Characterization and High-Level Periplasmic Expression of Thermostable α-Carbonic Anhydrase from Thermosulfurimonas Dismutans in Escherichia Coli for CO2 Capture and Utilization.

Byung Hoon Jo,In Seong Hwang

doi:10.3390/ijms21010103

Abstract

Carbonic anhydrase (CA) is a diffusion-controlled enzyme that rapidly catalyzes carbon dioxide (CO2) hydration. CA has been considered as a powerful and green catalyst for bioinspired CO2 capture and utilization (CCU). For successful industrial applications, it is necessary to expand the pool of thermostable CAs to meet the stability requirement under various operational conditions. In addition, high-level expression of thermostable CA is desirable for the economical production of the enzyme. In this study, a thermostable CA (tdCA) of Thermosulfurimonas dismutans isolated from a deep-sea hydrothermal vent was expressed in Escherichia coli and characterized in terms of expression level, solubility, activity and stability. tdCA showed higher solubility, activity, and stability compared to those of CA from Thermovibrio ammonificans, one of the most thermostable CAs, under low-salt aqueous conditions. tdCA was engineered for high-level expression by the introduction of a point mutation and periplasmic expression via the Sec-dependent pathway. The combined strategy resulted in a variant showing at least an 8.3-fold higher expression level compared to that of wild-type tdCA. The E. coli cells with the periplasmic tdCA variant were also investigated as an ultra-efficient whole-cell biocatalyst. The engineered bacterium displayed an 11.9-fold higher activity compared to that of the recently reported system with a halophilic CA. Collectively these results demonstrate that the highly expressed periplasmic tdCA variant, either in an isolated form or within a whole-cell platform, is a promising biocatalyst with high activity and stability for CCU applications.

Highlights

Carbonic anhydrase (CA) is a zinc-metalloenzyme that catalyzes carbon dioxide (CO2) hydration: CO2 + H2O → HCO3− + H+ [1]
The average sequence identity across the five sequences is 49.9%, while the highest identity is found between the sequences of Thermovibrio ammonificans whose α-CA (taCA) and pmCA (62.0%)
The solubility of tdCA was higher than taCA

Summary

Introduction

Carbonic anhydrase (CA) is a zinc-metalloenzyme that catalyzes carbon dioxide (CO2) hydration: CO2 + H2O → HCO3− + H+ [1]. CAs show diffusion-controlled kinetics with a kcat of up to 4.4 × 106 s−1 [2]. This feature makes them powerful and eco-friendly catalysts for bioinspired CO2 capture and utilization (CCU) which is one of the promising routes to the mitigation of greenhouse gas emissions. The hydration of CO2, which is the rate-determining step of CO2 capture into HCO3−, can be accelerated by the catalysis of CA [3]. The rapid formation of HCO3− can benefit CCU processes that utilize HCO3− as a feedstock for mineral carbonation [4,5], production of value-added chemicals [6,7], or cultivation of photoautotrophic microorganisms [8] by accelerating the reactions and reducing energy requirement, improving the efficiencies of CCU. The primary barriers to the industrial application of CA are the low stability of the enzymes and the relatively high enzyme production cost

Methods

Results

Conclusion