Enhancing isoprene production by supplementing mevalonate pathway expressed in E. coli with immobilized enzymes.

Isoprene is an important component in rubber production, which can be produced using the E. coli mevalonic acid (MVA) pathway, and this method has the advantage of green environmental protection and sustainable. However, due to the excessive accumulation of intermediates, the growth of cells was inhibited and the enzyme activity decreased gradually, so it was difficult to increase the yield of isoprene. The immobilized enzyme has the characteristics of high stability and strong reusability, so in this study, the immobilized enzyme was added to the fermentation process of isoprene production by mevalonate metabolizing bacteria (PT-P), to explore the effect on isoprene synthesis. Under the optimum conditions, compared with PT-P fermentation alone, the enzyme catalyzes the conversion of MVA with an efficiency of up to 50.86%, and the yield of isoprene increased by about 30%, reaching 234.47 mg/L.

Screening thrombin inhibitors from Yangxinshi tablets by online capillary electrophoresis-based immobilized enzyme microreactor and molecular docking.

INTRODUCTION: Yangxinshi tablet (YXST) is a effective traditional Chinese medicine in treating cardiovascular diseases such as heart failure and myocardial infarction. OBJECTIVES: This study aims to develop a method for screening thrombin inhibitors from YXST using an online immobilized enzyme microreactor (IMER) based on capillary electrophoresis (CE). MATERIALS AND METHODS: Thrombin (THR) was immobilized on the capillary's inner wall using polydopamine (PDA). The chromogenic substrate S-2238 was employed to assess thrombin (THR) activity and kinetic parameters. The stability and repeatability of the constructed thrombin-immobilized enzyme microreactor (THR-IMER) were evaluated over 40 runs, maintaining 85% of initial activity. The Michaelis-Menten constant (Km) for THR was determined to be 11.98 mM. The half-maximal inhibitory concentration (IC50) and inhibition constant (Ki) for argatroban on THR were calculated. Ten compounds in YXST were screened for THR inhibitory potency using the THR-IMER. RESULTS: Salvianolic acid B and caffeic acid were identified as potential THR inhibitors in YXST, with inhibition rates at 200 µg/mL of 55.06 ± 6.70% and 31.88 ± 4.79%, respectively, aligning with microplate reader assay results. Molecular docking analysis confirmed their interactions with key THR residues, verifying their inhibitory activity. CONCLUSION: The CE-based THR-IMER method was successfully developed for screening thrombin inhibitors from YXST, offering a reliable approach for identifying potential therapeutic compounds.

A new method to immobilize urease in silk fibroin membrane by unidirectional nanopore dehydration.

The immobilization of free enzymes is crucial for enhancing their stability in different environments, enabling reusability, and expanding their applications. However, the development of a straightforward immobilization method that offers stability, high efficiency, biocompatibility, and modifiability remains a significant challenge. Silk fibroin (SF) is a good carrier for immobilized enzymes and drugs. Here, we employed urease as a model enzyme and utilized our developed technology called unidirectional nanopore dehydration (UND) to efficiently dehydrate a regenerated SF solution containing urease in a single step, resulting in the preparation of a highly functionalized SF membrane immobilizing urease (UI-SFM). The preparation process of UI-SFM is based on an all-water system, which is mild, green and able to efficiently and stably immobilize urease in the membranes, maintaining 92.7% and 82.8% relative enzyme activity after 30 days of storage in dry and hydrated states, respectively. Additionally, we performed additional post-treatments, including stretching and cross-linking with polyethylene glycol diglycidyl ether (PEGDE), to obtain two more robust immobilized urease membranes (UI-SFMs and UI-SFMc). The thermal and storage stability of these two membranes were significantly improved, and the recovery ratio of enzyme activity reached more than 90%. After 10 repetitions of the enzymatic reaction, the activity recovery of UI-SFMs and UI-SFMc remained at 92% and 88%, respectively. The results suggest that both UND-based and post-treatment-developed membranes exhibit excellent urease immobilization capabilities. Furthermore, the enzyme immobilization method offers a straightforward and versatile approach for efficient and stable enzyme immobilization, while its flexible modifiability caters to diverse application requirements.

Magnetic nanoparticles-immobilized phospholipase LM and phospholipase 3G: Preparation, characterization, and application on soybean crude oil degumming.

Immobilization of enzymes improves their stability and recoverability and is therefore crucial for scientific research and industrial applications. In this study, phospholipase LM (PLLM) and phospholipase 3G (PL3G) were immobilized using Fe3O4@SiO2@CS-COOH polycarboxylated magnetic nanoparticles (MNPs-COOH) as carriers and then used for degumming soybean crude oil. The immobilization rates and relative enzyme activities of these immobilized phospholipases were evaluated to determine the optimal immobilization parameters. The enzyme activities of PLLM-MNPs-COOH and PL3G-MNPs-COOH were 2830.87 and 1162.25 U/g, respectively. Enzymatic properties of the free and immobilized enzymes were compared. Both immobilized phospholipases exhibited higher condition tolerance and stability after immobilization. After 30-day storage at 4 °C, both immobilized phospholipases retained approximately 1.3 times the residual activity of the corresponding free phospholipases. When the degumming conditions were optimized, the residual phosphorus contents of the PLLM-MNPs-COOH- and PL3G-MNPs-COOH-degummed oils were 4.91 and 7.41 mg/kg, respectively, which were consistent with the safety standards for oil products. After 6 cycles, PLLM-MNPs-COOH and PL3G-MNPs-COOH continued to preserve 71.88 % and 70.00 % of their initial activities, respectively. The immobilized phospholipases are thus suitable for degumming soybean crude oil, and the mixed enzymes exhibited better degumming potential.

Ultrasound assisted enzymatic synthesis of OPO structured lipids bycovalent organic framework immobilizedlipase.

In this study, the covalent organic framework immobilized Rhizomucor miehie lipase COF@RML as a novel biocatalyst was applied in the enzymatic synthesis of OPO structured lipids (1, 3-dioleoyl-2-palmitoylglycerol). The impact of reaction medium, substrate molar ratio, enzyme addition amount, reaction time and temperature on the enzymatic synthesis of OPO structured lipids were studied. Furthermore, the effects of ultrasonic power and ultrasonic time on the synthesis of OPO structural lipids were studied. The results showed that ultrasonication could increase the yeild of OPO structured lipids by improving substrate mass transfer and enzyme particle dispersion. The optimal process for the synthesis of OPO structured lipids was obtained. When the ultrasonic power was set at 90 W, ultrasonic time at 12 minutes, enzyme addition amount at 10 wt%, substrate molar ratio at 1:8, reaction temperature at 45 °C, and reaction time at 6 hours, the yield of OPO structured lipids reached a remarkable 51.27 %. Finally, the commercial lipase Lipozyme RM IM was compared with the COF@RML. The findings indicated that COF@RML immobilized enzyme had better application value in the synthesis of OPO structured lipids.

[Heterologous expression of bacterial tyrosinase and its applications in biological hair dyeing and DOPA modification of hydrolyzed silk fibroin].

Tyrosinase is a copper-containing polyphenol oxidase widely applied in the food, cosmetics, pharmaceutical, and other industries. Currently, the production of commercial tyrosinase primarily relies on extraction from fungi, which has high costs, low purity, low specific activity, and poor stability. The objective of this study is to obtain highly expressed bacterial tyrosinase with potential for industrial applications. The bacterial tyrosinases from five different sources were heterologously expressed in Escherichia coli BL21(DE3), and the tyrosinases TyrBm and TyrVs derived from Bacillus megaterium and Verrucomicrobium spinosum were obtained with the enzyme activities of (16.1±0.2) U/mL and (48.6±0.9) U/mL, respectively. After protein purification, we compared the enzymatic properties of TyrBm and TyrVs, which revealed that TyrVs exhibited better thermal stability and higher substrate specificity than TyrBm. On the basis of characterizing TyrVs with high catalytic performance, we established a biological hair dyeing system based on TyrVs catalysis to achieve in-situ catalytic hair dyeing. The color washing fastness test measured the ∆E value less than 7.38±0.64 after simulated 14-day cleaning. To facilitate the rapid separation of catalytic products and enzymes, we successfully constructed an immobilized enzyme TyrVs-CipA dependent on self-assembly label CipA and applied this enzyme in the DOPA modification of hydrolyzed silk fibroin (HSF). The immobilized enzyme continuously catalyzed HSF for more than seven cycles, resulting in a single DOPA modification degree exceeding 70.00%. Further investigations demonstrated that DOPA modification enhances the scavenging activity of HSF towards DPPH and O2- radicals by 507.80% and 78.23%, respectively. This study provides a technical foundation for the development of environmentally friendly biological hair dye based on tyrosinase and biomaterials for tissue engineering.

Effective Ethyl Carbamate Prevention in Red Wines by Treatment with Immobilized Acid Urease.

Climate change poses several challenges in the wine industry, including increasing risks related to chemical food contaminants such as biogenic amines and ethyl carbamate (EC). In this work, we focused on urea removal in red wines by immobilized acid urease aiming at limiting EC formation during wine storage. By considering separable kinetics of catalyst deactivation and urea hydrolysis, it was possible to model the time course of urea removal in repeated uses in stirred batch reactors. Treatments based on immobilized urease of red wine enriched with 30 mg/L of urea allowed the reduction in the contaminant concentration to <5 mg/L. After 28.5 h of treatment, the observed urea level was reduced to about 0.5 mg/L, corresponding to a decrease in the potential ethyl carbamate (PEC) from 1662 µg/L to 93 µg/L, below the level of the non-enriched wine (187 µg/L). As a comparison, when treating the same wine with the free enzyme at maximum doses allowed by the EU law, urea and PEC levels decreased to only 12 mg/L and 415 µg/L respectively, after 600 h of treatment. These results show that, for red wines, urease immobilization is an effective strategy for urea removal and, thus, effective reduction in ethyl carbamate as a process contaminant. This study provides the scientific background for the future scaling-up of the process at an industrial level.

Pancreatic lipase immobilization on cellulose filter paper for inhibitors screening and network pharmacology study of anti-obesity mechanism.

The discovery of pancreatic lipase (PL) inhibitors is an essential route to develop new anti-obesity drugs. In this experiment, chitosan was used to add amino groups to cellulose filter paper (CFP) and then glutaraldehyde was used to covalently combine PL with amino-modified CFP through the Schiff base reaction. Under optimal immobilization conditions, CFP immobilized PL has a wide range of pH and temperature tolerance, as well as excellent reproducibility, reusability and storage stability. Subsequently, 26 natural products (NPs) were screened by immobilized PL with black tea extract having the highest inhibition rate. Three compounds with binding effects on PL (epigallocatechin gallate, theaflavin-3-gallate and theaflavin-3,3'-digallate) were captured. Molecular docking proved that these three compounds have a strong binding affinity for PL. Fluorescence spectra further revealed that theaflavin-3,3'-digallate could statically quench the intrinsic fluorescence of pancreatic lipase. The molecular docking and thermodynamic parameters indicated that electrostatic interaction was considered as the main interaction force between PL and theaflavin-3,3'-digallate. Finally, the potential anti-obesity targets and pathways of the three compounds were discussed through network pharmacology. This study not only proposes a simple and efficient method for screening PL inhibitors, but also sheds light on the anti-obesity mechanism of active compounds in black tea.

Investigation of Enzyme Immobilization and Clogging Mechanisms in the Enzymatic Synthesis of Amoxicillin.

This study investigated the blocking mechanism of immobilized penicillin G acylase (PGA) during the enzymatic synthesis of amoxicillin. Laboratory observations revealed that the primary cause of clogging was the crystallization of the substrate and product on the enzyme surface. Adjusting key parameters can significantly reduce clogging and improve catalytic efficiency. Methanol can decrease enzyme activity, but isopropyl alcohol cleaners can effectively remove clogs and protect enzyme activity. These findings provide an experimental foundation for optimizing the PGA immobilization process, which is crucial for achieving high efficiency and sustainability in industrial production.

Heterologous expression, characterization, and application of recombinant thermostable α-amylase from Geobacillus sp. DS3 for porous starch production.

Novel Geobacillus sp. DS3, isolated from the Sikidang Crater in Dieng, exhibits promising characteristics for industrial applications, particularly in thermostable α-amylase production. Recombinant technology was used to express thermostable α-amylase in E. coli BL21(DE3) to overcome high-temperature production challenges. The study aimed to express, purify, characterize, and explore potential applications of this novel enzyme. The enzyme was successfully expressed in E. coli BL21(DE3) at 18 °C for 20 h with 0.5 mM IPTG induction. Purification with Ni-NTA column yielded 69.23 % from the initial crude enzyme, with a 3.6-fold increase in specific activity. The enzyme has a molecular weight of ±70 kDa (±58 kDa enzyme+11 kDa SUMO protein). It exhibited activity over a wide temperature range (30-90 °C) and pH range (6-8), with optimal activity at 70 °C and pH 6 with great stability at 60 °C. Kinetic analysis revealed Km and Vmax values of 324.03 mg/ml and 36.5 U/mg, respectively, with dextrin as the preferred substrate without cofactor addition. As a metalloenzyme, it showed the best activity in the presence of Ca2+. The enzyme was used for porous starch production and successfully immobilized with chitosan, exhibiting improved thermal stability. After the fourth reuse, the immobilized enzyme maintained 62 % activity compared to the initial immobilization.

Immobilized PAD4 enzyme on magnetic nanoparticles for screening natural inhibitors from traditional Chinese medicines.

Dysregulation of peptidyl arginine deiminase 4 (PAD4) is involved in a variety of diseases including rheumatoid arthritis (RA) and Alzheimer's disease (AD), and it has emerged as potential and promising therapeutic target. However, no PAD4 inhibitor is ready for clinical use. Immobilized enzyme screening technology has gained increasing attention due to its low cost, reusability, easy separation from the reaction mixture, and resistance to changes in environmental conditions. In this study, PAD4 was immobilized on the magnetic nanoparticles (MNP) to prolong its activity stability, and a simple and rapid screening strategy of traditional Chinese medicine inhibitors based on immobilized PAD4 was established. The PAD4 enzyme was immobilized on magnetic nanoparticles (MNP) via Schiff base reaction using glutaraldehyde (GA) as crosslinking agent. Compared with free PAD4, the resulting MNP@GA@PAD4 exhibited an enhanced tolerance to temperature and storage stability, and its reusability was greatly improved with 66 % of initial enzyme activity after being recycled 10 times. The inhibitory activity of the immobilized PAD4 was assessed using two known PAD4 inhibitors GSK484 and BB-Cl-amidine. The semi-maximum inhibitory concentrations (IC50) of GSK484 and BB-Cl-amidine for MNP@GA@PAD4 were 1.00 and 0.97 µM, respectively, for free PAD4 were 0.64 and 0.85 µM, respectively. Finally, the MNP@GA@PAD4 was employed to rapid screen of natural PAD4 inhibitors from forty traditional Chinese medicines (TCMs). Under the same conditions, the controlled experiment was conducted with free PAD4. The screening results of TCMs inhibitors on MNP@GA@PAD4 and free PAD4 were similar, the alcohol extracts of Cinnamomi Cortex and Caryophylli Flos had significant inhibitory effects on PAD4 enzyme activity. The IC50 values of Cinnamomi Cortex extract for MNP@GA@PAD4 and free PAD4 were determined as 27 and 48 µg/mL, respectively. The IC50 values of Caryophylli Flos extracts for MNP@GA@PAD4 and free PAD4 were determined as 48 and 32 µg/mL, respectively. For the first time, this study proposed a method to immobilize PAD4 on magnetic materials, and developed a rapid, reusable and feasible strategy to screening natural PAD4 inhibitors from TCMs.

Development of automated proteomic workflows utilizing silicon-based coupling agents.

Automated methods for enzyme immobilization via 4-triethoxysilylbutyraldehyde (TESB) derived silicone-based coupling agents were developed. TESB and its oxidized derivative, 4-triethoxysilylbutanoic acid (TESBA), were determined to be the most effective. The resulting immobilized enzyme particles (IEPs) displayed robustness, rapid digestion, and immobilization efficiency of 51 ± 8%. Furthermore, we automated the IEP procedure, allowing for multiple enzymes, and/or coupling agents to be fabricated at once, in a fraction of the time via an Agilent Bravo. The automated trypsin TESB and TESBA IEPs were shown to rival a classical in-gel digestion method. Moreover, pepsin IEPs favored cleavage at leucine (>50%) over aromatic and methionine residues. The IEP method was then adapted for an in-situ immobilized enzyme microreactor (IMER) fabrication. We determined that TESBA could functionalize the silica capillary's inner wall while simultaneously acting as an enzyme coupler. The IMER digestion of bovine serum albumin (BSA), mirroring IEP digestion conditions, yielded a 33-40% primary sequence coverage per LC-MS/MS analysis in as little as 15 min. Overall, our findings underscore the potential of both IEP and IMER methods, paving the way for automated analysis and a reduction in enzyme waste through reuse, thereby contributing to a more cost-effective and timely study of the proteome. SIGNIFICANCE: This research introduces 4-triethoxysilylbutyraldehyde (TESB) and its derivatives as silicon-based enzyme coupling agents and an automated liquid handling method for bottom-up proteomics (BUP) while streamlining sample preparation for high-throughput processing. Additionally, immobilized enzyme particle (IEP) fabrication and digestion within the 96-well plate allows for flexibility in protocol where different enzyme-coupler combinations can be employed simultaneously. By enabling the digestion of entire microplates and reducing manual labor, the proposed method enhances reproducibility and offers a more efficient alternative to classical in-gel techniques. Furthermore, pepsin IEPs were noted to favor cleavage at leucine residues which represents an interesting finding when compared to the literature that warrants further study. The capability of immobilized enzyme microreactors (IMER) for rapid digestion (in as little as 15 min) demonstrated the system's efficiency and potential for rapid proteomic analysis. This advancement in BUP not only improves efficiency, but also opens avenues for a fully automated, mass spectrometry-integrated proteomics workflow, promising to expedite research and discoveries in complex biological studies.

Application of hydrophobic eutectic solvent in efficient biotransformation of total flavonoids of Herba Epimedii.

Icaritin, a hydrolysate from total flavonoids of Epimedii (TFE), which has better anti-hepatoma activity than its glycosylated form. In this work, immobilized enzymes 4LP-Tpebgl3@Na-Y and DtRha@ES-107 were used to hydrolyze TFE to prepare icaritin. Five different hydrophobic deep eutectic solvents (HDES) were prepared and the most ideal HDES was successfully selected, which was composed of dodecyl alcohol and thymol with the molar ratio of 2:1. The relative enzyme activity of 4LP-Tpebgl3@Na-Y and DtRha@ES-107 was about 102.4 % and 112.5 %, respectively. In addition, the thermal and binding stability of 4LP-Tpebgl3@Na-Y and DtRha@ES-107 in HDES was not affected negatively. In the biphasic system composed of 50 % (v/v) HDES and Na2HPO4-citric acid buffer (50 mM, pH 5.5), 4LP-Tpebgl3@Na-Y (1.0 U/mL) and TFE (1 g/L) were reacted at 80 °C for 1 h, and then reacted with DtRha@ES-107 (20 U/mL) at 80 °C for 2 h. Finally, TFE was completely converted to 301.8 mg/L icaritin (0.82 mM). After 10 cycles, 4LP-Tpebgl3@Na-Y/DtRha@ES-107 still maintained 84.1 % original activity. In this study, we developed an efficient methodology for icaritin preparation through the integration of enzymatic catalysis and adsorption separation, presenting a viable approach for large-scale, cost-effective production of icaritin.

Enzymatic synthesis and sensory evaluation of the novel kokumi compound N-butyryl phenylalanine.

In this study, food-grade glutamine transaminase (TGase) was utilized for the green-catalyzed preparation of N-butyryl amino acids. For improving the reusability of the enzyme preparation, immobilized TG enzyme (94.23% immobilization rate) was prepared. Furthermore, the yield of N-butyryl phenylalanine (BP) synthesized by TGase was obtained as 20.73% by one-factor experiment. The BP synthesis yield of immobilized TGase was 95.03% of that of TGase and remained above 60% of the initial enzyme activity after five runs. The sensory evaluation and E-tongue results showed that the addition of BP significantly increased the umami, saltiness, and richness intensities of the samples, and decreased the intensities of sourness, bitterness, and aftertaste-B. The molecular docking results indicated that hydrogen bonding dominated the binding of BP to taste receptors in the taste presentation mechanism of BP. These results confirmed the potential of BP as a flavor enhancer with promising applications in the food industry.

Ultrasound-assisted intensification of Pickering interfacial biocatalysis preparation of vitamin A aliphatic esters.

A novel approach to ultrasound-assisted Pickering interfacial biocatalysis (PIB) has been proposed and implemented for the efficient enzymatic transesterification production of vitamin A fatty acid esters. This is the first instance of exploiting the synergistic effect of ultrasound and the bifunctional modification of enzyme supports to accelerate biocatalytic performance in PIB systems. The optimal conditions were determined to be ultrasound power of 70 W, on/off time of 5 s/5 s, substrate molar ratio of 1:1, enzyme addition of 2 %, and a volume ratio of n-hexane to PBS of 3:1, a temperature of 40 °C, and a time of 30 min. The application of ultrasound technology not only improved lipase activity but also allowed for a reduction in emulsion droplet size to enhance interfacial mass transfer.Bifunctional modification of silica-based supports enhanced stability of immobilized enzymes by increasing hydrogen bonding while maintaining the active interface microenvironment. Compared with a non-ultrasound-assisted PIB system stabilized by mono-modified immobilized enzyme particles, the catalytic efficacy (CE) of the novel system reached 8.18 mmol g-1 min-1, which was enhanced by 3.33-fold, while the interfacial area was found to have increased by 17.5-fold. The results facilitated the conversion of vitamin A palmitate (VAP), vitamin A oleate (VAO), vitamin A linoleate (VAL), and vitamin A linolenate (VALn), with conversion rates of approximately 98.2 %, 97.4 %, 96.1 %, and 94.7 %, respectively. This represents the most efficient example that has been reported to our knowledge. Furthermore, the system demonstrated improved reusability, with a conversion rate of 62.1 % maintained even after 10 cycles. The findings presented in this paper provide valuable insights into an efficient and conveniently promising protocol for the development of PIB systems.

Spatially selective catalysis of OSA starch for preparation of Pickering emulsions with high emulsification properties.

The traditional strategies of chemical catalysis and biocatalysis for producing octenyl succinic anhydride modified starch can only randomly graft hydrophobic groups on the surface of starch, resulting in unsatisfactory emulsification performance. In this work, a lipase-inorganic hybrid catalytic system with multi-scale flower like structure is designed and applied to spatially selective catalytic preparation of ocenyl succinic anhydride modified starch. With the appropriate floral morphology and petal density, lipases distributed in the "flower center" can selectively catalyze the grafting of hydrophobic groups in a spatial manner, the hydrophobic groups are concentrated on one side of starch particles. The obtaining OSA starch exhibits excellent emulsifying property, and the pickering emulsion has good protective effect on the embedded curcumin. This work provides a direction for the development of high-performance starch-based emulsifiers for the food and pharmaceutical industries, which is of great significance for improving the preparation and emulsification theory research of modified starch.

Immobilization of pancreatin based on ultrasound-assisted polydopamine functionalized magnetic porous chitosan for the detoxification of ochratoxin A in wine.

Ochratoxin A (OTA) is a mycotoxin that globally contaminates fruits and their products. Since OTA have a huge negative impact on health hazards and economic losses, it is imperative to establish an effective and safe strategy for detoxification. Here, pancreatin was immobilized on the surface of polydopamine functionalized magnetic porous chitosan (MPCTS@ PDA) for the degradation of OTA. Compared with free pancreatin, MPCTS@ PDA@ pancreatin displayed excellent thermal stability, acid resistance, storage stability and OTA detoxification in wine (>58%). Moreover, the MPCTS@ PDA@ pancreatin retained 43% initial activity after 8 reuse cycles. There was no significant change in the quality of wine after MPCTS@ PDA@ pancreatin treatment. Moreover, it did not exhibit cytotoxicity which facilitated its application in wine. These results demonstrated that MPCTS@ PDA@ pancreatin can be used as a highly effective biocatalysate for OTA detoxification in wine.

Bioremediation of Hazardous Pollutants Using Enzyme-Immobilized Reactors.

Bioremediation uses the degradation abilities of microorganisms and other organisms to remove harmful pollutants that pollute the natural environment, helping return it to a natural state that is free of harmful substances. Organism-derived enzymes can degrade and eliminate a variety of pollutants and transform them into non-toxic forms; as such, they are expected to be used in bioremediation. However, since enzymes are proteins, the low operational stability and catalytic efficiency of free enzyme-based degradation systems need improvement. Enzyme immobilization methods are often used to overcome these challenges. Several enzyme immobilization methods have been applied to improve operational stability and reduce remediation costs. Herein, we review recent advancements in immobilized enzymes for bioremediation and summarize the methods for preparing immobilized enzymes for use as catalysts and in pollutant degradation systems. Additionally, the advantages, limitations, and future perspectives of immobilized enzymes in bioremediation are discussed.

Chitosanase-immobilized magnetite-agar gel particles as a highly stable and reusable biocatalyst for enhanced production of physiologically active chitosan oligosaccharides.

A novel immobilized chitosanase was developed and utilized to produce chitosan oligosaccharides (COSs) via chitosan hydrolysis. Magnetite-agar gel particles (average particle diameter: 338 µm) were prepared by emulsifying an aqueous agar solution dispersing 200-nm magnetite particles with isooctane containing an emulsifier at 80 °C, followed by cooling the emulsified mixture. The chitosanase from Bacillus pumilus was immobilized on the magnetite-agar gel particles chemically activated by introducing glyoxyl groups with high immobilization yields (>80%), and the observed specific activity of the immobilized chitosanase was 16% of that of the free enzyme. This immobilized chitosanase could be rapidly recovered from aqueous solutions by applying magnetic force. The thermal stability of the immobilized chitosanase improved remarkably compared with that of free chitosanase: the deactivation rate constants at 35 °C of the free and immobilized enzymes were 8.1 × 10-5 and 3.9 × 10-8 s-1, respectively. This immobilized chitosanase could be reused for chitosan hydrolysis at 75 °C and pH 5.6, and 80% of its initial activity was maintained even after 10 cycles of use. COSs with a degree of polymerization (DP) of 2-7 were obtained using this immobilized chitosanase, and the product content of physiologically active COSs (DP ≥ 5) reached approximately 50%.

A magnetic beads-based ligand fishing method for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from Hunteria zeylanica.

In this study, a novel magnetic bead-based ligand fishing method was developed for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from natural products. In order to improve the screening efficiency, two different magnetic beads, i.e. amine and carboxyl terminated magnetic beads, were comprehensively compared in terms of their ability to immobilize monoamine oxidase A (MAOA), biocatalytic activity and specific adsorption rates for affinity ligands. Carboxyl terminated magnetic beads performed better for MAOA immobilization and demonstrated superior performance in ligand fishing. The MAOA immobilized magnetic beads were applied to screen novel monoamine oxidase inhibitors in an alkaloid-rich plant, Hunteria zeylanica. Twelve MAOA affinity ligands were screened out, and ten of them were identified as monoterpene indole alkaloids by HPLC-Obitrap-MS/MS. Among them, six ligands, namely geissoschizol, vobasinol, yohimbol, dihydrocorynanthenol, eburnamine and (+)-isoeburnamine which exhibited inhibitory activity against MAOA with low IC50 values. To further explore their inhibitory mechanism, enzyme kinetic analysis and molecular docking studies were conducted.