Engineering de novo disulfide bond in bacterial α-type carbonic anhydrase for thermostable carbon sequestration

Abstract

Exploiting carbonic anhydrase (CA), an enzyme that rapidly catalyzes carbon dioxide hydration, is an attractive biomimetic route for carbon sequestration due to its environmental compatibility and potential economic viability. However, the industrial applications of CA are strongly hampered by the unstable nature of enzymes. In this work, we introduced in silico designed, de novo disulfide bond in a bacterial α-type CA to enhance thermostability. Three variants were selected and expressed in Escherichia coli with an additional disulfide bridge. One of the variants showed great enhancement in terms of both kinetic and thermodynamic stabilities. This improvement could be attributed to the loss of conformational entropy of the unfolded state, showing increased rigidity. The variant showed an upward-shifted optimal temperature and appeared to be thermoactivated, which compensated for the lowered activity at 25 °C. Collectively, the variant constructed by the rapid and effective de novo disulfide engineering can be used as an efficient biocatalyst for carbon sequestration under high temperature conditions.

FoldX as Protein Engineering Tool: Better Than Random Based Approaches?

“…Using FRESCO, a temperature improvement of 35 °C was achieved due to the combination of single disulfide bridges. Jo et al increased the T m of the α-type carbonic anhydrase by 7.8 °C due to an introduction of a disulfide bond efficiently predicted by DD2 [ 60 ]. Albeit the promising examples, it has to be mentioned, that the extensive FRESCO strategy cannot be understood as an end-user script, but more or less as a blue script for improving thermostability.…”

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

“…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 .…”

“…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, 21 or to engineer protein by genetic approach. 22,23 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. 21 The CA from S. yellowstonense (donated as SyCA) has extremely heat resistance and activity, which can maintain around 60% activity for 3 hr at 100 C. This provides the opportunity of SyCA apply in CCS.…”

Crystal Structure of a Highly Thermostable α-Carbonic Anhydrase from Persephonella marina EX-H1

“…2A ). The disulfide bridge provides conformational and thermal stability by increasing structural rigidity, as supported by previous cysteine mutagenesis data showing that addition of an intramolecular disulfide bond markedly enhances the thermostability of hCAII and Neisseria gonorrhoeae NgCA ( Boone et al, 2013 ; Jo et al, 2016 ; Mårtensson et al, 2002 ).…”