Rational engineering of a mesohalophilic carbonic anhydrase to an extreme halotolerant biocatalyst

Warden, Andrew C.; Williams, Michelle R.; Peat, Thomas S.; Seabrook, Shane A.; Newman, Janet; Dojchinov, Greg; Haritos, Victoria S.

doi:10.1038/ncomms10278

Cited by 82 publications

(89 citation statements)

References 41 publications

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“…However, the difficulties are heat resistance and stability of CA. Thus, two different strategies to overcome the problem are isolated CA from species that survive in extreme environments, that is, hot springs or anaerobic conditions, or to engineer protein by genetic approach . A thermophilic bacterium named Sulfurihydrogenibium yellowstonense YO3AOP1 was isolated from the high spring in Yellowstone National Park at the temperatures up to 110°C in 2012 .…”

Section: Introductionmentioning

confidence: 99%

ARduino‐pH Tracker and screening platform for characterization of recombinant carbonic anhydrase in Escherichia coli

Hsu

Tan

Chiu

et al. 2019

Biotechnology Progress

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Carbonic anhydrase (CA, EC 4.2.1.1) is an ancient enzyme with zinc ion as its active site, which catalyzes the chemical reaction of carbon dioxide (CO2) to react with water and form bicarbonate ions. Due to its high catalytic efficiency on CO2 assimilation, CA is expected to use for carbon sequestration in industry. However, the protein expression level, thermostability and high‐throughput screening of an active CA are still with difficulty. In this study, the CA from Sulfurihydrogenibium yellowstonense (denoted as SyCA) was selected for overexpressed in Escherichia coli by different pET vectors. The enzymatic properties including thermo‐stability, pH tolerance, effect of metal ion, and kinetic parameters were characterized through a novel ARduino‐pH Tracker (ART) for monitoring online effectively. The SyCA is thermophilic and acidophilic as it maintains 100% activity at 50°C, while the residual activity is 34.8% after heating at 80°C for 150 min and the optimal pH is 3–5. The kinetic analysis by ART system showed that the k cat/K m of free enzyme was 4.4‐folds that that of whole cell. On the other hand, the screening platforms as Wilbur–Anderson unit, phenol red indicator and size of colony forming unit have been established to explore CA with higher activity. The high‐throughput screening platform is support in direct evolution of CA and further used in the industry.

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Section: Introductionmentioning

confidence: 99%

ARduino‐pH Tracker and screening platform for characterization of recombinant carbonic anhydrase in Escherichia coli

Hsu

Tan

Chiu

et al. 2019

Biotechnology Progress

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“…Higher catalytic activities and thermostability at high salt concentrations Lower activity at low salt concentration (Warden et al, 2015) Subtilisin protease Anionization Activity assay, thermal inactivation, pH optimization Increased proteolytic activity, thermal resistance; higher activity at lower pH N.A. (Jakob et al, 2013) Lipase Anionization Activity assay…”

Section: Surface Charge Modification Of Enzymes By Genetic Engineeringmentioning

confidence: 99%

“…Liu et al, 2017) lipase A (BsLA) contains 12 mutations, four of which involves the introduction of carboxyl amino acids to improve solvent interaction and formation of hydrogen bonds and salt bridges (Singh, Bulusu, & Mitra, 2015). Indeed, surface charge engineering is increasingly applied to enhancing enzyme properties, such as thermostability (Singh et al, 2015), IL tolerance (Nordwald, Armstrong, & Kaar, 2014), lignin tolerance (Whitehead et al, 2017), and halophilicity (Warden et al, 2015). Strikingly, the highly negative charged octuplet mutant BsLA8M displays excellent thermal stability and refolding (Y.…”

Section: Surface Charge Modification Of Enzymes By Genetic Engineeringmentioning

confidence: 99%

“…Control activity refers to the activity of the individual enzyme in the absence of added salt (adapted with permission from Warden et al, 2015). Experiments were performed in the presence of increasing concentrations of (a) Na 2 SO 4 , (b) NaCl, (c) NaNO 3 , and (d) the ionic liquid ethanol ammonium formate.…”

Section: Surface Charge Engineering By Chemical Modificationsmentioning

confidence: 99%

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Genetic and chemical approaches for surface charge engineering of enzymes and their applicability in biocatalysis: A review

Pedersen

Zhou

Guo

et al. 2019

Biotech & Bioengineering

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Surface charge engineering has received considerable interest from the scientific and industrial community in the last few decades. Although it was previously hypothesized that the surface charge-charge interactions were not a fundamental force to determine protein folding and stability, many studies today show that surface charge plays a key role determining protein structure and activity. This review aims to (a) highlight the value of surface charged engineering of proteins to improve enzyme stability and activity in aqueous media and in the presence of ionic liquids (ILs) and organic solvents, (b) describe the existing approaches (genetic engineering or chemical modifications) for surface charged engineering, and (c) demonstrate the applicability of these surface charged enzymes in biocatalysis. The review provides a new foundation for the scientific and research community to exploit the surface engineering of protein concept for the development of new enzymes that are more active and stable in the presence of ILs and organic solvents, thereby offering new opportunities for industrial biocatalysis. Furthermore, this review is a useful tool for researchers to decide the best available technology to improve their enzyme system/ process.

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“…Several computationally designed proteins with altered surface chargecharge interactions showed improvements in thermostability (2325). A rational modification of 18 surface residues of carbonic anhydrase yielded a variant with extreme halotolerance that is active at >3 M NaCl (26). A replacement of charged residues on the surface with hydrophobic residues improved stability of a protease in organic solvents (20).…”

mentioning

confidence: 99%

Protein Consensus-Based Surface Engineering (ProCoS): A Computer-Assisted Method for Directed Protein Evolution

et al. 2016

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Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: () identification of conserved and highly variable regions; () protein sequence design by substituting residues in the highly variable regions, and gene synthesis; () in vitro DNA recombination of synthetic genes; and () screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.

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Rational engineering of a mesohalophilic carbonic anhydrase to an extreme halotolerant biocatalyst

Cited by 82 publications

References 41 publications

ARduino‐pH Tracker and screening platform for characterization of recombinant carbonic anhydrase in Escherichia coli

ARduino‐pH Tracker and screening platform for characterization of recombinant carbonic anhydrase in Escherichia coli

Genetic and chemical approaches for surface charge engineering of enzymes and their applicability in biocatalysis: A review

Protein Consensus-Based Surface Engineering (ProCoS): A Computer-Assisted Method for Directed Protein Evolution

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