Biological carbon fixation is a feasible strategy to reduce atmospheric carbon dioxide levels (CO2). In this platform, carbonic anhydrase (CA) enzyme is employed to accelerate the sequestration of CO2. The present work explored the effect of chaperone GroELS and TrxA-tag on improving soluble expression of the recombinant Sulfurihydrogenibium yellowstonense CA which activity and biomineralization capability were taken into consideration. At first, the expression of GroELS using the inducible T7 promoter and constitutive J23100 promoter were investigated. The results indicated that 1.4 folds increment of soluble protein and 100% of CA activity enhancement were achieved with GroELS co-expression driven by J23100 promoter. Furthermore, the involvement of TrxA fusion tag displayed a significant enhancement of soluble protein production which was about 2.67 times higher than that of original SyCA. Besides, co-expression with GroELS intensified the thermostability of SyCA at 60°C owing to changes in the structural conformation of the protein, which was proved by an invitro assay. The SyCA was further entrapped and immobilized into polyacrylamide gel (i.e., PAGE-SyCA). The biomineralization capability of the PAGE-SyCA and whole-cell (WC) was compared in a two-column system. After 5 cycles of reuse, PAGE-SyCA maintained 29.8% activity and formed 774mg of CaCO3 solids in the B::JG strain. This study presents the recombinant engineering strategies to improve SyCA productivity, activity, thermostability, and effective carbon dioxide conversion.

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