Free Enzyme Research Articles

Engineering bio-brick protein scaffolds for organizing enzyme assemblies.

Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi-enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi-enzyme systems. Two SCAB systems are developed, one employing head-to-tail interactions with reversible covalent disulfide bonds, the other relying on non-covalent metal-driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non-reducing environment or by metal presence. A proof-of-concept with formate dehydrogenase (FDH) and L-alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6-fold compared to free enzymes, with the covalent stapling outperforming the metal-driven assembly. This enhancement likely stems from higher-order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial-organizing tools, for more efficient enzymatic cascade reactions.

Horseradish peroxidase immobilized in calcium alginate-gelatin hybrid beads with high stability against metallic ions and organic solvents

Horseradish peroxidase (HRP) was immobilized in hybrid calcium alginate-gelatin hydrogel beads by entrapment. Then, organic solubility, metal ion compatibility, pH/temperature studies of the synthesized materials have been carried out. Hybrid beads (Ca-Alg@gel-HRP) were synthesized under optimum synthesis conditions with high yield and specific activity. The synthesized bead size was determined to be approximately 0.29 mm in diameter, hydrogel structure and transparent appearance. Accordingly, the optimum pH of free HRP and Ca-Alg@gel HRP was found to be pH 6 and 5 and the optimum temperature was found to be 40 °C and 60 °C respectively. For all identified metal ions except Fe2+, the Ca-Alg@gel HRP had a higher activity than the free HRP. In the presence of 1 mM Na+, Ca2+, Mg2+, Ba2+, Cu2+, Ni2+, Mn2+, Hg2+ ions, the activity increased by more than 20%. The relative activity was also approximately 153%, 150% and 145% in 2.5 mM chloroform, glycerol and DMF. Free enzyme activity was reduced by more than 30%. The Ca-Alg@gel HRP at room temperature and 4 °C retained most of its activity after 14 days. In cycle 5, it had half the activity. As a conclusion, it may have significant potential in a number of industrial processes, particularly in organic synthesis, because it is tolerant to metal ions and organic solvents and is a cheap, practical and simple immobilization method.

Recent Progress on Conversion of Lignocellulosic Biomass by MOF-Immobilized Enzyme.

The enzyme catalysis conversion of lignocellulosic biomass into valuable chemicals and fuels showed a bright outlook for replacing fossil resources. However, the high cost and easy deactivation of free enzymes restrict the conversion process. Immobilization of enzymes in metal-organic frameworks (MOFs) is one of the most promising strategies due to MOF materials' tunable building units, multiple pore structures, and excellent biocompatibility. Also, MOFs are ideal support materials and could enhance the stability and reusability of enzymes. In this paper, recent progress on the conversion of cellulose, hemicellulose, and lignin by MOF-immobilized enzymes is extensively reviewed. This paper focuses on the immobilized enzyme performances and enzymatic mechanism. Finally, the challenges of the conversion of lignocellulosic biomass by MOF-immobilized enzyme are discussed.

Tailored chitosan integration in diatomaceous earth particles as a scaffold for fructosyltransferase immobilization in fructo-oligosaccharide production.

Fructo-oligosaccharide (FOS) belongs to the group of short inulin-type fructans and is one of the most important non-digestible bifid-oligosaccharides capable of biotransforming sucrose using fructosyltransferase (FTase). However, there are no immobilized FTase products that can be successfully used industrially. In this study, diatomite was subjected to extrusion, sintering and granulation to form diatomaceous earth particles that were further modified via chitosan aminomethylation for modification. FTase derived from Aspergillus oryzae was successfully immobilized on the modified support via covalent binding. The immobilized enzyme activity was 503 IU g-1 at an enzyme concentration of 0.6 mg mL-1, immobilization pH of 7.0 and contact time of 3 h. Additionally, the immobilization yield was 56.91%. Notably, the immobilized enzyme was more stable under acidic conditions. Moreover, the half-life of the immobilized enzyme was 20.80 and 10.96 times as long as that of the free enzyme at 45 and 60 °C, respectively. The results show good reusability, as evidenced by the 84.77% retention of original enzyme activity after eight cycles. Additionally, the column transit time of the substrate was 35.56 min when the immobilized enzyme was applied in a packed-bed reactor. Furthermore, a consistently high FOS production yield of 60.68% was achieved and maintained over the 15-day monitoring period. Our results suggest that immobilized FTase is a viable candidate for continuous FOS production on an industrial scale. © 2024 Society of Chemical Industry.

Identification and characterisation of moderately thermostable diisobutyl phthalate degrading esterase from a Great Artesian Basin Bacillus velezensis NP05

Phthalate esters are known to be endocrine disrupting chemicals and are documented to pollute environments. Enzymatic degradation of PAEs is a potential bioremedial strategy to manage contamination. Thermostable bioremedial enzymes have advantages in enzyme manufacturing and storage. In this study, we identified, overexpressed, and characterised a moderately thermostable para-nitrobenzyl esterase from whole genome sequencing of a Bacillus velezensis NP05 from the Great Artesian Basin, capable of sequential 2-step hydrolysis of diisobutyl phthalate. The pnbA enzyme has a molecular weight of 55.14 kDa and pI of 5.31. It preferentially degrades para-nitrophenyl butanoate and has an optimal pH of 7–8. The pnbA esterase has an optimal temperature of 55 °C with a half-life of 4 h. Using HPLC we found that pnbA (0.122 U) can hydrolyse 0.83 mM of DIBP within 25 min. Lastly, pnbA is potentially a more economically viable candidate for enzymatic bioremediation of diisobutyl phthalate as a free enzyme.

Boosted Enzyme Activity via Encapsulation within Metal-Organic Frameworks with Pores Matching Enzyme Size and Shape.

A novel and versatile approach called "physical imprinting" is introduced to modulate enzyme conformation using mesoporous materials, addressing challenges in achieving improved enzyme activity and stability. Metal-organic frameworks with tailored mesopores, precisely matching enzyme size and shape, are synthesized. Remarkably, enzymes encapsulated within these customized mesopores exhibit over 1670% relative activity compared to free enzymes, maintaining outstanding efficiency even under harsh conditions such as heat, exposure to organic solvents, wide-ranging pH extremes from acidic to alkaline, and exposure to a digestion cocktail. After 18 consecutive cycles of use, the immobilized enzymes retain 80% of their initial activity. Additionally, the encapsulated enzymes exhibit a substantial increase in catalytic efficiency, with a 14.1-fold enhancement in kcat/KM compared to native enzymes. This enhancement is among the highest reported for immobilized enzymes. The improved enzyme activity and stability are corroborated by solid-state UV-vis, electron paramagnetic resonance, Fourier-transform infrared spectroscopy, and solid-state NMR spectroscopy. The findings not only offer valuable insights into the crucial role of size and shape complementarity within confined microenvironments but also establish a new pathway for developing solid carriers capable of enhancing enzyme activity and stability.

DNA Framework-Programmed Nanoscale Enzyme Assemblies.

Multienzyme assemblies mediated by multivalent interaction play a crucial role in cellular processes. However, the three-dimensional (3D) programming of an enzyme complex with defined enzyme activity in vitro remains unexplored, primarily owing to limitations in precisely controlling the spatial topological configuration. Herein, we introduce a nanoscale 3D enzyme assembly using a tetrahedral DNA framework (TDF), enabling the replication of spatial topological configuration and maintenance of an identical edge-to-edge distance akin to natural enzymes. Our results demonstrate that 3D nanoscale enzyme assemblies in both two-enzyme systems (glucose oxidase (GOx)/horseradish peroxidase (HRP)) and three-enzyme systems (amylglucosidase (AGO)/GOx/HRP) lead to enhanced cascade catalytic activity compared to the low-dimensional structure, resulting in ∼5.9- and ∼7.7-fold enhancements over homogeneous diffusional mixtures of free enzymes, respectively. Furthermore, we demonstrate the enzyme assemblies for the detection of the metabolism biomarkers creatinine and creatine, achieving a low limit of detection, high sensitivity, and broad detection range.

Stabilizing enzymatic membrane reactor for precise production of oligodextran with tailored molecular weight

Enzyme membrane reactor (EMR) provides an effective strategy for controlling molecular weight (Mw) of oligodextrans. However, the synergistic effect between dextran hydrolysis and product separation as well as continuous operation stability remains still unclear. We observed that covalently immobilized dextranase exhibited more obvious exo-hydrolysis behavior compared to free enzymes probably due to enzyme conformational alterations. Using non-covalently immobilized enzyme, suitable ultrafiltration membrane, optimal substrate/enzyme ratio and matched permeate flux, the yield of the target product reached 62.8%. However, the non-covalently immobilized dextranase exhibited reduced hydrolytic activity and more obvious membrane fouling (caused by high-Mw dextran). When free dextranase with high activity was used together with suitable permeate flux and high shear rate, the yield increased to 72.0%. The obtained EMR maintained stable for 8 h with slight fouling formation because enzymatic hydrolysis and membrane separation were well matched. The polydispersity index of the target product reached 1.3 (analytical grade) following diafiltration purification.

A hydroesterification reaction system to produce octyl esters using degummed soybean oil as substrate: Combining reusable free and immobilized lipases

This study shows the enzymatic synthesis of octyl alcohol-based biolubricants carried out in two catalytic steps: degummed soybean oil (DSO) hydrolysis and esterification of the produced and purified free fatty acids (FFA). Several lipases were evaluated as catalysts in the hydrolysis of DSO. Under optimal conditions, lipase from Pseudomonas fluorescens (PFL) yielded 97 % conversion after 23 h using 50 U/g oil and DSO:water mass ratio of 1:0.5. Sequential batch strategies have been developed for the recovery and reuse of free enzyme in further hydrolysis cycles. When hexane was used in the separation between the FFA-rich phase and the phase composed of water, enzyme and glycerol, there was better PFL recovery and FFA-conversion of 65 % was still achieved after five 24-hour hydrolytic cycles. In the esterification step, the potential of four lipases (commercial Lipozym-435, and lipase B from Candida antarctica (CALB), lipase from porcine pancreas (PPL) and Eversa® Transform 2.0 (EV) immobilized on Purolite Lifetech EC8806F) were evaluated in the production of octyl esters in a solvent-free medium (FFA/octanol molar ratio of 1:2.5) using screw-capped glass bottles as reactor. The immobilized EV gave the best ester yield (∼87 wt%). When EV-Purolite was used in a vortex flow reactor, a yield of around 94 wt% was reached after 3 h of reaction using a biocatalyst load of 1 % (w/v) and FFA/octanol molar ratio of 1:2.5. The immobilized lipase could be reused for five consecutive 3 h-batches of esterification, maintaining the octyl esters yield of the first batch.

Covalent Organic Frameworks Based Photoenzymatic Nano‐reactor for Asymmetric Dynamic Kinetic Resolution of Secondary Amines

AbstractBio‐catalysis represents a highly efficient and stereoselective method for the synthesis of valuable chiral compounds, however, the poor stability and limited reaction types of free enzymes restrict their wide application in industrial production. In this work, to overcome these problems, a multifunctional photoenzymatic nanoreactor CALB@COF‐Ir was developed through the encapsulation of Candida antarctica lipase B (CALB) in a photosensitive covalent organic framework COF‐Ir. This bio‐nanocluster serves as efficient catalysts in asymmetric dynamic kinetic resolution (DKR) of secondary amines to give a series of chiral amines in high yields (up to 99 %) and enantioselectivities (up to 99 % ee). The well‐designed COF‐Ir not only acts as safety cover to prevent CALB from deactivation but promotes racemization of secondary amines via photo‐induced hydrogen atom transfer (HAT) process. Photoelectric characterization and TDDFT calculation revealed that (ppy)2Ir units in COF‐Ir play crucial role in this photocatalytic system which enhance its photo‐redox properties through facilitating the separation between photoelectrons (e−) and holes (h+). Furthermore, the heterogeneous photoenzymatic nanoreactor could be recycled for five rounds with slight decline of catalytic reactivity.

Functionalized magnetic nanoparticles for Cellic Ctec2 cellulase immobilization: Allowing reusability of enzyme in the conversion of cellulosic biomass

The method of enzyme immobilization can ameliorate the overall stability and restoration of enzymes, hence facilitating their broader application in several sectors. This investigation utilized cellulase as a hydrolytic enzyme. In order to enhance the stability and performance of the cellulase enzyme, the research employed immobilization technology to secure the Cellic Ctec2 cellulase to the synthesized Cs@Fe3O4 nanocomposites. Fe3O4 nanoparticles (NPs) were coated with chitosan obtained from co-precipitation method that served as enzyme carrier. The NPs (Cs@ Fe3O4) were observed under XRD; VSM (vibrating-sample magnetometer) shows saturation magnetizations (Ms), UV–vis, field emission scanning electron microscopy (FESEM), and Fourier transform infrared spectroscopy (FT-IR). Response surface approach was applied to optimize the conditions for immobilization of cellulase. The optimum immobilization of cellulase reaches to 99.1% of loading efficiency and 69.7% of recovery activity with 2.5% of glutaraldehyde concentration. Furthermore, under ideal circumstances the immobilized enzyme’s thermostability, pH stability, temperature tolerance and reusability, were studied with respect to free cellulase. Higher relative activity of cellulase enzyme was observed at pH 5 with 50 °C temperature than free enzyme. One percent CMC hydrolysis is considered for reusability of free and immobilized enzyme and releases 222 mg glucose/g substrate at 24 h, showing great quiescence in cellulosic biomass conversion. Immobilized cellulase demonstrated high reusability by retaining almost 61.2% up to the 5th cycles and 51.2% of activity-maintained 10th cycle of hydrolysis. Reusability of cellulase enzyme can attain a gradual decrease in relative activity as number of repeats of the cycle increases to 10 during hydrolysis and increases in glucose yield after hydrolysis.

Various Strategies for the Immobilization of a Phospholipase C from Bacillus cereus for the Modulation of Its Biochemical Properties.

In this study, the effect of various immobilization methods on the biochemical properties of phospholipase C (PLC) from Bacillus cereus obtained from the oily soil located in Sfax, Tunisia, was described. Different supports were checked: octyl sepharose, glyoxyl agarose in the presence of N-acetyl cysteine, and Q-sepharose. In the immobilization by hydrophobic adsorption, a hyperactivation of the PLCBc was obtained with a fold of around 2 times. The recovery activity after immobilization on Q-sepharose and glyoxyl agarose in the presence of N-acetyl cysteine was 80% and 58%, respectively. Furthermore, the biochemical characterization showed an important improvement in the three immobilized enzymes. The performance of the various immobilized PLCBc was compared with the soluble enzyme. The derivatives acquired using Q-sepharose, octyl sepharose, and glyoxyl agarose were stable at 50 °C, 60 °C, and 70 °C. Nevertheless, the three derivatives were more stable in a large range of pH than the soluble enzyme. The three derivatives and the free enzyme were stable in 50% (v/v) ethanol, hexane, methanol, and acetone. The glyoxyl agarose derivative showed high long-term storage at 4 °C, with an activity of 60% after 19 days. These results suggest the sustainable biotechnological application of the developed immobilized enzyme.

Study on one-step purification and immobilization of glycosyltransferase by modified Fe3O4 for the synthesis of rare ginsenoside Rh2

Enzymatic catalysis is the most suitable method for synthesizing the ginsenoside Rh2. However, challenges in the purification and reuse of the enzyme limited the applications of this method. In this study, Fe3O4/PMG/NTA-Ni2+ magnetic nanospheres modified with nano-Fe3O4 were employed to achieve the one-step purification and immobilization of glycosyltransferase. The resulting assembly catalyzed ginsenoside protopanaxadiol (PPD) to more valuable Rh2. This material successfully immobilized the recombinant enzyme on the carrier. Moreover, the good dispersibility and superparamagnetism of the immobilized enzyme were maintained without any alteration in its structure, and the maximum immobilized enzyme amount was 180 mg/g. Notably, the pH value, temperature and storage stability of the immobilized enzyme were significantly improved compared with the free enzyme. After reusing the immobilized enzyme six times, the relative enzyme activity was still maintained at 67.8%. Thus, this study specifically adsorbed, purified, and immobilized free glycosyltransferase by modifying nanometer-sized Fe3O4 particles. This reduced operating costs and enhanced the properties of the enzyme.

Elucidation of enhanced cellulase immobilization onto synthetic magnetic nickel nanomaterials for lignocellulosic biomass hydrolysis

The immobilization of cellulase enzymes for the conversion of lignocellulosic biomass into sustainable biochemical products is essential for the stability and recovery of the enzymes. In this study, nickel nanoparticles (NiNPs) were synthesized and coated with 3-aminopropyl triethoxysilane (APTES) to serve as cellulase enzyme carriers. Cellulase enzyme immobilization on the prepared NiNPs was achieved using glutaraldehyde as a cross-linker. The physicochemical properties of the carrier were determined before and after enzyme immobilization using X-ray Powder Diffraction (XRD), Transmission Electron Microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), Vibrating-Sample Magnetometer (VSM), and Field Emission Scanning Electron Microscopy (FESEM). The impacts of different experimental factors on the performance of the cellulase enzymes immobilized on the synthesized NiNPs and commercial nickel nanoparticles were compared. The results showed comparable optimal cellulase enzyme immobilization conditions on both substrates in terms of the immobilization time, pH, and cellulase enzyme concentration. However, the recommended temperatures for cellulase enzyme immobilization on the synthesized and commercial NiNPs were 50 °C and 40 °C, respectively. Under these optimum conditions, the immobilized cellulase enzyme on the synthesized NiNPs had an activity of about 99.1% (in comparison to the activity of free cellulase enzyme), while the activity of the enzyme upon immobilization on commercial NiNPs was about 93%. The particle size of the NiNPs was found to be crucial for enzyme immobilization efficiency and its magnetic strength. Therefore, cellulase enzyme immobilization on tunable NiNPs could be a sustainable and eco-friendly approach towards high recovery of cellulose from lignocellulosic materials.

Termostable and effective immobilized invertase for sucrose determination in fruit juices

In this study, immobilization of invertase enzyme was performed on a previously synthesized and characterized poly(N-vinylpyrrolidone-co-butylacrylate-co-N hydroxymethylacrylamide) terpolymer membranes by covalent bonding method. Glutaraldehyde(GA) was used as the crosslinker and Bovine Serum Albumin(BSA) was used as the binding agent. Optimum pH, temperature, amount of polymer, substrate concentration, amount of BSA and amount of GA values were determined for both free and immobilized enzyme. Optimum pH and temperature values were found as pH = 5.0, T = 55 °C, pH = 7.0 and T = 80 °C for free and immobilized enzyme, respectively. In particular, the optimum temperature of 80 °C for the immobilized enzyme provides its potential to be used commercially. The kinetic parameters of the free enzyme and the immobilized enzyme were determined using the well known Lineweaver-Burk method. The Vmax values for free (13.4 μM/min) and immobilized enzyme (12.2 μM/min) were found as close to each other, while the Km value of the immobilized enzyme (8.33 mM) was much lower than that of the free enzyme (29.41 mM). In reuse studies conducted with peach and orange juices, it was determined that the immobilized enzyme retained approximately 90% of its activity even after 30 reuses within 1 month.

Encapsulation of Enzymes into Hydrophilic and Biocompatible Metal Azolate Framework: Improved Functions of Biocatalyst in Cascade Reactions and its Sensing Applications.

Multiple enzyme-triggered cascade biocatalytic reactions are vital in vivo or vitro, considering the basic biofunction preservation in living organisms and signals transduction for biosensing platforms. Encapsulation of such enzymes into carrier endows a sheltering effect and can boost catalytic performance, although the selection and preparation of an appropriate carrier is still a concern. Herein, focusing on MAF-7, a category of metal azolate framework (MAF) with superiority against the topologically identical ZIF-8, this enzyme@MAF system can ameliorate the sustainability of encapsulating natural enzymes into carriers. The proposed biocatalyst composite AChE@ChOx@MAF-7/hemin is constructed via one-pot in situ coprecipitation method. Subsequently, MAF-7 is demonstrated to exhibit an excellent capacity of the carrier and protection against external factors in the counterpart of ZIF-8 through encapsulated and free enzymes. In addition, detections for specific substrates or inhibitors with favorable sensitivity are accomplished, indicating that the properties above expectation of different aspects of the established platform are successfully realized. This biofunctional composite based on MAF-7 can definitely provide a potential approach for optimization of cascade reaction and enzyme encapsulation.

Deciphering the immobilization of lipases on hydrophobic wrinkled silica nanoparticles

In this work, the adsorption of Candida antarctica B (CALB) and Rhizomucor miehei (RML) lipases into hydrophobic wrinkled silica nanoparticles (WSNs) is investigated. WSNs are hydrophobized by chemical vapor deposition. Both proteins are homogeneously distributed inside the pores of the nanoparticles, as confirmed by Transmission Electron Microscopy and Energy Dispersive X-ray measurements. The maximum enzyme load of CALB is twice that obtained for RML. Fourier Transform Infrared Spectroscopy confirms the preservation of the enzyme secondary structure after immobilization for both enzymes. Adsorption isotherms fit to a Langmuir model, resulting in a binding constant (KL) for RML 4.5-fold higher than that for CALB, indicating stronger binding for the former. Kinetic analysis reveals a positive correlation between enzyme load and RML activity unlike CALB where activity decreases along the enzyme load increases. Immobilization allows for enhancing the thermal stability of both lipases. Finally, CALB outperforms RML in the hydrolysis of ethyl-3-hydroxybutyrate. However, immobilized CALB yielded 20 % less 3-HBA than free lipase, while immobilized RML increases 3-fold the 3-HBA yield when compared with the free enzyme. The improved performance of immobilized RML can be explained due to the interfacial hyperactivation undergone by this lipase when immobilized on the superhydrophobic surface of WSNs.

Green synthesis of NiO NPs for metagenome-derived laccase stabilization: Detoxifying pollutants and wastes

Laccases play a crucial role in neutralizing environmental pollutants, including antibiotics and phenolic compounds, by converting them into less harmful substances via a unique oxidation process. This study introduces an environmentally sustainable remediation technique, utilizing NiO nanoparticles (NPs) synthesized through green chemistry to immobilize a metagenome-derived laccase, PersiLac1, enhancing its application in pollutant detoxification. Salvadora persica leaf extract was used for the synthesis of NiO nanoparticles, utilizing its phytochemical constituents as reducing and capping agents, followed by characterization through different analyses. Characterization of NiO nanoparticles revealed distinctive FTIR absorption peaks indicating the nanoparticulate structure, while FESEM showed structured NiO with robust interconnections and dimensionality of about 50nm, confirmed by EDX analysis to have a consistent distribution of Ni and O. The immobilized PersiLac1 demonstrated enhanced thermal stability, with 85.55 % activity at 80 °C and reduced enzyme leaching, retaining 67.93 % activity across 15 biocatalytic cycles. It efficiently reduced rice straw (RS) phenol by 67.97 % within 210 min and degraded 70–78 % of tetracycline (TC) across a wide pH range (4.0–8.0), showing superior performance over the free enzyme. Immobilized laccase achieved up to 71 % TC removal at 40–80 °C, significantly outperforming the free enzyme. Notably, 54 % efficiency was achieved at 500 mg/L TC by immobilized laccase at 120 min. This research showed the potential of green-synthesized NiO nanoparticles to effectively immobilize laccase, presenting an eco-friendly approach to purify pollutants such as phenols and antibiotics. The durability and reusability of the immobilized enzyme, coupled with its ability to reduce pollutants, indicates a viable method for cleaning the environment. Nonetheless, the production costs and scalability of NiO nanoparticles for widespread industrial applications pose significant challenges. Future studies should focus on implementation at an industrial level and examine a wider range of pollutants to fully leverage the environmental clean-up capabilities of this innovative technology.

Bioproduction, purification, partial characterization and phenol removal efficacy of tyrosinase enzyme from Streptomyces sp. strain MR28

The study focused on the production of the tyrosinase enzyme from Streptomyces sp. MR28 and its potency in removal of phenol content from water using free and immobilized tyrosinase enzyme. The tyrosinase was produced by Streptomyces sp. MR28 in liquid tyrosine broth medium, and it was further purified to near its homogeneity by employing, precipitation, dialysis, and column chromatography. After the purification, 44.49% yield with a 4 fold purification was achieved. The characterization of the purified enzyme showed a single major peak on HPLC and a solitary band on SDS-PAGE. The purified tyrosinase enzyme was active at a pH of 7.0 and a temperature of 30 °C. Further immobilization of purified tyrosinase was performed using the sodium alginate entrapment method. The capacity of the purified tyrosinase to remove phenol in water was evaluated by spectrophotometric method. The free tyrosinase enzyme-treated solutions showed a gradual decrease in the concentration of phenol with increased incubation time at 30 °C and 40 °C, at 90 min of the incubation time, it showed maximum efficacy in removing phenol from the solution. At 50 °C and 60 °C, the free tyrosinase enzyme exhibited very less capacity to remove the phenol. The immobilized enzyme showed good capacity for the removal of phenol from the solutions; the concentration of phenol in the solution decreased with an increase in the incubation time. At temperatures of 40 °C and 50 °C, the immobilized tyrosinase enzyme beads showed significant removal of phenol from the solution, and at temperatures of 30 °C and 60 °C, they also exhibited good capacity for the removal of phenol. At the end of the 90 min incubation period, it exhibited good capability. The current study suggests using immobilized microbial tyrosinase enzyme can be used for the removal of phenol from the contaminated water in a greener manner.

Magnetic nanoparticles immobilized thrombin ligand fishing to screen thrombin inhibitors in natural products

In this study, thrombin was immobilized with magnetic particles modified by glutaraldehyde. The changes in secondary structures of immobilized enzyme revealed an increment in conformational rigidity and stability, which can be reflected in temperature and pH stability as well as the tolerance of organic reagents. The optimal reutilization times of magnetic particle immobilized thrombin were 7 times, and the half-life of enzyme activity preserved at room temperature was 5 days, which was 2.5 times higher than that of free enzyme. Ligusticum chuanxiong and Anemarrhenae Rhizoma with high enzyme inhibitory activity were selected for primary screening, and six potential inhibitors of thrombin were identified by HPLC/MS. The results showed that three compounds in Anemarrhenae Rhizoma had better predictive thrombin inhibitory activity. Through the in vitro thrombin activity inhibition experiment, it was also verified that mangiferin and neo-mangiferin had an ideal thrombin activity inhibition effect, which was consistent with the results of molecular docking.