*3.1. Materials*

Nickel (II) acetate tetrahydrate was purchased from Yuanye Biotechnology Company. (Shanghai, China). Benzene-1, 3, 5-tricarboxylic acid (H3BTC) was purchased from J&K China Chemical Ltd. p-nitrophenyl acetate (p-NPA) and N, N-dimethylformamide (DMF) were purchased from Aladdin (Shanghai, China). Ampicillin and isopropyl β-D-1-thiogalactopyranoside (IPTG) were obtained from Beijing QXTD-Biotechnology Co., Ltd. (Beijing, China). Milli-Q water was used to prepare all the buffers and solutions. Bovine serum albumin (BSA) and methionine were purchased from Beijing Chemical Works (Beijing, China). All other reagents and solvents were of analytical grade.

#### *3.2. Expression and Purification of His-hCA II*

The human carbonic anhydrase (hCA II) gene was cloned to the pETDuet-1 vector with restriction sites BamH I and Kpn I. The recombinant plasmids were transformed into *E. coli* BL21 (DE3) for protein synthesis. The culture medium was incubated at 37 ◦C for 4 h, then isopropyl-β-Dthiogalactopyranoside (IPTG) was added for induction [37], and the culture continued at 18 ◦C for 20 h. The cells were then harvested by high-speed centrifugation (10,000 rpm, 30 min, 4 ◦C) and lysed by acoustic degradation. Subsequent cell lysate centrifugation (10,000 rpm, 30 min, 4 ◦C) removed the crude precipitate to obtain the crude protein solution and the protein concentration of the lysate was measured by the Bradford assay using BSA as a standard [38]. The protein fraction was purified with affinity chromatography using a Ni-NTA column (Qiagen) pre-equilibrated with the lysis buffer and eluted with the lysis buffer supplemented with 300 mM of imidazole. The purified protein was analyzed by sodium salt -Polyacrylamide gel electrophoresis (SDS-PAGE).

#### *3.3. Preparation of Ni-BTC*

H3BTC (0.5 g, 2.38 mmol) was dissolved in a DMF/ethanol (V/V = 1:1, 40 mL) mixture and moved to a metal reactor. Then 20 mL of nickel acetate solution (1.0725 g, 4.31 mmol) was added to the reaction mixture and stirred constantly. Then the reaction was finished after continuous stirring at 70 ◦C for 8 h. Finally, the product was separated by centrifugation and washed three times with Milli-Q water and ethanol solution (95%), successively.

#### *3.4. Assay of His-hCA II Immobilization*

#### 3.4.1. The Immobilization of His-hCA II and Determination of Activity

The activity of free and His-hCA II @Ni-BTC activity were based on p-NPA with some modifications, as described previously [10]. The 1.5 mL total reaction volume contained 600 μL of phosphate buffer (50 mM, pH 8.0), 300 μL Milli-Q water, 500 μL freshly prepared p-NPA solution (3 mM) and 100 μL crude cell lysate or immobilized enzyme which contained the same protein content of His-hCA II. The mixture was shaken at room temperature for 5 min and then centrifuged for 10 min at 12,000 rpm. The increase of absorbance was measured by employing a spectrophotometer (Metash, UV-5100, Shanghai, China) at 348 nm. The same procedure was performed without His-hCA II solution as a control.

To optimize the immobilization conditions, His-hCA II was incubated with Ni-BTC under various conditions. In detail, 100 μL cell lysate of His-hCA II (which contained 0.4 mg protein) and 900 mL Milli-Q water were incubated with Ni-BTC. For optimizing the bonding ratio between enzyme and carrier, 100 μL His-hCA II crude cell lysate (0.4 mg total protein) was incubated with different amounts of Ni-BTC in a 1 mL system. For optimization in regard to immobilization temperature, 3 mg Ni-BTC was oscillated with 100 μL His-hCA II crude cell lysate at different temperatures ranging from 20–50 ◦C for 30 min. For optimization in regard to the immobilization time, 3 mg Ni-BTC was incubated with 100 μL His-hCA II crude cell lysate at 25 ◦C over different lengths of time (15–120 min) with shaking. After separated by centrifugation, the standard curve of protein was determined using Bradford assay with BSA, then analyzed the supernatant and the original protein concentration. The activity of enzyme was detected by the above method. In all experiments, all of the experimental results were measured by three independent experimental groups.

#### 3.4.2. Enzyme Stability Detection

For the pH stability, the activity of free His-hCA II and His-hCA II @Ni-BTC was carried out in different phosphate buffer solutions (50 mM, pH 6–10) at 25 ◦C and the enzymatic activities were determined by the above method. Thermostability was also studied, free enzyme and His-hCA II @Ni-BTC were added to the phosphate buffer (50 mM, pH 8.0) over a range of temperatures, 20–50 ◦C, and incubated for 30 min. For the storage stability, the free enzyme and His-hCA II @Ni-BTC were kept in a phosphate buffer (50 mM, pH 8.0) at 25 ◦C. For the reusability of His-hCA II @Ni-BTC after the reaction, for 5 min, the immobilized enzyme was collected by centrifugation in the next cycle. Then the same substrate and buffer solution were added to the reaction system for the next cycle and the enzyme activity of each cycle was measured by the above method.

#### 3.4.3. Assay of Kinetics of the His-hCA II

For the kinetic analysis, the reaction system was detected by the same method as enzyme activity the concentrations of p-NPA were varied from 1.0 to 5.0 mM. The Km and Vmax were acquired by using a Lineweaver-Burk plot.

$$\frac{1}{\upsilon} = \left(\frac{Km}{Vm\infty}\,\,\frac{1}{[S]}\right) + \frac{1}{Vm\infty}$$
