Ni2+-NTA columns were purchased from Sunresin New Materials Co. Ltd. (Xian, China). Coomassie brilliant blue (CBB) G-250 was purchased from biosharp. BCA Protein Assay Kit was bought from Meilunbio (Dalian, China). The carboxyl Fe3O4 NPs were purchased from Neogene Biochemical Technology (Tieling, China). N-hydroxysuccinimide (NHS), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), p-nitrophenyl acetate (p-NPA), and p-nitrophenol (p-NP) were obtained from Aladdin (Shanghai, China). PET powder (<165μm, non-crystalline) was purchased from Kingfa SCI. & TECH. CO., LTD. (Guangzhou, China). We found the protein sequence of 4M and MG8 from NCBI. The gene sequences of 4M and MG8 were codon optimized and commercially synthesized by BGI's Gene (Beijing, China), and then cloned into the NcoI and XhoI sites of pET28a vector.
4M-pET28a was transformed into E. coli BLP. MG8-pET28a was transformed into E. coli Y01. Single colonies were isolated by picking up normal growth of the colony to culture in 10mL LB medium which contained 10μL kanamycin. After culturing at 150rpm, 37℃, overnight，the bacterial solution was transferred to 100mL medium at 1:100 to enlarge cultivation, until OD600 reached 0.6-0.8. Next, expression of 4M and MG8 protein by E. coli 4M-BLP and MG8-Y01 cells were induced by addition of 0.5 mM IPTG (isopropyl-β-D-1-thiogalactopyranoside) followed by incubation for 13h with shaking at 25°C to induce expression. Next, precipitation of E. coli 4M-BLP and MG8-Y01 were collected by centrifugation. Resuspend the bacteria with Tris-HCl buffer (pH 8.0) for 4M and Tris-HCl buffer (pH 8.0) with 5M NaCl for 4M. The bacteria were broken by ultrasound (200W, 10min for 10mL). After centrifugation, the clear supernatant was collected and subjected to affinity chromatography using a Ni2+-NTA column equilibrated with binding buffer (Tris-HCl buffer, pH 8.0). Apply the supernatant to the column in binding buffer for three times, followed by washing of the column with washing buffer (10 mM, 50 mM, 100mM, 200mM, 500Mm and 1M imidazole). Flowthroughs from the column during collected. 4M and MG8 were verified using 12% SDS–polyacrylamide gel electrophoresis (PAGE) followed by staining with Coomassie Blue R-250. Ultrafiltered in addition to desalt the purified 4M and MG8. Then the final 4M and MG8 concentration were determined using a BCA Protein Assay Kit.
The enzyme activities of the 4M and MG8 were determined by spectrophotometry at 55°C and 40°C.
The reaction system was 1000μL, including 5mM pNPA 20 μL, purified enzyme 100 μL, MES buffer 850 μL (pH 7.0). The formation rate of p-NP was recorded at 405 nm wavelength. One active unit (U) of the 4M was the amount of enzyme required to hydrolyze pNPA to produce 0.036g p-NP in 60s at 55°C and that of MG8 was 1.6g at 40°C. The evaluation of enzyme activity was carried out according to the calibration curve of p-NP.
EDC/NHS activation modifies Fe3O4 NPs and covalently immobilized 4M and MG8. 80μL Fe3O4 NPs (50mg/mL) were added in 2mL MES (50mm, pH 5.5) buffer containing 5mg EDC and 4mg NHS and oscillated at 25 °C for 1h at 150 rpm. Activated Fe3O4 NPs were magnetically recycled and washed three times with MES buffer. The activated carrier was re-suspended in 2mL 4M solution (0.1 mg/mL) or MG8 solution (0.1mg/mL) and incubated in MES buffer over-night at 4 °C. The immobilized 4M and MG8 was magnetized and recycled and washed three times with MES buffer. The supernatant was collected for remaining protein concentration analysis. The concentration of the remaining enzyme in the supernatant was determined by BCA.
The sizer and ζ-potential were measured by a Malvern zetasizer (Nano ZS, Malvern Instruments, U.
K.). The carboxyl Fe3O4 NPs solution was diluted to 1mg/mL as a control. 1mL of 4M@Fe3O4 and MG8@Fe3O4 were used for measurement of sizer and ζ-potential then recycled.
In order to investigate the reusability of MG8@Fe3O4 and 4M@Fe3O4, p-NPA was repeatedly used as the substrate for hydrolysis at 55°C and 40°C. After 2 min of the reaction, MG8@Fe3O4 and 4M@Fe3O4 were isolated from the reaction system by magnetic separation, washed three times with MES buffer, and added to the new reaction system for the next cycle. The activity after each cycle indicated the enzyme activity in the first cycle.