Protocols

1. Strains and plasmids

The amino acid sequence of CA was obtained from the genome sequence of Vibrio salmonicida, a strain of bacteria from the ocean. The nucleotide sequence coding the mature enzyme was optimized for expression in E. coli BL21(DE3) and expressed using the pETDuet-1 vector that contains the ColE1 as the origin of replication, an ampicillin resistance gene as the selection marker, the T7 promoter and the lac operon. The constructed plasmid was transformed into E. coli BL21(DE3) by receptive state transformation.

2. Gene cloning and expression vector construction

mASCA gene was amplified by PCR reaction system with 50μL solution using the synthesized mASCA gene as template. The reaction system is as follows:

	μL in 1 reaction
Mix(2×)	25μL
pR	1μL
pF	1μL
bacteria solution	3μL

Thermocycling Conditions For PCR:

	Temperature	Time	Cycle
Predenaturation	95℃	5min	1
Denaturation	95℃	30s	30
Annealing	58℃	30s	30
Extension	72℃	1min	30
Final Extension	72℃	10min	1
Hold	4℃	∞

The PCR products were examined by electrophoresis on 1% agarose gel.

The PCR product and plasmid pETDuet-1 were purified by DNA purification kit and double digested with BamH Ⅲ and EcoR Ⅰ . The reaction system was as follows:

	μL in 1 reaction
Gene	20μL
10× NB	5μL
BamH Ⅲ enzyme	1μL
EcoR Ⅰ enzyme	1μL

Rerformed at 37℃ for 1h
Terminated at 80℃ for 20min
Digested products were recovered by DNA purification kit and quantified
Products were reacted with T4 DNA ligase at room temperature for 30min or overnight at 16℃
Transformed into competent E. coli BL21(DE3) cells
Cultured overnight at 37℃ by LB(Amp+) screening plate

3. Determination of the activity of carbonic anhydrase

A single colony from the above LB(Amp+) screening plate was cultured in 100mL LB(Amp+) liquid medium for 12h
IPTG was added to induce expression for 12h
Lysozyme-freeze-thaw method was used to extract crude enzyme solution
- 10ml of bacterial solution was centrifuged at 5000rpm for 5min, discarded the supernatant
- The bacterial cells were resuspended in 0.01m PBS buffer and centrifuged at 5000rpm for 5min
- Add 5mL of 0.01M PBS buffer and 100 μl of 20mg/mL lysozyme solution
- React at 4℃ for 30min
- Freeze at -80℃ for 30min and then thaw at 4℃, repeat until the thawed liquid becomes sticky obviously
- Centrifuge at 12000rpm for 10min
- Collect supernatant for enzyme activity determination
Carbonic anhydrase activity was determined by p-NPA method.

Measuring the absorbance value at 348nm to calculated the enzyme activity.

Successively adding 5.4mL of fresh Tris buffer (100mM, pH7.6), 3mL of free enzyme, and 0.6mL of p-NPA dissolved in acetone (3mM) to a centrifuge tube.
React for 3minthe
Absorbance value at 348nm was measured by UV spectrophotometer, and the sample without enzyme was used as blank control. Relative enzyme activity was defined as the percentage of the ratio of any enzyme activity to the optimal enzyme activity under the same conditions.

4. Error prone PCR amplification

The reaction systems with mASCA template concentrations of 1ng/μL and 10ng/μL were set as follows:

	μL in 1 reaction
mASCA	1μL
Mix	3μL
dNTP	3μL
dGTP	2μL
MnCl₂	3μL
pR	1μL
pF	1μL

Thermocycling Conditions For PCR:

	Temperature	Time	Cycle
Predenaturation	94℃	3min	1
Denaturation	94℃	1min	45
Annealing	45℃	1min	45
Extension	72℃	1min	45
Hold	4℃	∞

The error-prone PCR products are examined by electrophoresis on 1% agarose gel.

5. Construction and of mutation libraries

Error-prone PCR product and plasmid pETDuet-1 were purified by DNA purification kit and double digested with BamH Ⅲ and EcoR Ⅰ . The reaction system was as follows:

	μL in 1 reaction
Gene	20μL
10× NB	5μL
BamH Ⅲ enzyme	1μL
EcoR Ⅰ enzyme	1μL
ddH₂O	38μL

Reaction was performed as follows:

Rerformed at 37℃ for 1h
Terminated at 80℃ for 20min
Digested products were recovered by DNA purification kit and quantified
Products were reacted with T4 DNA ligase at room temperature for 30min or overnight at 16℃
Transformed into competent E. coli BL21(DE3) cells
Cultured overnight at 37℃ by LB(Amp+) screening plate

6. Determination of enzyme activity of recombinant enzymes

Steps are same as “Determination of the activity of carbonic anhydrase”

7. Bioinformatics analysis

We used primers pR/pF to PCR recombinant strains exhibiting higher enzyme activity, and the PCR products were sent to Sangon Biological Company for sequencing. The gene sequence analysis and 3D structure prediction of mASCA was conducted with Phyer2 protein structure server. After finding the populations with high sequence similarity using Blastp server in NCBI, the sequences were aligned and the tree was built using the neighbor joining method in software Mega.