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    We first synthesized the sequence of the mutant CA2, and then cloned it into the expression vector pET-30a(+), and identified the correctness of the obtained recombinant vector by restriction enzyme digestion and sequencing (Fig. 1 and Fig. 2).

    

Fig. 1 Map of CA2 (L203K) recombinant vector

Fig. 2 Agarose Gel Electrophoresis of CA2(L203K) recombinant plasmid and its identification by enzyme digestion （NdeⅠand Hind Ⅲ）. Lane M: DNA marker; Lane 1: CA2(L203K) recombinant plasmid; Lane 2: enzyme digestion band of CA2(L203K) , the length was 825 bp (the arrow indicated).

Induced expression of CA2(L203K)

    The CA2(L203K) expression plasmid was transformed into E. coli BL21 (DE3), and the cultured liquid was subjected to IPTG-induced CA2 (L203K) expression, and the bacterial solution was sonicated, followed by SDS-PAGE(figure 3), the size of CA2(L203K) is known to be 30.6 kDa, which is compared with Marker. The position indicated by the arrow in the figure is the CA2(L203K) band. It can be seen from lanes 1 and 2 in the figure that the IPTG condition is significant to the expression of CA2 which was induced, and it can be seen from lanes 3-6 that the induced expression of CA2 was mainly expressed in soluble form in the supernatant of the bacterial liquid. The above results indicated that we successfully obtained E. coli expressing CA2(L203K).

    

Fig. 3 SDS-PAGE analysis for CA2(L203K) cloned in pET-30a(+) and expressed in BL21(DE3) strain.

Purification of CA2(L203K) protein

    After confirming that CA2(L203K) can be induced by E. coli BL21(DE3), we will further purify the crude protein extract by nickel column purification to obtain purified CA2(L203K) protein. Figure 4 shows the results. We have obtained a highly purified mutant CA2 protein.

    

Fig. 4 SDS-PAGE and Western blot analysis of CA2(L203K). Lane 1: Negative control; Lane 2: purified CA2(L203K) protein

Determination of protease activity of CA2 and CA2 (L203K)

    We determined the enzymatic activities of wild-type and mutant CA2 by colorimetric and esterase methods. As shown in Figure 5 and Figure 6, mutant CA2 (L203K) has higher enzymatic activity than wild-type CA2.

    

Fig. 5 Colorimetric assay of CA2 activity

    

Fig. 6 Esterase activity analysis of CA2 protein

Analysis of Thermal Stability of CA2 and CA2 (L203K)

    We examined the activity of carbonic anhydrase in wild-type and mutant CA2 at different times and temperatures by esterase method. The results are shown in Figure 7. As the temperature increases, especially at 55 ° C and 65 ° C, The enzymatic activity of wild type CA2 was significantly decreased, while the mutant CA2 still had higher activity, indicating that CA2 (L203K) has better thermal stability.

    

Fig. 7 Activity of purified CA2-WT and CA2 (L203K) under indicated temperatures and time points.