Engineering of Thermovibrio ammonificans carbonic anhydrase mutants with increased thermostability
Introduction
The development of technologies to mitigate CO2 release from human activities is of utmost importance for the well-being of our planet. Amongst these is the capture of CO2 from industrial processes, to be further stored or utilized [1]. The removal of CO2 from flue gases can be done by either absorption or adsorption, with the former employing basic aqueous solvents. In this context, the enzyme carbonic anhydrase (CA; EC 4.2.1.1) has been used to accelerate the hydration of CO2 during absorption in the liquid phase. Carbonic anhydrase is a ubiquitous metalloenzyme that maintains the balance of CO2 / HCO3- in cells, by the reversible hydration of carbon dioxide into bicarbonate ions. This function is important in processes of carbon fixation, which use metabolic pathways with either CO2 or bicarbonate as the carbon source [2]. Given their functionality, CAs have been used in carbon capture processes, to increase the efficiency of CO2 absorption and biomineralisation [3]. One example is the acceleration of the reaction rate during reactive absorption of CO2 by alkaline solvents such as amines or K2CO3 solutions, which results in increased absorber loading capacity and therefore has the potential to improve the energy efficiency of the carbon capture process [[4], [5], [6]]. On the other hand, the application of CAs in large scale industrial flue gas clean-up requires increased protein stability at the process conditions [7,8]. For this reason, many efforts have been directed to engineer carbonic anhydrase with increased tolerance to high temperatures [9], alkaline conditions [10] and high salt concentrations [11,12].
To meet the stability requirements, interest was developed in bioprospecting for thermostable CAs that dwell in ocean vents, soil and hot springs [13]. Thermophilic CAs from the α family, discovered over the past decade, have been shown to have some of the highest activities and thermal stabilities known to date (Table 1). In particular, CA from Thermovibrio ammonificans (TaCA hereafter) has been identified as a highly robust enzyme, with a half-life of ∼ 150 days at 40 °C [14]. The increased thermostability of this enzyme was attributed to the presence of an inter-subunit disulfide bond formed between two cysteines at position 67, which promoted the organization of this protein in a tetrameric structure [15]. Further engineering of TaCA by directed evolution led to a 3-fold improvement in the half-life compared to the wild-type (WT) recombinant TaCA in 1.45 M K2CO3 at pH 10 and > 70 °C, using mutations in the N-terminal region of the protein [16]. In the same α-CA family, genetic engineering of mesostable CAs has also been used to increase thermostability. For example, the thermostability of α-CA from Neisseria gonorrhoeae (NgCA) was 8-fold increased by engineering disulfides at the protein surface, whilst human carbonic anhydrase II (hCAII) was stabilized by either proline substitution, surface loop engineering or disulfide bond design [9,17,18].
In a previous theoretical study, we have shown how the high thermal stability of α-CAs was related to the high rigidity of their structures [19]. A systematic investigation of flexible sites within TaCA using molecular dynamics (MD) simulations led to the design of four mutants, which were suggested to stabilize the TaCA structure even further and therefore lead to ultra-thermostability. In the present contribution, we report on the experimental preparation of the previously designed TaCA mutants and on their activities and stabilities at high temperatures. Out of the four rigidifying mutations, two showed improved thermostability parameters compared to WT, confirming the predictions of the MD simulations (Table 1). Additionally, mutant C67 G was also investigated, where the inter-subunit disulfide bond was absent, in order to assess the impact of this feature on thermostability.
Section snippets
Cloning and site directed mutagenesis
The gene encoding for wild-type Thermovibrio ammonificans carbonic anhydrase (WT TaCA) without the N-terminal signal peptide sequence was synthesized by Integrated DNA Technologies, USA, with the codon usage optimized for E. coli using the GeneOptimizer software. The gene was cloned between the NdeI and XhoI restriction sites of the expression vector pET19b (Novagen inc., Life Science research, Washington, DC, USA), which confers a polyhistidine tag sequence at the N-terminus. The resulting
Design, expression and catalytic activity of TaCA variants
Previous molecular dynamic simulations showed that TaCA possessed increased flexibility at certain regions [19]. Rigidifying mutations were designed at flexible sites (Fig. 1), containing either stabilising amino acids or disulfide bonds. Four mutants with increased overall protein rigidity were further characterized by molecular dynamic simulations and showed a similar flexibility of the active site residues, when compared to WT TaCA: N140 G, T175 P, A242 P and P165C-Q170C. Based on this, we
Conclusions
The development of carbonic anhydrases with very high thermal stability is one important requirement for their implementation in post combustion carbon capture processes. We have previously used molecular dynamics simulations to rationally design stabilized mutants of α-carbonic anhydrase from Thermovibro ammonificans. Here, we produced these mutants in E. coli, and we evaluated their catalytic activities and thermostability parameters using CO2 hydrolysis and esterase assays. Remarkably, the
Declarations of interest
None.
Acknowledgements
The research was funded by the Malaysian Ministry of Higher Education through the Fundamental Research Grant Scheme, project no. FRGS/1/2014/TK05/UNIM/02/2. RPC was funded by a PhD studentship from the University of Nottingham. RPC and AP gratefully acknowledge support received from the University of Nottingham Research Beacon of Excellence: Green Chemicals.
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