Page 129 - IJB-9-3

International Journal of Bioprinting Programmable formaldehyde dehydrogenase for biodegradation formaldehyde

(CaCl ; AR, SINO Chemical), phosphate-buffered saline and 160 mg SA (1.6 wt%) was added to the supernatant
2
(PBS; Solarbio), β-nicotinamide adenine dinucleotide and stirred to mix well. CaCl (7.4 wt%) was prepared for
2
trihydrate (β-NAD ; Sangon Biotech), formaldehyde crosslinking.
+
solution (CH O; AR, HUSHI), ammonium acetate 2.6. 3D-printed PFDH/CA polymer
2
(CH COONH ; AR, HUSHI), acetylacetone (C H O ; AR, Direct ink writing method was adopted to print PFDH/
5
3
4
2
8
Aladdin), and acetic acid (CH COOH; AR, XILONG). CA microspheres in a layer-by-layer manner (Figure S2).
3
Deionized water was used throughout the experiment.
The needles with the diameters of 0.21 mm, 0.34 mm, and
2.2. Cloning and expression of the formaldehyde 0.41 mm were selected. To print PFDH/CA microspheres
dehydrogenase with uniform ink, the appropriate pressure is regulated
The GeneScript (NanJing, China) synthesized and by computer to suit different needle diameters, i.e., 0.038
cloned the PFDH gene into the pET-28a (+) vector. The MPa for the 0.21-mm needle, 0.034 MPa for the 0.34-mm
constructed plasmid was introduced into E. coli BL21 needle, and 0.027 MPa for the 0.41-mm needle. The printed
(DE3). The bacterial cells were cultured in 10 mL LB PFDH/CA microspheres were crosslinked by 7.4 wt%
medium for 12 h, then cultured in 200 mL LB medium at CaCl for 8 min. The prepared PFDH/CA microspheres
2
37°C for 4 h, and finally induced with 0.5 mM isopropyl were then used in the subsequent experiments.
β-D-1-thiogalactopyranoside (IPTG) and cultured at 20°C 2.7. Recycling performance test of
for 20 h. After the culture, the cells were suspended in the immobilized PFDH
cold PBS (1×, pH 7.2–7.4). The cells were lysed and the Immobilized PFDH/CA microspheres with different
crude enzyme solution was collected after centrifugation at diameters (1.5 mm, 2.5 mm, and 3.5 mm) produced by 3D
11,000 rpm for 35 min. Pure enzyme was obtained by Ni - printing technology were tested for the number of cycles in
2+
Sepharose affinity column (1.5 × 8 cm, Smart-Lifesciences, the same reaction system. The PFDH activity was measured
Changzhou, China). The purity and concentration of using the method described above, and the highest enzyme
PFDH were determined by sodium dodecyl-sulfate activity was selected as 100% at the first cycle. The relative
polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme activity was calculated. The above experiment has
Bradford method. been repeated three times.
2.3. Enzyme assay 2.8. Catalyst characterization
In the presence of PFDH, formaldehyde is biodegraded The surface morphology of PFDH/CA microspheres was
to formic acid and produces NADH and H , in which observed by scanning electron microscopy (SEM, Hitachi,
+
β-NAD is required . PFDH activity reaction system (1.47 SU5500). The dried sample is placed on conductive
[36]
+
mL):formaldehyde (0.29 mM): Formaldehyde solution (0.29 adhesive and sprayed with gold. Testing was carried out at
mM, 60 μL), glycine-NaOH (50 mM, pH = 8.9, 0.97 mL), the end of sample preparation. Fourier transform infrared
β-NAD+(5.7 mM, 0.25mL). About 200 μL of the reaction spectrometer (FT-IR) was used to analyze the changes
mixture was transferred into a 96-well microtiter plate and of chemical bonds or functional groups in samples. The
the absorbance at 340 nm was measured using a microplate samples were placed in the diamond ATR module to
reader. The enzyme activity was calculated based on the carry out FT-IR (Nicolet iS5) test, with a wavelength
above conditions. The activity of PFDH as: the amount range of 4000–400 cm . The number of scans was 32,
−1
of enzyme required per minute to reduce or oxidize 1 μL and the resolution was 4 cm . The samples were put on
−1
coenzyme NADH is defined as a unit of enzyme activity. the conductive adhesive. The morphologies of PFDH/CA
The maximum enzyme activity was defined as 100% and microspheres and the corresponding energy dispersive
the relative enzyme activity was calculated. X-ray (EDS) elemental maps were obtained by using SEM
(TESCAN MIRA LMS). We used the Oxford Quorum
2.4. Optimization of immobilization conditions SC7620 sputtering coating by gold. The morphology
for PFDH of the sample was characterized at the voltage of 3 kV,
In this experiment, we first investigated the effects of SA and the spectra of C, N, Zn, Ca, and O elements in the
concentration, CaCl concentration, crosslinking time, and samples were analyzed at 15 kV. The thermal degradation
2
reaction temperature on PFDH activity through single-factor behaviors of the CA control and PFDH/CA microspheres
experiments. Then, the optimal printing conditions were were investigated by using Rigaku TG-DTA 8122
analyzed by RSM on the basis of single-factor experiments. thermogravimetric analyzer in a temperature range of
[37]
2.5. Ink preparation 30°C–800°C at a constant heating rate of 10°C/min.
The ink for 3D printing was prepared based on the results of Compressive stress–strain curve was obtained by
RSM analysis. The 0.4 mg/L PFDH solution was prepared, compression test with Texture Analyzer (TA, SMA) to

Volume 9 Issue 3 (2023) 121 https://doi.org/10.18063/ijb.695