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.

Bacterial nanocellulose loaded with bromelain and nisin as a promising bioactive material for wound debridement.

The bacterial nanocellulose (BnC) membranes were produced extracellularly by a novel aerobic acetic acid bacterium Komagataeibacter melomenusus. The BnC was modified in situ by adding carboxymethyl cellulose (CMC) into the culture media, obtaining a BnC-CMC product with denser fibril arrangement, improved rehydration ratio and elasticity in comparison to BnC. The proteolytic enzyme bromelain (Br) and antimicrobial peptide nisin (N) were immobilized to BnC matrix by ex situ covalent binding and/or adsorption. The optimal Br immobilization conditions towards the maximized specific proteolytic activity were investigated by response surface methodology as factor variables. At optimal conditions, i.e., 8.8 mg/mL CMC and 10 mg/mL Br, hyperactivation of the enzyme was achieved, leading to the specific proteolytic activity of 2.3 U/mg and immobilization efficiency of 39.1 %. The antimicrobial activity was observed against Gram-positive bacteria (S. epidermidis, S. aureus and E. faecalis) for membranes with immobilized N and was superior when in situ modified BnC membranes were used. N immobilized on the BnC or BnC-CMC membranes was cytocompatible and did not cause changes in normal human dermal fibroblast cell morphology. BnC membranes perform as an efficient carrier for Br or N immobilization, holding promise in wound debridement and providing antimicrobial action against Gram-positive bacteria, respectively.

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.

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.

Support Materials of Organic and Inorganic Origin as Platforms for Horseradish Peroxidase Immobilization: Comparison Study for High Stability and Activity Recovery.

In the presented study, a variety of hybrid and single nanomaterials of various origins were tested as novel platforms for horseradish peroxidase immobilization. A thorough characterization was performed to establish the suitability of the support materials for immobilization, as well as the activity and stability retention of the biocatalysts, which were analyzed and discussed. The physicochemical characterization of the obtained systems proved successful enzyme deposition on all the presented materials. The immobilization of horseradish peroxidase on all the tested supports occurred with an efficiency above 70%. However, for multi-walled carbon nanotubes and hybrids made of chitosan, magnetic nanoparticles, and selenium ions, it reached up to 90%. For these materials, the immobilization yield exceeded 80%, resulting in high amounts of immobilized enzymes. The produced system showed the same optimal pH and temperature conditions as free enzymes; however, over a wider range of conditions, the immobilized enzymes showed activity of over 50%. Finally, a reusability study and storage stability tests showed that horseradish peroxidase immobilized on a hybrid made of chitosan, magnetic nanoparticles, and selenium ions retained around 80% of its initial activity after 10 repeated catalytic cycles and after 20 days of storage. Of all the tested materials, the most favorable for immobilization was the above-mentioned chitosan-based hybrid material. The selenium additive present in the discussed material gives it supplementary properties that increase the immobilization yield of the enzyme and improve enzyme stability. The obtained results confirm the applicability of these nanomaterials as useful platforms for enzyme immobilization in the contemplation of the structural stability of an enzyme and the high catalytic activity of fabricated biocatalysts.

Optimizing the enzymatic production of biolubricants by the Taguchi method: Esterification of the free fatty acids from castor oil with 2-ethyl-1-hexanol catalyzed by Eversa Transform 2.0.

The solvent-free esterification of the free fatty acids (FFAs) obtained by the hydrolysis of castor oil (a non-edible vegetable oil) with 2-ethyl-1-hexanol (a branched fatty alcohol) was catalyzed by different free lipases. Eversa Transform 2.0 (ETL) features surpassed most commercial lipases. Some process parameters were optimized by the Taguchi method (L16'). As a result, a conversion over 95% of the FFAs of castor oil into esters with lubricants properties was achieved under optimized reaction conditions (15 wt% of biocatalyst content, 1:4 molar ratio (FFAs/alcohol), 30 °C, 180 rpm, 96 h). The substrates molar ratio had the highest influence on the dependent variable (conversion at 24 h). FFAs/2-ethyl-1-hexanol esters were characterized regarding the physicochemical and tribological properties. Interestingly, the modification of the FFAs with 2-ethyl-1-hexanol by ETL increased the oxidative stability of the FFAs feedstock from 0.18 h to 16.83 h. The biolubricants presented a lower friction coefficient than the reference commercial mineral lubricant (0.052 ± 0.07 against 0.078 ± 0.04). Under these conditions, ETL catalyzed the oligomerization of ricinoleic acid (a hydroxyl fatty acid) into estolides, reaching a conversion of 25.15% of the initial FFAs (for the first time).

Covalent immobilization of ß-galactosidase using a novel carrier alginate/tea waste: statistical optimization of beads modification and reusability.

ß-galactosidase has been immobilized onto novel alginate/tea waste gel beads (Alg/TW) via covalent binding. Alg/TW beads were subjected to chemical modification through amination with polyethyleneimine (PEI) followed by activation with glutaraldehyde (GA). Chemical modification parameters including PEI concentration, PEI pH, and GA concentration were statistically optimized using Response Surface methodology (RSM) based on Box-Behnken Design (BBD). Analysis of variance (ANOVA) results confirmed the great significance of the model that had F value of 37.26 and P value < 0.05. Furthermore, the R2 value (0.9882), Adjusted R2 value (0.9617), and predicted R2 value (0.8130) referred to the high correlation between predicted and experimental values, demonstrating the fitness of the model. In addition, the coefficient of variation (CV) value was 2.90 that pointed to the accuracy of the experiments. The highest immobilization yield (IY) of ß-galactosidase (75.1%) was given under optimized conditions of PEI concentration (4%), PEI pH (9.5), and GA concentration (2.5%). Alg/TW beads were characterized by FT-IR, TGA, and SEM techniques at each step of immobilization process. Moreover, the immobilized ß-galactosidase revealed a very good reusability as it could be reused for 15 and 20 consecutive cycles keeping 99.7 and 72.1% of its initial activity, respectively. In conclusion, the environmental waste (tea waste) can be used in modern technological industries such as the food and pharmaceutical industry.

Methyl oleate for plant protection products formulations: Enzymatic synthesis, reaction kinetics and application testing.

This study presents a solvent-free enzymatic approach for the synthesis of fatty acid methyl esters (FAMEs), such as methyl oleate, for their application as adjuvant in plant protection products (PPP) formulations. The direct esterification between free fatty acid and methanol was optimized to achieve 98% acid conversion. The kinetics of this conversion was accurately described by a simple second order mechanism and non-linear regression was applied to calculate the rate constants of the forward and backward reactions based on full progress curves data. The rate constant of the forward reaction (synthesis) was one order of magnitude higher than the backward reaction (hydrolysis) and favored formation of the target methyl ester product, rendering the removal of water unnecessary. Enzymatically synthesized methyl oleate was benchmarked against the chemically synthesized compound, showing matching results in terms of stability, spreadability and emulsifying capacity in plant care formulations. The enzymatic synthesis of FAMEs under solvent free conditions allows to achieve a safer and more sustainable character for carrier solvents in PPP formulations.

Enzymatic production process of capric acid-rich structured lipids: Development of formulation as a new therapeutic approach.

The present work reports an optimization of the synthesis of MLM-type (medium, long, medium) structured lipids (SL) through an acidolysis reaction of grape seed oil with capric acid catalyzed by Rhizopus oryzae lipase immobilized. At first, tests were carried out by preparing the biocatalysts using enzyme loadings (0.15 to 1 g of enzymatic powder) for each gram of support. Enzyme loading was used 0.3 g of enzymatic powder, and hydrolytic activity of 1860 ± 23.4 IU/g was reached. Optimized conditions determined by the Central Composite Rotatable Design (CCRD) revealed that the acidolysis reaction reached approximately 59 % incorporation degree (%ID) after 24 h, in addition to the fact that the biocatalyst could maintain the incorporation degree in five consecutive cycles. From this high incorporation degree, cell viability assays were performed with murine fibroblast cell lines and human cervical adenocarcinoma cell lines. Concerning the cytotoxicity assays, the concentration of MLM-SL to 1.75 and 2 % v/v were able to induce cell death in 56 % and 64 % of adenocarcinoma cells, respectively. Human cervical adenocarcinoma cells showed greater sensitivity to the induction of cell death when using emulsions with MLM-SL > 1.75 % v/v compared to emulsions with lower content indicating a potential for combating carcinogenic cells.

Preparation of magnetic dialdehyde starch-immobilized phospholipase A1 and acyl transfer in reflection.

In this paper, using a coprecipitation method to prepare Fe3O4 magnetic nanoparticles (Fe3O4 MNPS), magnetic dialdehyde starch nanoparticles with immobilized phospholipase A1 (MDSNIPLA) were successfully prepared by using green dialdehyde starch (DAS) instead of glutaraldehyde as the crosslinking agent. The Fe3O4 MNPS was characterized by infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), the Brunauer-Emmett-Teller (BET) surface area analysis method, thermogravimetric analysis (TGA), and transmission electron microscopy (TEM) et al. The results showed that the alkaline resistance and acid resistance of the enzyme were improved after the crosslinking of DAS. After repeated use (seven times), the relative activity of MDSNIPLA reached 56 %, and the magnetic dialdehyde starch nanoparticles (MDASN) had good carrier performance. MDSNIPLA was applied to enzymatic hydrolysis of phospholipids in the soybean oil degumming process. The results showed that the acyl transfer rate of sn-2-HPA was 14.01 %, and the content of free fatty acids was 1.144 g/100 g after 2 h reaction at 50 °C and pH 5.0 with appropriate boric acid. The immobilized enzyme has good thermal stability and storage stability, and its application of soybean oil improves the efficiency of the oil.

Catechol-tetraethylenepentamine co-deposition modified cellulose filter paper for α-glucosidase immobilization and inhibitor screening from traditional Chinese medicine.

Cellulose filter paper (CFP) is expected to be an ideal carrier for enzyme immobilization due to its sustainability and biocompatibility. However, the interaction between the carrier and enzyme might change the spatial conformation of the enzyme and its microenvironment, and thus the flexibility of the enzyme molecule or the transport of the substrate to the active site would be hampered. In this work, a two-component system of catechol and tetraethylene pentamine was used to replace dopamine, and a polydopamine-like composite layer was deposited on the surface of CFP to introduce amino groups, which was similar to the self-polymerization-adhesion behavior of dopamine. Using polyethylene glycol diglycidyl ether with flexible spacer arms as the cross-linking agent, α-glucosidase was covalently bonded to amino-modified CFP through an epoxy ring-opening reaction. The immobilized α-glucosidase exhibited greater tolerance to pH and high temperature. After 10 repeated uses, the immobilized α-glucosidase maintained relatively high enzyme activity. Its kinetic behavior was investigated to illustrate the reliability for enzyme inhibitor screening. Finally, a screening method combining an immobilized enzyme and capillary electrophoresis analysis was proposed and applied to screening inhibitors from 11 kinds of traditional Chinese medicines, among which Chebulae Fructus, Phyllanthi Fructus and Terminaliae Relliricae Fructus exhibited strong enzyme inhibitory activities.

Enzyme immobilized on magnetic fluorescent bifunctional nanoparticles for α-glucosidase inhibitors virtual screening from Agrimonia pilosa Ledeb extracts accompanied with molecular modeling.

Agrimonia pilosa Ledeb (APL) is a significant source of inhibitors for α-glucosidase, which is an essential target enzyme for the treatment of type 2 diabetes, cancer and acquired immune deficiency syndrome. Ligand fishing is a suitable approach for the highly selective screening of bioactive substances in complex mixtures. Yet it is unable to conduct biomedical imaging screening, which is crucial for real-time identification. In this case, a bioanalytical platform combining magnetic fluorescent ligand fishing and in-situ imaging technique was established for the screening and identification of α-glucosidase inhibitors (AGIs) from APL crude extract, utilizing α-glucosidase coated CuInS2/ZnS-Fe3O4@SiO2 (AG-CIZSFS) nanocomposites as extracting material and fluorescent tracer. The AG-CIZSFS nanocomposites prepared through solvothermal and crosslinking methods displayed fast magnetic separation, excellent fluorescence performance and high enzyme activity. The tolerance of immobilized enzyme to temperature and pH was stronger than that of free enzyme. Prior to proof-of-concept with APL crude extract, a number essential parameters (glutaraldehyde concentration, immobilized time, enzyme amount, reaction solution pH, incubation temperature, incubation time, percentage of methanol in eluen, elution times and eluent volume) were optimized using an artificial test mixture. The fished ligands were identified by UPLC-MS/MS and their biological activities were preliminarily evaluated by real-time cellular morphological imaging of human colon carcinoma (HCT-116) cells based on confocal laser scanning microscope (CLSM). Their α-glucosidase inhibitory activities were further verified and studied by classical pNPG method and molecular docking. The isolated compounds exhibited significant α-glucosidase inhibitory activities with a IC50 value of 11.57 µg·mL-1. Six potential AGIs including tribuloside, ivorengenin A, tormentic acid, 1ß, 2ß, 3ß, 19α-Tetra hydroxyurs-12-en-28-oic acid, corosolic acid and pomolic acid were ultimately screened out and identified from APL crude extracts. The proposed approach, which combined highly specific screening with in-situ visual imaging, provided a powerful platform for discovering bioactive components from multi-component and multi-target traditional Chinese medicine (TCM).

Biodiesel Production Using Palm Oil with a MOF-Lipase B Biocatalyst from Candida Antarctica: A Kinetic and Thermodynamic Study.

This research presents the results of the immobilization of Candida Antarctica Lipase B (CALB) on MOF-199 and ZIF-8 and its use in the production of biodiesel through the transesterification reaction using African Palm Oil (APO). The results show that the highest adsorption capacity, the 26.9 mg·g-1 Lipase, was achieved using ZIF-8 at 45 °C and an initial protein concentration of 1.20 mg·mL-1. The results obtained for the adsorption equilibrium studies allow us to infer that CALB was physically adsorbed on ZIF-8 while chemically adsorbed with MOF-199. It was determined that the adsorption between Lipase and the MOFs under study better fit the Sips isotherm model. The results of the kinetic studies show that adsorption kinetics follow the Elovich model for the two synthesized biocatalysts. This research shows that under the experimental conditions in which the studies were carried out, the adsorption processes are a function of the intraparticle and film diffusion models. According to the results, the prepared biocatalysts showed a high efficiency in the transesterification reaction to produce biodiesel, with methanol as a co-solvent medium. In this work, the catalytic studies for the imidazolate, ZIF-8, presented more catalytic activity when used with CALB. This system presented 95% biodiesel conversion, while the biocatalyst formed by MOF-199 and CALB generated a catalytic conversion percentage of 90%. Although both percentages are high, it should be noted that CALB-MOF-199 presented better reusability, which is due to chemical interactions.

Immobilized lipase for sustainable hydrolysis of acidified oil to produce fatty acid.

Acidified oil is obtained from by-product of crops oil refining industry, which is considered as a low-cost material for fatty acid production. Hydrolysis of acidified oil by lipase catalysis for producing fatty acid is a sustainable and efficient bioprocess that is an alternative of continuous countercurrent hydrolysis. In this study, lipase from Candida rugosa (CRL) was immobilized on magnetic Fe3O4@SiO2 via covalent binding strategy for highly efficient hydrolysis of acidified soybean oil. FTIR, XRD, SEM and VSM were used to characterize the immobilized lipase (Fe3O4@SiO2-CRL). The enzyme properties of the Fe3O4@SiO2-CRL were determined. Fe3O4@SiO2-CRL was used to catalyze the hydrolysis of acidified soybean oil to produce fatty acids. Catalytic reaction conditions were studied, including amount of catalyst, reaction time, and water/oil ratio. The results of optimization indicated that the hydrolysis rate reached 98% under 10 wt.% (oil) of catalyst, 3:1 (v/v) of water/oil ratio, and 313 K after 12 h. After 5 cycles, the hydrolysis activity of Fe3O4@SiO2-CRL remained 55%. Preparation of fatty acids from high-acid-value by-products through biosystem shows great industrial potential.

Immobilization of lipase on hydrophobic MOF synthesized simultaneously with oleic acid and application in hydrolysis of natural oils for improving unsaturated fatty acid production.

The hydrolysis of natural oils (vegetable oils and fats) by lipase has significant applications in food and medicine. However, free lipases are usually sensitive to temperature, pH and chemical reagents in aqueous solutions, which hinders their widespread industrial application. Excitingly, immobilized lipases have been widely reported to overcome these problems. Herein, inspired by lipase interface activation, a hydrophobic Zr-MOF (UiO-66-NH2-OA) with oleic acid was synthesized for the first time in an emulsion consisting of oleic acid and water, and the Aspergillus oryzae lipase (AOL) was immobilized onto the UiO-66-NH2-OA through hydrophobic interaction and electrostatic interaction to obtain immobilized lipase (AOL/UiO-66-NH2-OA). 1H NMR and FT-IR data indicated that oleic acid was conjugated with the 2-amino-1,4-benzene dicarboxylate (BDC-NH2) by amidation reaction. As a result, the Vmax and Kcat values of AOL/UiO-66-NH2-OA were 179.61 µM﹒min-1 and 8.27 s-1, which were 8.56 and 12.92 times higher than those of the free enzyme, respectively, due to the interfacial activation. After treated at 70 °C for 120 min, the immobilized lipase maintained 52 % of its original activity, but free AOL only retained 15 %. Significantly, the yield of fatty acids by the immobilized lipase reached 98.3 % and still exceeded 82 % after seven times of recycling.

Covalent immobilization of tyrosinase on magnetic multi-walled carbon nanotubes for inhibitor screening.

The inhibition of tyrosinase is considered to be a common therapeutic strategy for some hyperpigmentation disorders. Screening of tyrosinase inhibitors is of great significance to the treatment of pigmentation diseases. In this study, tyrosinase was covalently immobilized on magnetic multi-walled carbon nanotubes for the first time, and the immobilized tyrosinase was applied for ligand fishing of tyrosinase inhibitors from complex medicinal plants. The immobilized tyrosinase was characterized by transmission electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, vibrating sample magnetometry, and thermo-gravimetric analyzer, which indicated that tyrosinase was immobilized onto magnetic multi-walled carbon nanotubes. The immobilized tyrosinase showed better thermal stability and reusability than the free one. The ligand was fished out from Radix Paeoniae Alba and identified as 1,2,3,4,6-pentagalloylglucose by ultra-performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry. 1,2,3,4,6-pentagalloylglucose was found to be a tyrosinase inhibitor with similar half maximal inhibitory concentration values of 57.13 ± 0.91 µM compared to kojic acid (41.96 ± 0.78 µM). This work not only established a new method for screening tyrosinase inhibitors but also holds considerable potential for exploring the new medicinal value of medicinal plants.

Fabrication of a Magnetic Porous Organic Polymer for α-Glucosidase Immobilization and Its Application in Inhibitor Screening.

The technology based on immobilized enzymes was employed to screen the constituents inhibiting disease-related enzyme activity from traditional Chinese medicine, which is expected to become an important approach of innovative drug development. Herein, the Fe3O4@POP composite with a core-shell structure was constructed for the first time with Fe3O4 magnetic nanoparticles as the core, 1,3,5-tris (4-aminophenyl) benzene (TAPB) and 2,5-divinylterephthalaldehyde (DVA) as organic monomers, and used as the support for immobilizing α-glucosidase. Fe3O4@POP was characterized by transmission electron microscopy, energy-dispersive spectrometry, Fourier transform infrared, powder X-ray diffraction, X-ray photoelectron spectroscopy, and vibrating sample magnetometry. Fe3O4@POP exhibited a distinct core-shell structure and excellent magnetic response (45.2 emu g-1). α-Glucosidase was covalently immobilized on core-shell Fe3O4@POP magnetic nanoparticles using glutaraldehyde as the cross-linking agent. The immobilized α-glucosidase possessed improved pH stability and thermal stability as well as good storage stability and reusability. More importantly, the immobilized enzyme exhibited a lower Km value and enhanced affinity for the substrate than the free one. The immobilized α-glucosidase was subsequently used for inhibitor screening from 18 traditional Chinese medicines in combination with capillary electrophoresis analysis among which Rhodiola rosea exhibited the highest enzyme inhibitory activity. These positive results demonstrated that such magnetic POP-based core-shell nanoparticles were a promising carrier for enzyme immobilization and the screening strategy based on immobilized enzyme provided an effective way to rapidly explore the targeted active compounds from medicinal plants.

Continuous Production of Dietetic Structured Lipids Using Crude Acidic Olive Pomace Oils.

Crude olive pomace oil (OPO) is a by-product of olive oil extraction. In this study, low-calorie structured triacylglycerols (TAGs) were produced by acidolysis of crude OPO with medium-chain fatty acids (caprylic, C8:0; capric, C10:0) or interesterification with their ethyl ester forms (C8EE, C10EE). These new TAGs present long-chain fatty acids (L) at position sn-2 and medium-chain fatty acids (M) at positions sn-1,3 (MLM). Crude OPO exhibited a high acidity (12.05-28.75% free fatty acids), and high contents of chlorophylls and oxidation products. Reactions were carried out continuously in a packed-bed bioreactor for 70 h, using sn-1,3 regioselective commercial immobilized lipases (Thermomyces lanuginosus lipase, Lipozyme TL IM; and Rhizomucor miehei lipase, Lipozyme RM IM), in solvent-free media at 40 °C. Lipozyme RM IM presented a higher affinity for C10:0 and C10EE. Lipozyme TL IM preferred C10:0 over C8:0 but C8EE over C10EE. Both biocatalysts showed a high activity and operational stability and were not affected by OPO acidity. The New TAG yields ranged 30-60 and the specific productivity ranged 0.96-1.87 g NewTAG/h.g biocatalyst. Lipozyme RM IM cost is more than seven-fold the Lipozyme TL IM cost. Therefore, using Lipozyme TL IM and crude acidic OPO in a continuous bioreactor will contribute to process sustainability for structured lipid production by lowering the cost of the biocatalyst and avoiding oil refining.

Activity and stability of lipase from Candida Antarctica after treatment in pressurized fluids.

Lipase B from Candida antarctica (CalB) is one of the biocatalysts most used in organic synthesis due to its ability to act in several medium, wide substrate specificity and enantioselectivity, tolerance to non-aqueous environment, and resistance to thermal deactivation. Thus, the objective of this work was to treat CalB in supercritical carbon dioxide (SC-CO2) and liquefied petroleum gas (LPG), and measure its activity before and after high-pressure treatment. Residual specific hydrolytic activities of 132% and 142% were observed when CalB was exposed to SC-CO2 at 35 â„ƒ, 75 bar and 1 h and to LPG at 65 â„ƒ, 30 bar and 1 h, respectively. Residual activity of the enzyme treated at high pressure was still above 100% until the 20th day of storage at low temperatures. There was no difference on the residual activity loss of CalB treated with LPG and stored at different temperatures over time. Greater difference was observed between CalB treated with CO2 and flash-frozen in liquid nitrogen (- 196 â„ƒ) followed by storage in freezer (- 10 â„ƒ) and CalB stored in freezer at - 10 â„ƒ. Such findings encourage deeper studies on CalB as well as other enzymes behavior under different types of pressurized fluids aiming at industrial application.

The evaluation of the performance of rice husk and rice straw as potential matrix to obtain the best lipase immobilized system: creating wealth from wastes.

India generates 126.6 and 42 million tons of Rice straw (RS) and Rice husk (RH) annually, respectively. These agro-processing wastes feedstock are dumped in landfills or burnt, releasing toxic gases and particulate matter into the environment. This paper explores the valorization of these wastes feedstock into sustainable, economic products. We compare these wastes as matrices for lipase immobilization. These matrices were characterized, different parameters (pH, temperature, ionic strength, and metal ion cofactors) were checked, and the selected matrix was analyzed for reusability and hydrolysis of vegetable oils. Lipase immobilized Rice straw (LIRS) showed the highest activity with 72.84% protein loading. Field emission scanning electron microscopy (FESEM) demonstrated morphological changes after enzyme immobilization. FTIR showed no new bond formation, and immobilization data was fitted to Freundlich adsorption isotherm (with K = 12.18 mg/g, nF = 4.5). The highest activity with protein loading, 91.05%, was observed at pH 8, 37 °C temperature, 50 mM ionic strength, and lipase activity doubled in the presence of Mg2+ ions. The LIRS retained 75% of its initial activity up to five cycles and efficiently hydrolyzed different oils. The results reflected that the LIRS system performs better and can be used to degrade oily waste.