Inquiry lipaseoring the mechanism of pancreatic lipase inhibition by isovitexin based on multispectral method and enzyme inhibition assay.

Isovitexin is a main natural flavonoid component in various plants. Currently, the inhibitory effect of isovitexin on pancreatic lipase (PL) and its mechanism have not been elucidated yet. In the present study, we investigated the inhibitory effect of isovitexin on PL, as well as its interaction mechanism, using enzyme inhibition methods, spectroscopic analysis, and molecular simulations. Results showed that isovitexin possessed significant PL inhibitory activity, with IC50 values of 0.26 ± 0.02 mM. The interaction between isovitexin and PL was dominated by static quenching, and mainly through hydrogen bonding and hydrophobic interaction forces. Analysis of fluorescence spectroscopy confirmed that isovitexin binding altered the conformation of the PL. Circular dichroism (CD) spectrum indicated that isovitexin altered the secondary structure of PL by decreasing the α-helix content and increasing the ß-fold content. Molecular simulations further characterize the conformational changes produced by the interaction between isovitexin with PL. The performed study may provide a new insight into the inhibitory mechanism of isovitexin as a novel PL inhibitor.

Engineered polymer nanoparticles as artificial chaperones facilitating the selective refolding of denatured enzymes.

Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these designs often have a limited range of substrates they can work with. In this paper, we present molecularly imprinted flexible polymer nanoparticles (nanoMIPs) designed as customizable biomimetic chaperones. We used model proteins such as cytochrome c, laccase, and lipase to screen polymeric monomers and identify the most effective formulations, offering tunable charge and hydrophobic properties. Utilizing a dispersed phase imprinting approach, we employed magnetic beads modified with destabilized whole-protein as solid-phase templates. This process involves medium exchange facilitated by magnetic pulldowns, resulting in the synthesis of nanoMIPs featuring imprinted sites that effectively mimic chaperone cavities. These nanoMIPs were able to selectively refold denatured enzymes, achieving up to 86.7% recovery of their activity, significantly outperforming control samples. Mechanistic studies confirmed that nanoMIPs preferentially bind denatured rather than native enzymes, mimicking natural chaperone interactions. Multifaceted analyses support the functionality of nanoMIPs, which emulate the protective roles of chaperones by selectively engaging with denatured proteins to inhibit aggregation and facilitate refolding. This approach shows promise for widespread use in protein recovery within biocatalysis and biomedicine.

Integrated Process for Schizochytrium Oil Extraction, Enzymatic Modification of Lipids and Concentration of DHA Fatty Acid Esters Using Alternative Methodologies.

Marine microalgae Schizochytrium sp. have a high content of docosahexaenoic acid (DHA), an omega-3 fatty acid that is attracting interest since it prevents certain neurodegenerative diseases. The obtention of a bioactive and purified DHA fatty acid ester using a whole-integrated process in which renewable sources and alternative methodologies are employed is the aim of this study. For this reason, lyophilized Schizochytrium biomass was used as an alternative to fish oil, and advanced extraction techniques as well as enzymatic modification were studied. Microalgal oil extraction was optimized via a surface-response method using pressurized liquid extraction (PLE) obtaining high oil yields (29.06 ± 0.12%) with a high concentration of DHA (51.15 ± 0.72%). Then, the enzymatic modification of Schizochytrium oil was developed by ethanolysis using immobilized Candida antarctica B lipase (Novozym® 435) at two reaction temperatures and different enzymatic loads. The best condition (40 °C and 200 mg of lipase) produced the highest yield of fatty acid ethyl ester (FAEE) (100%) after 8 h of a reaction attaining a cost-effective and alternative process. Finally, an enriched and purified fraction containing DHA-FAEE was obtained using open-column chromatography with a remarkably high concentration of 93.2 ± 1.3% DHA. The purified and bioactive molecules obtained in this study can be used as nutraceutical and active pharmaceutical intermediates of marine origin.

Switchable CO2-Responsive Janus Nanoparticle for Lipase Catalysis in Pickering Emulsion.

The use of convertible immobilized enzyme carriers is crucial for biphasic catalytic reactions conducted in Pickering emulsions. However, the intense mechanical forces during the conversion process lead to enzyme leakage, affecting the stability of the immobilized enzymes. In this study, a CO2-responsive switchable Janus (CrSJ) nanoparticle (NP) was developed using silica NP, with one side featuring aldehyde groups and the other side adsorbing N,N-dimethyldodecylamine. A switchable Pickering emulsion catalytic system for biphasic interface reactions was prepared by covalently immobilizing lipase onto the CrSJ NPs. The CO2-responsive nature of the CrSJ NPs allowed for rapid conversion of the Pickering emulsion, and covalent immobilization substantially reduced lipase leakage while enhancing the stability of the immobilization during the conversion process. Impressively, after repeated transformations, the Pickering emulsion still maintains its original structure. Following 10 consecutive cycles of esterification and hydrolysis reactions, the immobilized enzyme's activity remains at 77.7 and 79.5% of its initial activity, respectively. The Km of the CrSJ catalytic system showed no significant change compared to the free enzyme, while its Vmax values were 1.2 and 1.6 times that of the free enzyme in esterification and hydrolysis reactions, respectively.

Hydrophobic Clusters Regulate Surface Hydration Dynamics of Bacillus subtilis Lipase A.

The surface hydration diffusivity of Bacillus subtilis Lipase A (BSLA) has been characterized by low-field Overhauser dynamic nuclear polarization (ODNP) relaxometry using a series of spin-labeled constructs. Sites for spin-label incorporation were previously designed via an atomistic computational approach that screened for surface exposure, reflective of the surface hydration comparable to other proteins studied by this method, as well as minimal impact on protein function, dynamics, and structure of BSLA by excluding any surface site that participated in greater than 30% occupancy of a hydrogen bonding network within BSLA. Experimental ODNP relaxometry coupling factor results verify the overall surface hydration behavior for these BSLA spin-labeled sites similar to other globular proteins. Here, by plotting the ODNP parameters of relative diffusive water versus the relative bound water, we introduce an effective "phase-space" analysis, which provides a facile visual comparison of the ODNP parameters of various biomolecular systems studied to date. We find notable differences when comparing BSLA to other systems, as well as when comparing different clusters on the surface of BSLA. Specifically, we find a grouping of sites that correspond to the spin-label surface location within the two main hydrophobic core clusters of the branched aliphatic amino acids isoleucine, leucine, and valine cores observed in the BSLA crystal structure. The results imply that hydrophobic clustering may dictate local surface hydration properties, perhaps through modulation of protein conformations and samplings of the unfolded states, providing insights into how the dynamics of the hydration shell is coupled to protein motion and fluctuations.

Optical Fiber Surface Plasmon Resonance Sensor for Glyceryl Tributyrate Detection Based on the PAA/CS Composite Hydrogel Embedding Protease Method.

Glycerol tributyrate as a low-density lipoprotein plays a crucial role in drug development and food safety. In this work, a novel high-stability fiber optic sensor for glyceryl tributyrate based on the poly(acrylic acid) (PAA) and chitosan (CS) composite hydrogel embedding method is first proposed. Compared with traditional functionalization, the lipase in a polymer network structure used in this article can not only avoid chemical reactions that cause damage to the enzyme structure but also avoid the instability of ionic bonds and physical adsorption. Therefore, the PAA/CS hydrogel method proposed in this article can effectively retain enzyme structure. First, the impact of different layers (one to five layers) of PAA/CS on pH sensing performance was explored, and it was determined that layers 1-3 could be used for subsequent sensing experiments. Within the linear detection range of 0.5-10 mM, the detection sensitivities of the one to three layers of the biosensor are divided into 0.65, 0.95, and 1.51 nm/mM, respectively, with the three layers having the best effect. When the number of coating layers is three, the detection limit of the sensor is 0.47 mM, meeting the millimole level detection standard for anticancer requirement. Furthermore, the stability and selectivity of the sensor (in the presence of hemoglobin, urea, cholesterol, acetylcholine, and glucose) were analyzed. The three-layer sensor is used for sample detection. At concentrations of 1-10 mM, the absolute value of the recovery percentage (%) is 82-99%, which can accurately detect samples. The sensor proposed in this paper has the advantages of low sample consumption, high sensitivity, simple structure, and label-free measurement. The enzyme-embedding method provides a new route for rapid and reliable glyceryl tributyrate detection, which has potential applications in food safety as well as the development of anticancer drugs.

Tailoring the interfacial microenvironment of magnetic metal-organic frameworks using amino-acid-based ionic liquids for lipase immobilization.

Modifying the carrier interface is a promising method to improve the microenvironment of immobilized enzymes and enhance their activity and stability. In this work, using proline as amino acid, magnetic metal-organic frameworks (MOFs) were modified with an amino-acid-based ionic liquid (AAIL) with two hydroxyl groups followed by adsorption of porcine pancreatic lipase (PPL). The activity recovery of the prepared immobilized lipase (MMOF-AAIL/PPL) was up to 162 % higher than that of MMOF-PPL (70.8 %). The Michaelis constant of MMOF-AAIL/PPL was 0.0742 mM lower than that of MMOF-PPL, but the catalytic efficiency was 0.0223 min-1 which was higher than MMOF-PPL. Furthermore, MMOF-AAIL/PPL maintained 85.6 % residual activity after stored for 40 days and its residual activity was 71.9 % while that for MMOF-PPL was 58.8 % after incubated in 6 M urea for 2 h. Particularly, after ten consecutive cycles, the residual activity of MMOF-AAIL/PPL still reached 84.4 %. In addition, the magnetic properties of the support facilitate the separation process which improves the utilization efficiency of immobilized enzymes.

Enzymatic Synthesis of Copolyesters with the Heteroaromatic Diol 3,4-Bis(hydroxymethyl)furan and Isomeric Dimethyl Furandicarboxylate Substitutions.

Polyesters from furandicarboxylic acid derivatives, i.e., dimethyl 2,5-furandicarboxylate (2,5-DMFDCA) and 2,4-DMFDCA, show interesting properties among bio-based polymers. Another potential heteroaromatic monomer, 3,4-bis(hydroxymethyl)furan (3,4-BHMF), is often overlooked but holds promise for biopolymer synthesis. Cleaning and greening synthetic procedures, i.e., enzymatic polymerization, offer sustainable pathways. This study explores the Candida antarctica lipase B (CALB)-catalyzed copolymerization of 3,4-BHMF with furan dicarboxylate isomers and aliphatic diols. The furanic copolyesters (co-FPEs) with higher polymerization degrees are obtained using 2,4-isomer, indicating CALB's preference. Material analysis revealed semicrystalline properties in all synthesized 2,5-FDCA-based co-FPEs, with multiple melting temperatures (Tm) from 53 to 124 °C and a glass-transition temperature (Tg) of 9-10 °C. 2,4-FDCA-based co-FPEs showed multiple Tm from 43 to 61 °C and Tg of -14 to 12 °C; one of them was amorphous. In addition, all co-FPEs showed a two-step decomposition profile, indicating aliphatic and semiaromatic segments in the polymer chains.

Alloyed Trimetallic Nanocomposite as an Efficient and Recyclable Solid Matrix for Ideonella sakaiensis Lipase Immobilization.

In this work, a trimetallic (Ni/Co/Zn) organic framework (tMOF), synthesized by a solvothermal method, was calcinated at 400 and 600 °C and the final products were used as a support for lipase immobilization. The material annealed at 400 °C (Ni-Co-Zn@400) had an improved surface area (66.01 m2/g) and pore volume (0.194 cm3/g), which showed the highest enzyme loading capacity (301 mg/g) with a specific activity of 0.196 U/mg, and could protect the enzyme against thermal denaturation at 65 °C. The optimal pH and temperature for the lipase were 8.0 and 45 °C but could tolerate pH levels 7.0-8.0 and temperatures 40-60 °C. Moreover, the immobilized enzyme (Ni-Co-Zn@Lipase, Ni-Co-Zn@400@Lipase, or Ni-Co-Zn@600@Lipase) could be recovered and reused for over seven cycles maintaining 80, 90, and 11% of its original activity and maintained a residual activity >90% after 40 storage days. The remarkable thermostability and storage stability of the immobilized lipase suggest that the rigid structure of the support acted as a protective shield against denaturation, while the improved pH tolerance toward the alkaline range indicates a shift in the ionization state attributed to unequal partitioning of hydroxyl and hydrogen ions within the microenvironment of the active site, suggesting that acidic residues may have been involved in forming an enzyme-support bond. The high enzyme loading capacity, specific activity, encouraging stability, and high recoverability of the tMOF@Lipase indicate that a multimetallic MOF could be a better platform for efficient enzyme immobilization.

Molecular simulations guide immobilization of lipase on nest-like ZIFs with regulatable hydrophilic/hydrophobic surface.

The catalytic performance of immobilized lipase is greatly influenced by functional support, which attracts growing interest for designing supports to achieve their promotive catalytic activity. Many lipases bind strongly to hydrophobic surfaces where they undergo interfacial activation. Herein, the behavioral differences of lipases with distinct lid structures on interfaces of varying hydrophobicity levels were firstly investigated by molecular simulations. It was found that a reasonable hydrophilic/hydrophobic surface could facilitate the lipase to undergo interfacial activation. Building on these findings, a novel "nest"-like superhydrophobic ZIFs (ZIFN) composed of hydrophobic ligands was prepared for the first time and used to immobilize lipase from Aspergillus oryzae (AOL@ZIFN). The AOL@ZIFN exhibited 2.0-folds higher activity than free lipase in the hydrolysis of p-Nitrophenyl palmitate (p-NPP). Especially, the modification of superhydrophobic ZIFN with an appropriate amount of hydrophilic tannic acid can significantly improve the activity of the immobilized lipase (AOL@ZIFN-TA). The AOL@ZIFN-TA exhibited 30-folds higher activity than free lipase, and still maintained 82% of its initial activity after 5 consecutive cycles, indicating good reusability. These results demonstrated that nanomaterials with rational arrangement of the hydrophilic/hydrophobic surface could facilitate the lipase to undergo interfacial activation and improve its activity, displaying the potential of the extensive application.

Extending the computational and experimental analysis of lipase active site selectivity.

Molecular docking is an important computational analysis widely used to predict the interaction of enzymes with several starting materials for developing new valuable products from several starting materials, including oils and fats. In the present study, molecular docking was used as an efficient in silico screening tool to select biocatalysts with the highest catalytic performance in butyl esters production in a solvent-free system, an eco-friendly approach, via direct esterification of free fatty acids from Licuri oil with butanol. For such purpose, three commercial lipase preparations were used to perform molecular docking studies such as Burkholderia cepacia (BCL), Porcine pancreatic (PPL), and Candida rugosa (CRL). Concurrently, the results obtained in BCL and CRL are the most efficient in the esterification process due to their higher preference for catalyzing the esterification of lauric acid, the main fatty acid found in the licuri oil composition. Meanwhile, PPL was the least efficient because it preferentially interacts with minor fatty acids. Molecular docking with the experimental results indicated the better performance in the synthesis of esters was BCL. In conclusion, experimental results analysis shows higher enzymatic productivity in esterification reactions of 1294.83 µmol/h.mg, while the CRL and PPL demonstrated the lowest performance (189.87 µmol / h.mg and 23.96 µmol / h.mg, respectively). Thus, molecular docking and experimental results indicate that BCL is a more efficient lipase to produce fatty acids and esters from licuri oil with a high content of lauric acid. In addition, this study also demonstrates the application of molecular docking as an important tool for lipase screening to achieve more sustainable production of butyl esters with a view synthesis of biolubricants.

Calcium-based MOFs as scaffolds for shielding immobilized lipase and enhancing its stability.

The enzyme immobilization technology has become a key tool in the field of enzyme applications; however, improving the activity recovery and stability of the immobilized enzymes is still challenging. Herein, we employed a magnetic carboxymethyl cellulose (MCMC) nanocomposite modified with ionic liquids (ILs) for covalent immobilization of lipase, and used Ca-based metal-organic frameworks (MOFs) as the support skeleton and protective layer for immobilized enzymes. The ILs contained long side chains (eight CH2 units), which not only enhanced the hydrophobicity of the carrier and its hydrophobic interaction with the enzymes, but also provided a certain buffering effect when the enzyme molecules were subjected to compression. Compared to free lipase, the obtained CaBPDC@PPL-IL-MCMC exhibited higher specific activity and enhanced stability. In addition, the biocatalyst could be easily separated using a magnetic field, which is beneficial for its reusability. After 10 cycles, the residual activity of CaBPDC@PPL-IL-MCMC could reach up to 86.9%. These features highlight the good application prospects of the present immobilization method.

Enzymes in "Green" Synthetic Chemistry: Laccase and Lipase.

Enzymes play an important role in numerous natural processes and are increasingly being utilized as environmentally friendly substitutes and alternatives to many common catalysts. Their essential advantages are high catalytic efficiency, substrate specificity, minimal formation of byproducts, and low energy demand. All of these benefits make enzymes highly desirable targets of academic research and industrial development. This review has the modest aim of briefly overviewing the classification, mechanism of action, basic kinetics and reaction condition effects that are common across all six enzyme classes. Special attention is devoted to immobilization strategies as the main tools to improve the resistance to environmental stress factors (temperature, pH and solvents) and prolong the catalytic lifecycle of these biocatalysts. The advantages and drawbacks of methods such as macromolecular crosslinking, solid scaffold carriers, entrapment, and surface modification (covalent and physical) are discussed and illustrated using numerous examples. Among the hundreds and possibly thousands of known and recently discovered enzymes, hydrolases and oxidoreductases are distinguished by their relative availability, stability, and wide use in synthetic applications, which include pharmaceutics, food and beverage treatments, environmental clean-up, and polymerizations. Two representatives of those groups-laccase (an oxidoreductase) and lipase (a hydrolase)-are discussed at length, including their structure, catalytic mechanism, and diverse usage. Objective representation of the current status and emerging trends are provided in the main conclusions.

Design of a gold nanoparticles site in an engineered lipase: an artificial metalloenzyme with enantioselective reductase-like activity.

The conjugation of gold complexes with proteins has proved to be interesting and effective in obtaining artificial metalloenzymes as catalysts with improved properties such as higher stability, activity and selectivity. However, the design and precise regulation of their structure as protein nanostructured forms level remains a challenge. Here, we have designed and constructed a gold nanoparticles-enzyme bioconjugate, by tailoring the in situ formation of gold nanoparticles (AuNPs) at two specific sites on the structure of an alkalophilic lipase from Geobacillus thermocatenulatus (GTL). For this purpose, two genetically modified variants of GTL were created by inserting a unique cysteine residue into the catalytic active site by replacing the active serine (GTL-114) and into the lid site (GTL-193). The enzyme, after a first protein-gold coordination, induced the in situ formation of AuNPs, generating a homogeneous artificial enzyme. The size and morphology of the nanoparticles in the AuNPs-enzyme conjugate have been controlled by specific pH conditions in synthesis and the specific protein region where they are formed. Reductase activity of all of them was confirmed in the hydrogenation of nitroarenes in aqueous media. The protein area seemed to be key for the AuNPs, with the best TOF values obtained for the bioconjugates with AuNPs in the lid site. Finally, the protein environment and the asymmetric properties of the AuNPs were tested in the reduction of acetophenone to 1-phenylethanol in aqueous medium at room temperature. A high reductive conversion and an enantiomeric excess of up to 39% towards (R)-1-phenylethanol was found using Au-Mt@GTL-114 pH 10 as a catalyst. Moderate enantioselectivity towards the opposite isomer was also observed using the Au-Mt@GTL-193 pH 10 conjugate.

Enhancing biocatalyst performance through immobilization of lipase (Eversa® Transform 2.0) on hybrid amine-epoxy core-shell magnetic nanoparticles.

Magnetic nanoparticles were functionalized with polyethylenimine (PEI) and activated with epoxy. This support was used to immobilize Lipase (Eversa® Transform 2.0) (EVS), optimization using the Taguchi method. XRF, SEM, TEM, XRD, FTIR, TGA, and VSM performed the characterizations. The optimal conditions were immobilization yield (I.Y.) of 95.04 ± 0.79 %, time of 15 h, ionic load of 95 mM, protein load of 5 mg/g, and temperature of 25 °C. The maximum loading capacity was 25 mg/g, and its stability in 60 days of storage showed a negligible loss of only 9.53 % of its activity. The biocatalyst demonstrated better stability at varying temperatures than free EVS, maintaining 28 % of its activity at 70 °C. It was feasible to esterify free fatty acids (FFA) from babassu oil with the best reaction of 97.91 % and ten cycles having an efficiency above 50 %. The esterification of produced biolubricant was confirmed by NMR, and it displayed kinematic viscosity and density of 6.052 mm2/s and 0.832 g/cm3, respectively, at 40 °C. The in-silico study showed a binding affinity of -5.8 kcal/mol between EVS and oleic acid, suggesting a stable substrate-lipase combination suitable for esterification.

Kinetic and thermodynamic-based studies on the interaction mechanism of novel R. roxburghii seed peptides against pancreatic lipase and cholesterol esterase.

Pancreatic lipase (PL) and cholesterol esterase (CE) are vital digestive enzymes that regulate lipid digestion. Three bioactive peptides (LFCMH, RIPAGSPF, YFRPR), possessing enzyme inhibitory activities, were identified in the seed proteins of R. roxburghii. It is hypothesized that these peptides could inhibit the activities of these enzymes by binding to their active sites or altering their conformation. The results showed that LFCMH exhibited superior inhibitory activity against these enzymes compared to the other peptides. The inhibition mechanisms of the three peptides were identified as either competitive or mixed, according to inhibition models. Further studies have shown that peptides could bind to the active sites of enzymes, thus affecting their spatial conformation and restricting substrate entry into the active site. Molecular simulation further proved that hydrogen bonds and hydrophobic interactions played a vital role in the binding of peptides to enzymes. This study enriches our understanding of interaction mechanisms of peptides on PL and CE.

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.

Kinetic modeling and optimization of the mono- and diglycerides synthesis mediated by the lipase Lipozyme® TL 100 L immobilized on clayey support.

Mono- and diglycerides play a crucial role in the food industry as multifunctional food additives and emulsifiers. Their importance stems from their unique properties, which allow them to improve the quality, texture, and stability of various food products. Here, results of the kinetic modeling of the mono- and diglycerides synthesis mediated by the lipase Lipozyme® TL 100 L immobilized on the clayey support Spectrogel® type C are reported. The support was characterized by TEM, SEM, and FTIR. Firstly, the influence of pH and lipase load on the immobilization process was analyzed, resulting in an enzymatic activity of 93.2 ± 0.7 U g-1 under optimized conditions (170.9 U g-1 of lipase and pH of 7.1). Afterward, the effects of reaction temperature and concentration of immobilized biocatalyst in the feedstock conversion were evaluated. At optimized parameters, a triglycerides conversion of 97% was obtained at 36.5 °C, 7.9 vol.% of enzyme, a glycerol to feedstock molar ratio of 2:1, and 2 h. The optimized conditions were used to determine the kinetic constants of the elementary reactions involved in the glycerolysis, where a fit superior to 0.99 was achieved between experimental values and predicted data.

Assessment of the efficiency and stability of enzymatic membrane reaction utilizing lipase covalently immobilized on a functionalized hybrid membrane.

Enzyme immobilization in membrane bioreactors has been considered as a practical approach to enhance the stability, reusability, and efficiency of enzymes. In this particular study, a new type of hybrid membrane reactor was created through the phase inversion method, utilizing hybrid of graphene oxide nanosheets (GON) and polyether sulfone (PES) in order to covalently immobilize the Candida rugosa lipase (CRL). The surface of hybrid membrane was initially modified by (3-Aminopropyl) triethoxysilane (APTES), before the use of glutaraldehyde (GLU), as a linker, through the imine bonds. The resulted enzymatic hybrid membrane reactors (EHMRs) were then thoroughly analyzed by using field-emission scanning electron microscopy (FE-SEM), contact angle goniometry, surface free energy analysis, X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, attenuated total reflection (ATR), and energy-dispersive X-ray (EDX) spectroscopy. The study also looked into the impact of factors such as initial CRL concentration, storage conditions, and immobilization time on the EHMR's performance and activity, which were subsequently optimized. The results demonstrated that the CRLs covalently immobilized on the EHMRs displayed enhanced pH and thermal stability compared to those physically immobilized or free. These covalently immobilized CRLs could maintain over 60% of their activity even after 6 reaction cycles spanning 50 days. EHMRs are valuable biocatalysts in developing various industrial, environmental, and analytical processes.

Lipase in-situ immobilized in covalent organic framework: Enzymatic properties and application in the preparation of 1, 3-dioleoyl-2-palmitoylglycerol.

In this study, the critical importance of designing an appropriate immobilized carrier and method for free lipase to ensure exceptional biological catalytic activity and stability was emphasized. Covalent organic frameworks (COF-1) were synthesized as a novel porous carrier with an azine structure (-CN-NC-) through the condensation of hydrazine hydrate and benzene-1,3,5-tricarbaldehyde at room temperature. Simultaneously, Rhizomucor miehei lipase (RML) was immobilized within the COF-1 carrier using an in-situ aqueous phase method. Characterization of the carrier and RML@COF-1 and evaluation of the lipase properties of RML and RML@COF-1 through p-Nitrophenyl palmitate hydrolysis were conducted. Additionally, application in the synthesis of 1, 3-dioleoyl-2-palmitoylglycerol (OPO) was explored. The results showed that RML@COF-1 exhibited a high enzymatic loading of 285.4 mg/g. Under 60℃ conditions, the activity of RML@COF-1 was 2.31 times higher than that of free RML, and RML@COF-1 retained 77.25% of its original activity after 10 cycles of repeated use, indicating its excellent thermal stability and repeatability. Under the optimal conditions (10%, 1:8 PPP/OA, 45℃, 5 h), the yield of OPO reached 47.35%, showcasing the promising application prospects of the novel immobilized enzyme synthesized via in-situ aqueous phase synthesis for OPO preparation.