Engineering of Thermovibrio ammonificans carbonic anhydrase mutants with increased thermostability

Highlights

• Thermostable mutants of carbonic anhydrase from Thermovibrio ammonificans were prepared and characterized.

• Four mutants were identified with improved stability at 90 ᵒC, compared to the wild-type enzyme.

• Ultra-thermostable mutant N140 G exhibited a 3-fold increased time of half-life at 60 ᵒC.

• The most thermostable mutants showed a significantly enhanced esterase activity at high temperatures, up to 95 ᵒC.

Abstract

Carbonic anhydrase can be used as an additive to improve the efficiency of carbon capture and utilisation processes, due to its ability to increase the rate of CO₂ absorption into solvents. Successful industrial application requires robust carbonic anhydrases, able to withstand process conditions and to perform consistently over long periods of time. Tolerance of high temperatures, pH and salt concentrations are particularly desirable features. We have previously used molecular dynamics simulations to rationally design four mutants of Thermovibrio ammonificans carbonic anhydrase with increased rigidity, and we hypothesized that this will result in an increased thermostability. Herein, we report on the successful recombinant expression and characterization of these mutants. Four of the TaCA variants showed increased stability at 90 ᵒC during 1 h, compared to wild-type. Two out of the four mutations predicted by the theoretical studies resulted in marked stabilization of the protein, with up to 3-fold higher time of half-life for mutant N140 G compared to the wild-type enzyme at 60 ᵒC. A significantly 50-fold increased ester hydrolysis activity was also observed with the most thermostable variant at 95 ᵒC compared to 25 ᵒC, suggesting an increased flexibility of the active site at high temperatures.

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.