exhibits favorable enzymatic properties over most reported glucanases, i.e., remarkable stability over a wide pH range from 1.0 to 10.0 and superior activity on glucan substrates (up to 15,197 U/mg). The only weakness of TlGlu16A is the thermolability at 65 °C and higher. To improve the thermostability, the enzyme thermal stability system was then used to engineer TlGlu16A through optimization of residual charge–charge interactions. Eleven mutants were constructed and compared to the wild-type TlGlu16A. Four mutants, H58D, E134R, D235G and D296K, showed longer half-life time at 80 °C (31, 7, 25, 22 vs. 0.5 min), and two mutants, D235G and D296K, had greater specific activities (158.2 and 122.2 %, respectively) and catalytic efficiencies (kcat/Km, 170 and 114 %, respectively).ConclusionsThe engineered TlGlu16A has great application potentials from the perspectives of enzyme yield and properties. Its thermostability and activity were apparently improved in the engineered enzymes through charge optimization. This study spans the genetic, functional and structural fields, and provides a combination of gene mining and protein engineering approaches for the systematic improvement of enzyme performance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0544-8) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Improvement of the thermostability and catalytic efficiency of a highly active β-glucanase from Talaromyces leycettanus JCM12802 by optimizing residual charge–charge interactions

You

Zhang

et al. 2016

Biotechnol Biofuels

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“…This formalism calculates the work required to put a charge at locations based on the three dimensional structure of a protein. Though using a simple treatment of Coulobmic interactions, the approach has been successfully applied to protein engineering . The calculation showed a handful of charges in RNase H * that experience favorable or unfavorable Coulombic potential (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Though using a simple treatment of Coulobmic interactions, the approach has been successfully applied to protein engineering. 32,33 The calculation showed a handful of charges in RNase H* that experience favorable or unfavorable Coulombic potential ( Fig. S1 in Supporting Information).…”

Section: Computational Analysis Of the Electrostatic Environment Of Lmentioning

confidence: 99%

Salt bridge as a gatekeeper against partial unfolding

2016

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Salt bridges are frequently observed in protein structures. Because the energetic contribution of salt bridges is strongly dependent on the environmental context, salt bridges are believed to contribute to the structural specificity rather than the stability. To test the role of salt bridges in enhancing structural specificity, we investigated the contribution of a salt bridge to the energetics of native-state partial unfolding in a cysteine-free version of Escherichia coli ribonuclease H (RNase H*). Thermolysin cleaves a protruding loop of RNase H* through transient partial unfolding under native conditions. Lys86 and Asp108 in RNase H* form a partially buried salt bridge that tethers the protruding loop. Investigation of the global stability of K86Q/D108N RNase H* showed that the salt bridge does not significantly contribute to the global stability. However, K86Q/D108N RNase H* is greatly more susceptible to proteolysis by thermolysin than wild-type RNase H* is. The free energy for partial unfolding determined by native-state proteolysis indicates that the salt bridge significantly increases the energy for partial unfolding by destabilizing the partially unfolded form. Double mutant cycles with single and double mutations of the salt bridge suggest that the partially unfolded form is destabilized due to a significant decrease in the interaction energy between Lys86 and Asp108 upon partial unfolding. This study demonstrates that, even in the case that a salt bridge does not contribute to the global stability, the salt bridge may function as a gatekeeper against partial unfolding that disturbs the optimal geometry of the salt bridge.

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“…Thus, improvement of PG thermostability is of great importance to broaden its industrial applications. In this study, we first employed the combination of open-source software ETSS (28), four residue selection criteria, and site-directed mutagenesis to identify two key residues, D244 and D299, to be crucial contributors to thermostability of PG8fn from Achaetomium sp. strain Xz8.…”

Section: Discussionmentioning

confidence: 99%

“…Tanford and Kirkwood (24) initially set up the Tanford-Kirkwood (TK) model for calculating the contribution of a single charged residue to the overall stability, which was further improved by introducing solvent accessibility (SA) (25), Gibbs free energy (26), and electrostatic interactions (27). Recently, we released a suite of enzyme redesign algorithms, the enzyme thermal stability system (ETSS) (28), in order to refine the protocol of TK-SA model calculation and surface charge-charge interaction analysis. The ETSS is a general protocol for the rational improvement of enzyme thermostability based on the charge of the enzyme.…”

Section: Improvement In Thermostability Of An Achaetomium Sp Strain mentioning

confidence: 99%

Improvement in Thermostability of an Achaetomium sp. Strain Xz8 Endopolygalacturonase via the Optimization of Charge-Charge Interactions

Luo

Meng

et al. 2015

Appl Environ Microbiol

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bImproving enzyme thermostability is of importance for widening the spectrum of application of enzymes. In this study, a structure-based rational design approach was used to improve the thermostability of a highly active, wide-pH-range-adaptable, and stable endopolygalacturonase (PG8fn) from Achaetomium sp. strain Xz8 via the optimization of charge-charge interactions. By using the enzyme thermal stability system (ETSS), two residues-D244 and D299 -were inferred to be crucial contributors to thermostability. Single (D244A and D299R) and double (D244A/D299R) mutants were then generated and compared with the wild type. All mutants showed improved thermal properties, in the order D244A < D299R < D244A/D299R. In comparison with PG8fn, D244A/D299R showed the most pronounced shifts in temperature of maximum enzymatic activity (T max ), temperature at which 50% of the maximal activity of an enzyme is retained (T 50 ), and melting temperature (T m ), of about 10, 17, and 10.2°C upward, respectively, with the half-life (t 1/2 ) extended by 8.4 h at 50°C and 45 min at 55°C. Another distinguishing characteristic of the D244A/D299R mutant was its catalytic activity, which was comparable to that of the wild type (23,000 ؎ 130 U/mg versus 28,000 ؎ 293 U/mg); on the other hand, it showed more residual activity (8,400 ؎ 83 U/mg versus 1,400 ؎ 57 U/mg) after the feed pelleting process (80°C and 30 min). Molecular dynamics (MD) simulation studies indicated that mutations at sites D244 and D299 lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity. This study reveals the importance of charge-charge interactions in protein conformation and provides a viable strategy for enhancing protein stability. P ectin, the third most abundant polysaccharide of the plant cell wall, is the most structurally complex polysaccharide consisting of covalently linked galacturonic acid (1, 2). Pectinases-microbial enzymes which are capable of depolymerizing pectinplay an important role in wide biotechnological applications, including the fruit juice, vinification, paper, and textile industries, as well as the extraction of oils (3). Polygalacturonase (PG) is the most widely studied pectinase due to its high activity, mesophilic and acidic properties, and satisfactory performance in juice clarification, extraction, viscosity reduction, and yield improvement (4). Based on sequence similarity and action mode (5), PGs are classified into family 28 of glycoside hydrolase (GH) and endoand exo-types. Most endo-PGs are highly active and stable at 30 to 50°C (6; http://www.brenda-enzymes.org/), which are far from the requirements of thermophilic processing in the feed industry (7).To obtain a thermostable, highly active PG, either mining new genetic resources of thermophiles (8) or engineering the protein and application environment (9, 10) is the most popular practice. Thermophilic fungi Thielavia arenaria XZ7 (60°C) (7), Thermoascus aurantiacus CBMAI-756 (60 to 65°C) (11), Penicillium sp. strain SPC-F 20 (60°C) ...

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A method to rationally increase protein stability based on the charge–charge interaction, with application to lipase LipK107

Cited by 37 publications

References 49 publications

Improvement of the thermostability and catalytic efficiency of a highly active β-glucanase from Talaromyces leycettanus JCM12802 by optimizing residual charge–charge interactions

Improvement of the thermostability and catalytic efficiency of a highly active β-glucanase from Talaromyces leycettanus JCM12802 by optimizing residual charge–charge interactions

Salt bridge as a gatekeeper against partial unfolding

Improvement in Thermostability of an Achaetomium sp. Strain Xz8 Endopolygalacturonase via the Optimization of Charge-Charge Interactions

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