**4. Conclusions**

In conclusion, a simple and effective single pot immobilization strategy was developed to isolate histidine marker enzymes expressed in *E. coli* BL21 (DE3). Ni-BTC could be easily compounded by a hydrothermal process and showed the ability to efficiently immobilize the His-hCA II from cell lysate at high load and high activity recovery under optimal conditions. The His-hCA II @Ni-BTC exhibited excellent biocatalytic activity and reusability in the hydrolysis of p-nitrophenyl acetate. All of these results revealed the good potential of His-hCA II @Ni-BTC for biomineralization of CO2.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4344/10/4/401/s1, Figure S1: XPS spectra of Ni-BTC nanorods: (a) full scan, (b) Ni 2p, (c) N 1s, Figure S2: TG curves of Ni-BTC, Figure S3: The plasmid map of pETDuet-1-His-hCA II, Figure S4: SDS-PAGE of purified the recombinant hCA II from the culture supernatant. (Lane 1: His6-tagged hCA II purified using Ni-NTA column, Lane 2: supernatant of hCA II cell lysate, Lane 3: molecular weight marker), Figure S5: The protein shedding rate of immobilized enzyme at different pH and (B) the protein shedding rate of immobilized enzyme at different temperatures, Figure S6: The catalytic activity of His-hCA II@Ni-BTC and free enzyme at the same protein concentration was studied at 40 ◦C and 50 ◦C, Figure S7: The protein shedding rate of cycle process. Table S1: The amount of immobilized His-hCA II, Table S2: Michaelis-Menten kinetics parameters of immobilized and free enzymes.

**Author Contributions:** Conceptualization, methodology, formal analysis, validation, writing manuscript, and supervision, project management, H.L. Review, F.V. H.L. Experimental work and draft writing, M.J. Other auxiliary work, J.H., S.S., Q.Y., Y.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** This work was supported by the National Natural Science Foundation of China (No. 21606014 and 21878014) and the Double First-rate Program (No. ylkxj03).

**Conflicts of Interest:** The authors declare no competing financial interests.
