Magnetically responsive horseradish peroxidase@ZIF-8 for biocatalysis

A biocatalytic system based on the zeolitic imidazolate framework-8 (ZIF-8) is obtained in a one-pot process by directly combining the enzyme horseradish peroxidase (HRP), iron oxide magnetic nanoparticles, the ligand and metal ions, in water at room temperature. The resulting system provides a useful platform for the next generation of reusable/repositionable biocatalysts.

. Time-dependant yields for scale-up synthesis  Table S2. Data of loading and enzymatic activity of HRP in the composites  Table S3. Literature comparison of the catalytic activities of HRP-immobilized MOF composites  References Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2020 at 90 °C, then solid trisodium citrate (8.80 g, 0.030 mol) was added in one step and the stirring continued for additional 30 minutes at the same temperature. After removal of the stir bar and natural cooling at room temperature, the dark brown mixture was transferred in a cylindrical container, and a strong ND45 magnet (fitted into a glass tube) was immersed at the approximate centre of the solution for 5 minutes to collect the MNPs. The liquid part was removed and replaced by deionized (DI) water, then the magnet extracted. The suspension was briefly sonicated in a bath for 10 minutes, and the magnetic collection repeated. The process was repeated with water (3 x 100 mL), ethanol (3 x 100 mL) and water again (3 x 100 mL), then the suspension diluted to 250 mL and stored at room temperature in a polythene bottle. Before use, few millilitres were collected, the water removed and the MNPs dried under vacuum at 60 °C overnight, then re-suspended to the desired concentration.

Synthesis of ZIF-8 and MNP@ZIF-8
The method has been adapted from literature. 3 The procedure involves the preliminary preparation of three stock solutions, all in DI water: MNP suspension is prepared at a concentration of 6 mg/mL, and sonicated in a bath for 15 minutes to ensure homogeneous dispersion; zinc acetate stock solution is prepared with a concentration of 240 mM, whereas HmIm stock solution is prepared with a concentration of 1.92 M. To ensure the constant reproducibility of the sodalite (sod) phase, the metal-to-ligand ratio is kept as 1:16, 4 their final concentrations being 40 mM and 640 mM, respectively. The amount of MNP is varied within a final concentration range of 0 ÷ 1 mg/mL, precisely 0.33, 0.67, and 1.00 mg/mL. For pure ZIF-8, the MNP is omitted and substituted with an equivalent amount of DI water. In a typical procedure, HmIm stock (600 µL), MNP stock (0, 100, 200, or 300 µL) and water (filling up to the 1.5 mL volume mark) where mixed in a 2 mL plastic vial with cap and briefly vortexed for few seconds. Zn acetate stock (300 µL) is then added: the solution containing MNPs becomes milky almost instantaneously, whereas for pure ZIF-8 this will occur in approximately 20 minutes. The mixture is briefly vortexed one more time for few seconds, and the vial put in a tube rotator (20 rpm, 16 hours).

General workup procedure
The reaction mixture is centrifuged (Eppendorf Minispin with rotor F-45-12-11, 12000 x g, 60 sec) and the supernatant separated for further analysis. An aqueous solution of SDS (1 % w/v, 1 mL) is added and the pellet vortexed until dispersion, with the aid of a brief step of sonication in bath (3-5 sec) if necessary. The suspension is left in the tube rotator for 2 hours, then centrifuged again (12000 x g, 60 sec). After removing the supernatant, the pellet is washed with water (3 x 1 mL), vortexed, and centrifuged as previously described. The pellet is then re-dispersed in water (or a suitable buffer), and stored at room temperature (or 4 °C if enzyme is present). The samples were stored in water because ZIF-8 is highly hydrophobic in its dried form. 5,6 A portion (300 µL) of suspension is placed in pre-weighed vials and centrifuged (12000 x g, 5 min), the supernatant discarded and the pellet left to dry at room temperature overnight. The solid is weighed again to determine the amount of material. The final amount of composite is eventually adjusted to 10 mg/mL.

Synthesis of HRP@ZIF-8
The same procedure for the synthesis of MNP@ZIF-8 is followed, but using a stock solution of HRP in DI water (6 mg/mL) in place of the MNP suspension, with a final concentration in the range 0 ÷ 1 mg/mL, precisely 0.33, 0.67, and 1.00 mg/mL (0, 100, 200, 300 µL). The general workup protocol is then applied.

Synthesis of HRP/MNP@ZIF-8
The same procedure for the synthesis of MNP@ZIF-8 is followed, adding a stock solution of HRP in DI water (6 mg/mL) along with the MNP suspension, with a final concentration in the range 0 ÷ 1 mg/mL, precisely 0.33, 0.67, and 1.00 mg/mL (0, 100, 200 300 µL). Before adding HmIm, the suspension is left standing for few minutes. The general workup protocol is then applied.

Scale-up synthesis
In a 100 mL glass bottle with cap and magnetic stirring, 30 mL of BSA or HRP solution (3 mg/mL), 15 mL of MNP suspension (6 mg/mL) and 30 mL of 2 mIm solution (1.92 M) were stirred for one minute, then 15 mL of Zn acetate solution (240 mM) were added in one step. If protein and/or MNP were omitted, water was added as replacement. The reaction mixture become turbid within one minute. After 18 hours, the mixture was centrifuged (3750 rcf, 10 minutes) and washed three times with water (ca. 30 mL each time), and dried at room temperature overnight, then in a vacuum oven (ca. 200 mbar) at 60 °C to determine the weight and the yield.

Bradford assay 7
A calibration curve with HRP in water was determined in the 0 ÷ 1 mg/mL range. The resulting equation is: a.] -0.00268 where A is the absorbance and [p.a.] the protein amount in mg/mL (see Figure S10). To perform the assay, the supernatant of the reaction mixture after centrifugation (20 µL) is mixed with the Bradford Reagent (600 µL) in a plastic cuvette and the absorbance at 595 nm measured with a Nanodrop One C (Thermofisher Scientific Inc.). The quantity is then calculated by difference with the initial amount of protein present in the reaction medium (measured with the Bradford assay as well) and then applying the above equation. Protein loading in mg/g was determined from the amount of composite used in the assay.

Enzymatic activity
The assay is performed using pyrogallol and hydrogen peroxide as substrates. 8 Activity is determines by monitoring the purpurogallin formation via UV-Vis spectrophotometry (λ = 420 nm). The assay procedure was modified by replacing PBS buffer with DI water (see Figures S11 and S12). 9 In a typical procedure, HRP@ZIF-8 (or ZIF-8), is suspended in DI water (10 mg/mL) and left to mix on a tube rotator (20 rpm, 2 hours) to ensure homogeneity. 100 µL of the biocomposite suspension (1 mg), is added to 2420 µL of DI water, 160 µL of hydrogen peroxide (0.5 % v/v), and 320 µL of pyrogallol (50 mg/mL, in DI water) in a 4 mL cuvette with stirring bar, monitoring the increase of the absorbance at 420 nm within 10 minutes. The enzymatic activity obtained by the assay is expressed as U mL -1 , where U is defined as µmol min -1 substrate converted (pyrogallol to purpurogallin) at the defined reaction conditions. From this, it is possible to calculate the specific activity of the enzyme bound, by applying the formula: U mg bound HRP -1 = U mL -1 / mg mL -1 HRP (referred to the HRP in the composites, knowing the mg of enzyme per g of composite as previously determined).

Recyclability test
The assay is performed in DI water. The final volume is 1 mL. In a typical procedure, 100 µL of HRP@ZIF-8 or HRP/MNP@ZIF-8 suspension (20 mg/mL) are added to a mixture containing 420 µL of DI water and 160 µL of hydrogen peroxide (0.5 % w/v). After adding 320 µL of pyrogallol (50 mg/mL), the reaction is left for 15 minutes, then the suspension is centrifuged (12000 x g, 60 sec) or collected with a magnet for 2 minutes, and the absorbance of the supernatant measured at 420 nm. The material is washed and centrifuged three times with water before every cycle.

Recirculator setup
The system is illustrated in the image below, along with a photo of the real setup taken at the Elettra synchrotron: A 50 mL plastic reactor (1) with magnetic stirring (2) is charged with an aqueous solution containing HmIm, the enzyme and/or the magnetic nanoparticles. The reactor has an inlet (3), an outlet drawing out from the bottom of the reactor (4), and an additional inlet (5) for the injection (6) of the zinc acetate solution at the given time. The reactor is then connected with plastic tubing to a quartz capillary (7) put on the path of the synchrotron beam (8), with SAXS (9) and WAXS (10) detectors downstream the beam. The recirculation of the liquid is ensured by a peristaltic pump (11), withdrawing the liquid from the capillary. The green arrows illustrate the direction of flow in the system; the peristaltic pump is set so the mixture takes approx. 10 seconds to reach the capillary. The tubing volume is ca.            Legend: EE = encapsulation efficiency SA = Specific activity of the encapsulated enzyme (U mg immobilized enzyme -1 ) SAc = Specific activity of the whole composite (U g material -1 ) ƞ = effectiveness factor: 100 x (U mg immobilized enzyme -1 ) / (U mg free enzyme -1 ). 10 Legend: U/mg f = Units per mg of free enzyme U/mg i = Units per mg of immobilized enzyme U/g m = Units per g of material ABTS = 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oPDA = o-phenylenediamine PBS = phosphate buffer saline cont. = continuous process disc. = discontinuous process Notes: *: The Specific activity (in bracket) was calculated from k cat , considering HRP mol. wt of 44.000; **: Apparent Specific activity of GOx & HRP.