Catalytic Specificity Research Articles

Adaptation of the electron transport chain improves the biocatalytic efficiency of progesterone 17α hydroxylation

17α hydroxylase is a key enzyme for the conversion of progesterone to prepare various progestational drug intermediates. To improve the specific hydroxylation capability of this enzyme in steroid biocatalysis, the CYP260A1 derived from cellulose-mucilaginous bacteria Sorangium cellulosum Soce56 and the Fpr and bovine adrenal-derived Adx4-108 derived from Escherichia coli str. K-12 were used to construct a new electron transfer system for the conversion of progesterone. Selective mutation of CYP260A1 resulted in a mutant S276I with significantly enhanced 17α hydroxylase activity, and the yield of 17α-OH progesterone reached 58% after optimization of the catalytic system in vitro. In addition, the effect of phosphorylation of the ferredoxin Adx4-108 on 17α hydroxyl activity was evaluated using a targeted mutation technique, and the results showed that the mutation Adx4-108T69E transferred electrons to S276I more efficiently, which further enhanced the catalytic specificity in the C17 position of progesterone, and the yield of 17α-OH progesterone was eventually increased to 74%. This study provides a new option for the production of 17α-OH progesterone by specific transformation of bacterial-derived 17α hydroxylase, and lays a theoretical foundation for the industrial production of progesterone analogs using biotransformation method.

Electrochemical carbon–carbon coupling with enhanced activity and racemate stereoselectivity by microenvironment regulation

Enzymes are characteristic of catalytic efficiency and specificity by maneuvering multiple components in concert at a confined nanoscale space. However, achieving such a configuration in artificial catalysts remains challenging. Herein, we report a microenvironment regulation strategy by modifying carbon paper with hexadecyltrimethylammonium cations, delivering electrochemical carbon–carbon coupling of benzaldehyde with enhanced activity and racemate stereoselectivity. The modified electrode–electrolyte interface creates an optimal microenvironment for electrocatalysis—it engenders dipolar interaction with the reaction intermediate, giving a 2.2-fold higher reaction rate (from 0.13 to 0.28 mmol h−1 cm−2); Moreover, it repels interfacial water and modulates the conformational specificity of reaction intermediate by facilitating intermolecular hydrogen bonding, affording 2.5-fold higher diastereomeric ratio of racemate to mesomer (from 0.73 to 1.82). We expect that the microenvironment regulation strategy will lead to the advanced design of electrode–electrolyte interface for enhanced activity and (stereo)selectivity that mimics enzymes.

Biosynthesis of maltodextrin-derived glucan dendrimer using microbial branching enzyme.

Glucan dendrimers were developed with microbial branching enzyme (BE) treated maltodextrin. The molecular weight (Mw) of recombinant BE was 79.0kDa, and its optimum activity was observed at pH 7.0 and 70°C. BE converted different maltodextrins with dextrose equivalent value of 6 (MD6), 12 (MD12), or 19 (MD19) into the given glucan dendrimers, along with the marked increment of the molecular density (approximately 30-60 folds) and α-1,6 linkage percentage (up to 7.3-9.7%). Among three glucan dendrimers, the enzyme-treated MD12 showed a more homogeneous Mw distribution with the maximum Mw of 5.5×106 g/mol, indicating that higher substrate catalytic specificity of BE for MD12 substrate. During transglycosylation with MD12 for 24h, the shorter chains (degree of polymerization, DP<13) increased from 73.9% to 83.0%, accompanying by a reduction of medium chains (DP13-24) and long chains (DP>24). Moreover, the slowly digestible and resistant nutritional fractions were increased by 6.2% and 12.5%, respectively. The results suggested that the potentiality of BE structuring glucan dendrimer with tailor-made structure and functionality for industrial application.

Confined synthesis of ternary FeCoMn single-atom nanozyme in N-doped hollow mesoporous carbon nanospheres for synergistic chemotherapy and chemodynamic cancer therapy

Recently, nanozymes show increasing biological applications and promising possibilities for therapeutic intervention, while their mediated therapeutic outcomes are severely compromised due to their insufficient catalytic activity and specificity. Herein, ternary FeCoMn single atoms were incorporated into N-doped hollow mesoporous carbon nanospheres by in situ confinement pyrolysis at 800 °C as high-efficiency nanozyme. The confinement strategy endows the as-prepared nanozyme with the enhanced catalase- and oxidase-like activities. Specifically, the FeCoMn TSAs/N-HCSs nanozyme can decompose intracellular H2O2 to generate O2 and subsequently convert O2 to cytotoxic superoxide radicals (O2∙–), which can initiate cascade enzymatic reactions in tumor microenvironment (TME) for chemodynamic therapy (CDT). Moreover, the cancer therapy was largely enhanced by loading with doxorubicin (DOX). Impressively, the FeCoMn TSAs/N-HCSs nanozyme-mediated CDT and the DOX-induced chemotherapy endow the DOX@FeCoMn TSAs/N-HCSs with effective tumor inhibition, showing the superior therapeutic efficacy.

Enzyme immobilization on covalent organic framework supports.

Enzymes are natural catalysts with high catalytic activity, substrate specificity and selectivity. Their widespread utilization in industrial applications is limited by their sensitivity to harsh reaction conditions and difficulties relating to their removal and re-use after the reaction is complete. These limitations can be addressed by immobilizing the enzymes in solid porous supports. Covalent organic frameworks (COFs) are ideal candidate carriers because of their good biocompatibility, long-term water stability and large surface area. In post-synthetic immobilization, the enzyme is added to an existing COF; this has had limited success because of enzyme leaching and pore blockage by enzymes that are too large. Direct-immobilization methods-building the COF around the enzyme-allow tailored incorporation of proteins of any size and result in materials with lower levels of leaching and better mass transport of reactants and products. This protocol describes direct-immobilization methods that can be used to fabricate enzyme@COF (@ = engulfing) biocomposites with rationally programmed structures and functions. If COF construction requires harsh reaction conditions, the enzyme can be protected by using a removable metal-organic framework. Alternatively, a direct in situ approach, in which the enzyme and the COF monomers assemble under very mild conditions, can be used. Examples of both approaches are described: enzyme@COF-42-B/43-B capsules (enzymes including catalase, glucose oxidase, etc.) with ZIF-90 or ZPF-2 as protectors, and lipase@NKCOF-98/99 via in situ direct-immobilization methods (synthesis timing: 30-100 min). Example assays for physical and functional characterization of the COF and enzyme@COF materials are also described.

Self‐Assembled Asymmetric Electrodes for High‐Efficiency Thermogalvanic Cells

AbstractThe thermogalvanic cell (TGC) is considered a promising thermoelectric device for directly converting low‐grade waste heat into electricity due to its low‐cost and scalable properties. However, the low output and conversion efficiency limit its practical application. Herein, record‐high thermoelectric conversion performances are achieved in the aqueous ferri/ferrocyanide ([Fe(CN)6]3−/[Fe(CN)6]4−) based TGC by using the electrode of cobaltous oxide nanowires array on carbon cloth fiber. Because of the temperature‐dependence reaction activity between CoO with [Fe(CN)6]4−, the asymmetric electrodes of Co2Fe(CN)6 and CoO nanowires array on carbon cloth fiber are constructed at the hot anode and cold cathode, respectively. These self‐assembled asymmetric electrodes exhibit specific catalysis toward electrode reactions at both ends of TGC, leading to a significant reduction in electronic activation energy. It is demonstrated that the electrodes have high catalytic activity and high specific surface area, enabling the construction of a high‐efficiency TGC with a Carnot‐relative efficiency (ηr) of 14.8% and a maximum output power density (Pmax) of 24.5 W m−2. This work offers an asymmetric electrode engineering pathway for the continuous evolution of TGCs.

Improving the position specificity of Themomyces lanuginosus lipase based on semi-rational design

Diacylglycerol (DAG) is an intermediate product in lipid metabolism and plays an important physiological role in human body. It is mainly prepared by hydrolyzing lipid with lipase. However, research on the detection method of 1, 2-diacylglycerol (1, 2-DAG) and 1, 3-diacylglycerol (1, 3-DAG) and catalytic specificity of lipase was not enough, which limits its wide application. To address these challenges, an efficient quantitative detection method was first established for 1, 2-DAG (0.025-0.200 g/L) and 1, 3-DAG (0.025-0.150 g/L) by combining supercritical fluid chromatography with evaporative light scattering detector and optimizing the detection and analysis parameters. Based on the molecular docking between Thermomyces lanuginosus lipase (TLL) and triolein, five potential substrate binding sites were selected for site-specific saturation mutation to construct a mutation library for enzyme activity and position specificity screening. The specificity of sn-1, 3 of the I202V mutant was the highest in the library, which was 11.7% higher than the specificity of the wild type TLL. In summary, the position specificity of TLL was modified based on a semi-rational design, and an efficient separation and detection method of DAG isomers was also established, which provided a reference for the study of the catalytic specificity of lipase.

SARS main protease: structural insights on catalytic mechanism, substrate specificity and inhibition by the oral antiviral AG7404

SARS main protease: structural insights on catalytic mechanism, substrate specificity and inhibition by the oral antiviral AG7404

THE INFLUENCE OF PROLONGED STRESS ON THE ACTIVITY OF BLOOD ENZYMES IN THE CONDITIONS OF WAR IN UKRAINE

The article presents an analysis of scientific works on the effect of long-term stress on blood enzymes. It has been determined that enzymopathies include a wide range of diseases, the cause of which is a malfunction of enzymes. Understanding the mechanisms of the occurrence of such disorders is necessary for the search for new, effective methods of their treatment. In the course of the work, twenty scientific works in English, published in the period 2000-2021, were analyzed, which investigated the mechanisms of human enzyme disorders. It has been established that non-genetic factors, such as injuries or stress, can lead to enzyme malfunctions. Stress factors, such as physical injuries, can affect enzyme dysfunction. One of the ways in which stress affects the functioning of enzymes is a change in their activity in biochemical mechanisms. Mutations in the three-dimensional structure of enzymes are one of the main mechanisms underlying the disruption of enzyme activity under the influence of stress. Since enzymes are large proteins that often have a complex tertiary structure that is important for their functioning, mutations that affect this structure can change their catalytic activity, substrate specificity, and stability, leading to enzymopathies. The stress factor of war is not similar to everyday stress factors, because it exceeds a person's ability to adapt to the action of the stress factor. The results of the research and analysis of research indicate the significant role of stress in disrupting the work of antioxidant blood enzymes. As a result of the analysis of scientific research, it was established that these enzymes include superoxidedismutase, catalase, glutathioneperoxidase, and glutathionetransferase.&#x0D; Superoxidedismutase is an antioxidant enzyme that plays a crucial role in protecting cells from the harmful effects of reactive oxygen species. ROS are produced during normal cellular metabolism and can also be produced in response to stressful conditions. A decrease in the activity of superoxidedismutase in the erythrocytes of combatants may occur due to epigenetic changes in the regulation of this enzyme, which were caused by long-term stressful conditions.&#x0D; Catalase is another antioxidant enzyme that plays an important role in protecting cells from oxidative stress. Another possible cause may be a change in the expression of the genes that code for catalase, which leads to a decrease in the level of the enzyme.&#x0D; Glutathioneperoxidase is an antioxidant enzyme that helps protect cells from oxidative stress by catalyzing the reduction of hydrogen peroxide and organic hydroperoxides to less harmful compounds. Stressful conditions, in particular combat operations, can lead to an increase in the formation of reactive oxygen species and a decrease in antioxidant defense mechanisms, including glutathioneperoxidase.&#x0D; Glutathionetransferase catalyzes the conjugation of glutathione, a tripeptide consisting of glutamate, cysteine, and glycine, with electrophilic substrates, resulting in the formation of less reactive and more water-soluble products that are suitable for excretion. Prolonged exposure to stress can lead to depletion of glutathione and a decrease in the activity of this enzyme.&#x0D; The obtained results indicate that wartime conditions can be a significant factor in the development of disorders of these enzymes.

The peroxidase-like cleaning strategy for organic fouling of water treatment membranes based on MoS2 functional layers

Efficient cleaning strategies play a key role in implementing sustainable and economic membrane operations during water treatments and purification. We proposed in this paper to construct the functional layers on the surface of PES substate using MoS2 nanosheets synthesized by a hydrothermal method, namely MoS2/PES membrane. The membrane structure and properties were characterized by SEM, XRD, EDS and FTIR. This is then followed by an evaluation of the membrane fouling of the functional layers, and in turn exploring the membrane cleaning strategy. The specific catalytic effects between MoS2 nanosheets with peroxidase-like activity, and the cleaning agent (H2O2) was subtly combined to endow MoS2 functional layers with specific catalysis. Humic acid (HA, as a representative organic foulant) having the filtration efficiency (~91 %) of MoS2/PES membrane was ~6 % higher than that of the pristine PES membrane. The flux recovery rate of MoS2/PES membrane (~75 %) was significantly larger than the PES membrane (~16 %) under optimized conditions. Results of 7 cycles of consecutive membrane fouling and cleaning demonstrated MoS2 functional layers not only relieved membrane fouling but also exhibited higher water flux. The outstanding performance of MoS2 functional layers could be explained by their stacked honeycomb pore-like structure and the catalytic characteristics of MoS2-like peroxidases. H2O2 was used to trigger the in-situ catalytic function of MoS2 layers, generating strong ROS and oxygen for the enhanced removal of organic foulants. This study provides new insights into devising a novel membrane cleaning strategy using advanced functional nanomaterials.

Photoexcited Ru single-atomic sites for efficient biomimetic redox catalysis

The unsatisfactory catalytic activity of nanozymes owing to their inefficient electron transfer (ET) is the major challenge in biomimetic catalysis-related biomedical applications. Inspired by the photoelectron transfers in natural photoenzymes, we herein report a photonanozyme of single-atom Ru anchored on metal-organic frameworks (UiO-67-Ru) for achieving photoenhanced peroxidase (POD)-like activity. We demonstrate that the atomically dispersed Ru sites can realize high photoelectric conversion efficiency, superior POD-like activity (7.0-fold photoactivity enhancement relative to that of UiO-67), and good catalytic specificity. Both insitu experiments and theoretical calculations reveal that photoelectrons follow the cofactor-mediated ET process of enzymes to promote the production of active intermediates and the release of products, demonstrating more favorable thermodynamics and kinetics in H2O2 reduction. Taking advantage of the unique interaction of the Zr-O-P bond, we establish a UiO-67-Ru-based immunoassay platform for the photoenhanced detection of organophosphorus pesticides.

Redesign of γ-glutamyl transpeptidase from Bacillus subtilis for high-level production of L-theanine by cavity topology engineering

L-Theanine is a multifunctional nonprotein amino acid found naturally in tea leaves. It has been developed as a commercial product for a wide range of applications in the food, pharmaceutical, and healthcare industries. However, L-theanine production catalyzed by γ-glutamyl transpeptidase (GGT) is limited by the low catalytic efficiency and specificity of this class of enzymes. Here, we developed a strategy for cavity topology engineering (CTE) based on the cavity geometry of GGT from B. subtilis 168 (CGMCC 1.1390) to obtain an enzyme with high catalytic activity and applied it to the synthesis of L-theanine. Three potential mutation sites, M97, Y418, and V555, were identified using the internal cavity as a probe, and residues G, A, V, F, Y, and Q, which may affect the shape of the cavity, were obtained directly by computer statistical analysis without energy calculations. Finally, 35 mutants were obtained. The optimal mutant Y418F/M97Q showed a 4.8-fold improvement in catalytic activity and a 25.6-fold increase in catalytic efficiency. The recombinant enzyme Y418F/M97Q exhibited a high space-time productivity of 15.4 g L-1 h-1 by whole-cell synthesis in a 5 L bioreactor, which was one of the highest concentrations reported so far at 92.4 g L-1. Overall, this strategy is expected to enhance the enzymatic activity associated with the synthesis of L-theanine and its derivatives.Key points • Cavity topology engineering was used to modify the GGT for L-theanine biocatalysis. • The catalytic efficiency of GGT was increased by 25.6-fold. • Highest productivity of L-theanine reached 15.4 g L -1 h-1 (92.4 g L-1) in a 5 L bioreactor.

Structures of L-proline trans-hydroxylase reveal the catalytic specificity and provide deeper insight into AKG-dependent hydroxylation.

L-Proline hydroxylase is a member of the non-heme Fe2+/α-ketoglutarate (AKG)-dependent hydroxylase family that catalyzes the reaction from L-proline to hydroxy-L-proline, which is widely used in drug synthesis, biochemistry, food supplementation and cosmetic industries. Here, the first crystal structure of L-proline trans-hydroxylase and its complexes with substrate and product are reported, which reveal the structural basis of trans-cis proline hydroxylation selectivity. Structure comparison with other AKG-dependent hydroxylases identifies conserved amino acid residues, which may serve as signatures of in-line or off-line AKG binding modes in the AKG-dependent enzyme family.

Lactate-Oxidase-Instructed Cancer Diagnosis and Therapy.

Lactate oxidase (LOx) has attracted extensive interest in cancer diagnosis and therapy in recent years owing to its specific catalysis on l-lactate; its catalytic process consumes oxygen (O2 ) and generates a large amount of hydrogen peroxide (H2 O2 ) and pyruvate. Given high levels of lactate in tumor tissues and its tight correlation with tumor growth, metastasis, and recurrence, LOx-based biosensors including H2 O2 -based, O2 -based, pH-sensitive, and electrochemical have been designed for cancer diagnosis, and various LOx-based cancer therapy strategies including lactate-depletion-based metabolic cancer therapy/immunotherapy, hypoxia-activated chemotherapy, H2 O2 -based chemodynamic therapy, and multimodal synergistic cancer therapy have also been developed. In this review, the lactate-specific catalytic properties of LOx are introduced, and the recent advances on LOx-instructed cancer diagnostic or therapeutic platforms and corresponding biological applications are summarized. Additionally, the challenges and potential of LOx-based nanomedicines are highlighted.

A Single Active-Site Mutagenesis Confers Enhanced Activity and/or Changed Product Distribution to a Pentalenene Synthase from Streptomyces sp. PSKA01.

Pentalenene is a ternary cyclic sesquiterpene formed via the ionization and cyclization of farnesyl pyrophosphate (FPP), which is catalyzed by pentalenene synthase (PentS). To better understand the cyclization reactions, it is necessary to identify more key sites and elucidate their roles in terms of catalytic activity and product specificity control. Previous studies primarily relied on the crystal structure of PentS to analyze and verify critical active sites in the active cavity, while this study started with the function of PentS and screened a novel key site through random mutagenesis. In this study, we constructed a pentalenene synthetic pathway in E. coli BL21(DE3) and generated PentS variants with random mutations to construct a mutant library. A mutant, PentS-13, with a varied product diversity, was obtained through shake-flask fermentation and product identification. After sequencing and the functional verification of the mutation sites, it was found that T182A, located in the G2 helix, was responsible for the phenotype of PentS-13. The site-saturation mutagenesis of T182 demonstrated that mutations at this site not only affected the solubility and activity of the enzyme but also affected the specificity of the product. The other products were generated through different routes and via different carbocation intermediates, indicating that the 182 active site is crucial for PentS to stabilize and guide the regioselectivity of carbocations. Molecular docking and molecular dynamics simulations suggested that these mutations may induce changes in the shape and volume of the active cavity and disturb hydrophobic/polar interactions that were sufficient to reposition reactive intermediates for alternative reaction pathways. This article provides rational explanations for these findings, which may generally allow for the protein engineering of other terpene synthases to improve their catalytic efficiency or modify their specificities.

SARS-CoV-2 Mpro Protease Variants of Concern Display Altered Viral Substrate and Cell Host Target Galectin-8 Processing but Retain Sensitivity toward Antivirals.

The main protease of SARS-CoV-2 (Mpro) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in Mpro may alter the structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOCs) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and the rate of cleavage of a viral peptide. Crystal structures of 11 Mpro mutants provided structural insight into their altered functionality. Additionally, we show Mpro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and a subsequent significant decrease in cytokine secretion, providing evidence for alterations in the escape of host-antiviral mechanisms. Accordingly, mutations associated with the Gamma VOC and highly virulent Delta VOC resulted in a significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting Mpro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.

Allelic variation of terpene synthases drives terpene diversity in the wild species of the Freesia genus.

Terpene synthases (TPSs) play pivotal roles in conferring the structural diversity of terpenoids, which are mainly emitted from flowers, whereas the genetic basis of the release of floral volatile terpenes remains largely elusive. Though quite similar in sequence, TPS allelic variants still function divergently, and how they drive floral terpene diversity in closely related species remains unknown. Here, TPSs responsible for the floral scent of wild Freesia species were characterized, and the functions of their natural allelic variants, as well as the causal amino acid residues, were investigated in depth. Besides the 8 TPSs previously reported in modern cultivars, 7 additional TPSs were functionally evaluated to contribute to the major volatiles emitted from wild Freesia species. Functional characterization of allelic natural variants demonstrated that allelic TPS2 and TPS10 variants changed the enzymatic capacity while allelic TPS6 variants drove the diversity of floral terpene products. Further residue substitution analysis revealed the minor residues determining the enzyme catalytic activity and product specificity. The clarification of TPSs in wild Freesia species reveals that allelic TPS variants evolved differently to determine the interspecific floral volatile terpenes in the genus and might be used for modern cultivar improvement.

Activity and Characterization of Tocopherol Oxidase in Corn Germs and Its Relationship with Oil Color Reversion.

Color reversion has long been a major problem for the vegetable oil industry, and the enzymatic oxidation of γ-tocopherol is thought to trigger this phenomenon. In this study, first, the extraction, purification, and detailed characterization of tocopherol oxidase from fresh corn germs were performed. Then, the relationship between the enzyme reaction of γ-tocopherol and oil color reversion was verified. The results showed that the membrane-free extracts of raw corn germ performed specific catalysis of tocopherol in the presence of lecithin. In terms of the oxidation product, tocored (the precursor of color reversion) was detected in the mixture after the catalytic reactions, indicating that this anticipated enzyme reaction was probably correlated with the color reversion. Furthermore, the optimal pH and temperature for the tocopherol oxidase enzyme were 4.6 and 20 °C, respectively. In addition, ascorbic acid at 1.0 mM completely inhibited the enzymatic reaction.

Experimental and computational approaches to characterize a novel amidase that initiates the biodegradation of the herbicide propanil in Bosea sp. P5

The herbicide propanil and its major metabolite 3,4-dichloroaniline (3,4-DCA) are difficult to biodegrade and pose great health and environmental risks. However, studies on the sole or synergistic mineralization of propanil by pure cultured strains are limited. A two-strain consortium (Comamonas sp. SWP-3 and Alicycliphilus sp. PH-34), obtained from a swep-mineralizing enrichment culture that can synergistically mineralize propanil, has been previously reported. Here, another propanil degradation strain, Bosea sp. P5, was successfully isolated from the same enrichment culture. A novel amidase, PsaA, responsible for initial propanil degradation, was identified from strain P5. PsaA shared low sequence identity (24.0–39.7 %) with other biochemically characterized amidases. PsaA exhibited optimal activity at 30 °C and pH 7.5 and had kcat and Km values of 5.7 s-1 and 125 μM, respectively. PsaA could convert the herbicide propanil to 3,4-DCA but exhibited no activity toward other herbicide structural analogs. This catalytic specificity was explained by using propanil and swep as substrates and then analyzed by molecular docking, molecular dynamics simulation and thermodynamic calculations, which revealed that Tyr138 is the key residue that affects the substrate spectrum of PsaA. This is the first propanil amidase with a narrow substrate spectrum identified, providing new insights into the catalytic mechanism of amidase in propanil hydrolysis

Programmable and printable formaldehyde dehydrogenase as an excellent catalyst for biodegradation of formaldehyde.

As an environmental pollutant, formaldehyde can cause serious harm to the human body. Among many degradation methods, formaldehyde dehydrogenase from Pseudomonas putida (PFDH) exhibits broad potential because of its strong catalytic specificity and high degradation efficiency. However, the real application of PFDH in industry is limited by its instability and difficulties in recycling. In this work, the suitable printing conditions for immobilizing PFDH by three-dimensional (3D) printing technology were studied: the concentration of sodium alginate (SA) was 1.635 wt%, the concentration of CaCl2 was 7.4 wt%, the crosslinking time with CaCl2 was 8 min, and the temperature of the reaction was 31.5°C. 3D-printed PFDH/calcium alginate (CA) microspheres have 210% relative enzyme activity after seven repeated uses. Dried PFDH/CA particles were characterized by scanning electron microscope (SEM), Fourier transform infrared spectrometer (FT-IR), EDS elemental mapping, and thermogravimetric analysis (TGA) which proved that the enzyme was immobilized by the material. In addition, the recycling ability of 3D printing to immobilize different objects was explored and different shapes were designed by computer-aided design (CAD). In conclusion, 3D printing technology was applied to immobilize PFDH in this work, which provides a new idea to biodegrade formaldehyde in a green way.