Page 130 - IJB-9-3

International Journal of Bioprinting Programmable formaldehyde dehydrogenase for biodegradation formaldehyde

study the mechanical properties of 3D-printed PFDH/CA a protein composed of 399 amino acid residues with a
microspheres. calculated molecular weight of 42082 Da. After SDS-
PAGE, the purity of the protein was also determined to be
2.9. Swelling analysis 81% by ImageJ software and was ready for use in further
The swelling efficiency refers to the hydration ability experiments.
and stability inside the biological systems . In order to
[38]
calculate the swelling rate of 3D-printed samples, samples 3.2. Optimization of the conditions of
of different sizes were freeze-dried or dried. Then, the immobilized PFDH
samples were incubated at 4°C in 1 mL PBS for 5 min, 0.5 h, In this study, we studied the biocompatibility of SA
1 h, 4 h, 24 h, and 48 h, respectively. The swollen weight of before performing other experiments. Then, the effect
the samples was measured after different time periods. The of four factors (SA concentration, CaCl concentration,
2
formula for calculating swelling rate is as follows: crosslinking time, and temperature) on the relative activity
Wwet Wdry of PFDH was explored by single-factor experiments.
Swelling ratio % 100 (I) Finally, the optimal printing conditions to immobilize
Wdry
PFDH by RSM were selected. We mixed 2 wt% SA with
2.10. Formaldehyde decomposition by PFDH /CA the PFDH solution for 6 h to study the biocompatibility of
microspheres SA. The relative enzyme activity of PFDH was measured
The 3D-printed PFDH/CA microspheres with a diameter 1 h per cell, and the maximum enzyme activity was defined
of 2.5 mm were put into a reaction system containing 50 μg as 100%. Based on the results shown in the Figure S1, the
formaldehyde in 250 mL system. The concentration of relative enzyme activity maintained 96% after incubation
formaldehyde in the reaction system was measured at the for 6 h. This indicates that the biocompatibility of PFDH
interval of 10 min, 30 min, 1 h, 2 h, 4 h, and 24 h. The acetyl and SA is excellent, which can be further studied. Therefore,
acetone method was applied for the measurement . The single-factor experiments were conducted to explore
[39]
experiment was repeated three times. the effects of CaCl concentration, SA concentration,
2
crosslinking time and temperature on PFDH activity. The
2.11. Expansion of 3D printing immobilization results are presented in Figure 1. With the increase of the
technology concentration of SA and CaCl , we observed an initial
2
As the first step in translating the method into industrial increase in the activity of PFDH, which was followed by
application, we first attempted to encapsulate different a reduction. In the SA concentration range of 0–4 wt%,
objects Of course, they all function to biodegrade the enzyme activity of PFDH showed an increasing trend,
formaldehyde, including recombinant E. coli expressing followed by a decreasing trend. When 8 wt% SA was used,
formaldehyde dehydrogenase protein, crude enzyme the relative enzyme activity of PFDH was maintained
solution by crushing and centrifuging recombinant E. coli at 81%. Similar trend was also observed for the effect of
to remove precipitate, and purified PFDH solution. Based CaCl on PFDH activity: when the concentration of CaCl
2
2
on optimal printing conditions, we printed pure enzyme/ increases to 10 wt%, the relative enzyme activity of PFDH
CA microspheres, crude enzyme/CA microspheres, and E. is 82%, and the maximum enzyme activity is reached when
coli/CA microspheres to explore reusability performance. the CaCl concentration is 6 wt%. The results of the other
2
The diameter of the printing needle is 0.34 mm, and the two factors showed that the most suitable crosslinking
printing pressure is 0.035 MPa. The highest enzyme activity time is 7 min, and the most suitable reaction temperature
was designed to be 100%, and the relative enzyme activity is 30°C. The Na on α-L-guluronic acid (G) unit reacts
+
was calculated. The experiment was repeated three times. with divalent cation in ion exchange reaction, and G unit
Then, we used CAD to build the model we wanted to print. accumulates to form crosslinked network structure, thus
The ink was prepared in the right proportions , and the forming hydrogel . The influence of SA concentration,
[28]
[40]
print pressure was adjusted until the ink can flow evenly CaCl concentration, and crosslinking time on relative
2
and continuously from the nozzle. Printing was performed enzyme activity of PFDH/CA microspheres may be
on a three-axis micro-positioning stage, a process which is attributed to the amount of enzyme immobilized during
computer-controlled. immobilization, the space required by the enzyme to
maintain the active structure, as well as the number and
3. Results and discussion size of pores, which affect the mass transfer and diffusion
of the substrate [1,41] .
3.1. Preparation of PFDH strains
The PFDH gene, selected from Pseudomonas putida, RSM is a statistical method, which solves optimal
contains an open reading frame of 1197 bp and encodes process parameters and multivariate problems [42,43] . Based

Volume 9 Issue 3 (2023) 122 https://doi.org/10.18063/ijb.695