Immobilization Of Porcine Pancreatic Lipase Research Articles

Carboxy-Functionalized Covalent Organic Framework as a Carrier for Lipase Immobilization and Its Application in Inhibitors Screening.

Covalent organic frameworks (COFs) with large specific surface areas, high porosity, good stability, and designable structure are promising carriers for immobilized enzymes. It is important to explore lipase inhibitors from natural foods as lipase inhibitors are closely related to the treatment of obesity. In this work, a carboxyl functionalized covalent organic framework (TpBD-3COOH) was prepared by solvothermal method for covalent immobilization of porcine pancreatic lipase (PPL) and obtained the enzyme-decorated COF (PPL@COF). The immobilized lipase showed wider pH and temperature tolerance with the same optimal pH and temperature of 7.5 and 50 ℃ compared to free lipase. After 6 successive reuses, the PPL@COF maintained 53.0% of its original activity. Immobilized lipase also displayed enhanced storage stability (55.4% after 14days at 4 ℃). When p-nitrophenyl acetate was applied as the substrate, the calculated Michaelis constant was 3.57mM and the half maximal inhibitory concentration of orlistat was 3.20μM. Finally, the PPL@COF was used for enzyme inhibitors screening from natural foods combined with UV spectrophotometry, and Hawthorn was screened for excellent lipase inhibitory activity.

Immobilization of porcine pancreatic lipase on Celite: optimization using a full factorial experimental design

In this study, Celite was used as a stable, low-cost and eco-friendly support for the immobilization of porcine pancreatic lipase (PPL). A full factorial design (24) was applied to examine the effect of four operating factors affecting the immobilization process, namely medium pH, contact time, immobilization temperature and Celite/PPL ratio. The hydrolytic activity of the immobilized PPL was chosen as the response variable. The optimal immobilization parameters for maximum activity: y=0.2064 mmol.min-1.g-1 were obtained at pH=5 for 30 min at 4°C and a PPL/Celite ratio of 1/1 (w/w). Stability in terms of pH is in the range of 8.0-9.0, the optimal temperature that endangers the proper enzyme activity is 40°C.

Rapid Screening of Lipase Inhibitors in Scutellaria baicalensis by Using Porcine Pancreatic Lipase Immobilized on Magnetic Core–Shell Metal–Organic Frameworks

As for ligand fishing, the current immobilization approaches have some potential drawbacks such as the small protein loading capacity and difficult recycle process. The core–shell metal–organic frameworks composite (Fe3O4-COOH@UiO-66-NH2), which exhibited both magnetic characteristics and large specific surface area, was herein fabricated and used as magnetic support for the covalent immobilization of porcine pancreatic lipase (PPL). The resultant composite Fe3O4-COOH@UiO-66-NH2@PPL manifested a high loading capacity (247.8 mg/g) and relative activity recovery (101.5%). In addition, PPL exhibited enhanced tolerance to temperature and pH after immobilization. Then, the composite Fe3O4-COOH@UiO-66-NH2@PPL was incubated with the extract of Scutellaria baicalensis to fish out the ligands. Eight lipase inhibitors were obtained and identified by UPLC-Q-TOF-MS/MS. The feasibility of the method was further confirmed through an in vitro inhibitory assay and molecular docking. The proposed ligand fishing technique based on Fe3O4-COOH@UiO-66-NH2@PPL provided a feasible, selective, and effective platform for discovering enzyme inhibitors from natural products.

Immobilization of porcine pancreatic lipase on activated carbon by adsorption and covalent bonding and its application in the synthesis of butyl butyrate

Porcine pancreatic lipase (PPL) extract have been immobilized on activated carbon (ACT) and functionalized activated carbon (FCT). The effects of immobilization solution pH, addition of surfactant Triton X-100, and pH and temperature in the catalytic properties of the biocatalyst were evaluated. The functionalization led to an increase in the point of zero charge (5.13–6.11), reduction in the oxygenated groups (2.15 meq.g−1 to 1.99 meq.g−1) and a specific surface area (1028 m2. g−1 to 132 m2. g−1). The optimum immobilization pH was 5.0, resulting in the PPL extract loading of 71.80 mg/g (ACT) and 55.2 mg/g (FCT). A better hydrolytic activity (239.6 U/g) was observed for ACT to 0.6 mM Triton-X concentration, while FCT exhibited higher activity (208.63 U/g) in the absence of surfactant. For the PPL extract immobilized on ACT, the activity at pH 8.0 was 240.95 U/g, while a greater activity was observed at pH 7.0 for the PPL extract immobilized on FCT (221.94 U/g). The temperature of 35 °C led the highest activity values. The esterification yield of butyl butyrate was 82 % and 66 % for the PPL extract immobilized on ACT and FCT, respectively. Both biocatalysts showed residual esterification yield greater than 86 % after five cycles of use.

Development and characterization of enzymatic biosensor based on lipase from porcine pancreas for propylparaben detection

Purpose The purpose of this paper is to study the immobilization of porcine pancreatic lipase (PPL), in an organic matrix by a covalent cross-linking method to sense propylparaben (PP) present in aqueous solution. Design/methodology/approach PPL immobilization was performed by the covalent cross-linking method, using bovine serum albumin (BSA) in the presence of saturated glutaraldehyde vapor (GA). The preparation of the enzymatic membrane involves the incorporation of porcine pancreatic lipase (PPL), bovine serum albumin (BSA) and glycerol into a phosphate buffer solution (PBS). Characterization of this sensor was performed by impedance spectroscopy (EIS) and scanning electron microscope (SEM). The effect of experimental conditions such as PPL activity, potential, scan rate, PP concentration, pH and presence of interfering elements were studied by cyclic voltammetry. Findings Under the optimal experimental conditions, a number of significant factors were optimized. The method exhibited good linearity in the range of 10–14 to 10–9 mol/L with a good correlation coefficient of 0.957, detection limit (LOD) of 3.66 × 10–15 mol/L and high sensitivity of 1.086 mA mol−1L. The authors also obtained a very good coverage rate of the surface equal to 91.44%, and hydrolytic activity of lipase is evaluated to 26.64 mmol min−1. The stability and the interference were also evaluated. The equivalent circuit used to explain the electrochemical behavior of modified electrode is a Randle circuit. Practical implications The main application of biosensors is the detection of biomolecules that are either indicators of a disease. For example, electrochemical biosensing techniques can be used as clinical tools to detect breast tumors, because these compounds (PP) were found in breast tumors. Originality/value The result registered in this paper indicates that the developed sensor is an efficient, fast, simple and inexpensive analytical tool that can be used for the analysis of water containing PP.

Lipase@zeolitic imidazolate framework ZIF-90: A highly stable and recyclable biocatalyst for the synthesis of fruity banana flavour

A zeolitic imidazolate framework (ZIF-90) has been synthesized through solvothermal method. The structure was characterized by means of FT-IR spectroscopy, X-ray diffraction, thermogravimetric analysis (TGA), and scanning electron microscopy (SEM)/energy dispersive X-ray spectroscopy (EDS). The synthesized ZIF-90 was applied as a support for immobilization of porcine pancreatic lipase (PPL). The immobilized enzyme (PPL@ZIF-90) exhibited immobilization yield and efficiency of 66 ± 1.8% and 89 ± 1.4%, respectively. The pH and thermal stability of PPL was improved after immobilization and the initial activity was retained at about 57% after 20 days of storage at 4 °C for PPL@ZIF-90. Moreover, about 57% of the original activity was remained following 10 cycles of application. In Michaelis-Menten kinetic studies, Km value for PPL@ZIF-90 was lower, while, the Vmax was higher than free PPL. Moreover, optimized conditions to produce fruity banana flavour upon esterification of butyric acid were investigated. The optimum esterification yield was 73.79 ± 1.31% in the presence of 245 mg PPL@ZIF-90, alcohol/acid ratio of 2.78 and 39 h reaction time. PPL@ZIF-90 showed 39% relative esterification yield after six cycles of reuse. The results suggested that PPL@ZIF-90 can be used as a potential effective biocatalyst for synthesis of isoamyl butyrate.

Immobilization of lipase on the modified magnetic diatomite earth for effective methyl esterification of isoamyl alcohol to synthesize banana flavor.

The present study was designed to propose a simple, cost-effective, and efficient method for the preparation of a biocompatible composite made from magnetic diatomaceous earth (mDE) coated by aminopropyltriethoxysilane (APTES) and its application for immobilization of porcine pancreatic lipase (PPL). The produced mDE-APTES was instrumentally characterized and the obtained results of FTIR analysis and scanning electron microscopy equipped by energy-dispersive X-ray spectroscopy (SEM-EDS) showed successful coating of APTES on mDE surface. PPL was then immobilized onto mDE to obtain the biocatalyst of PPL@mDE (immobilization yield and efficiency of 78.0 ± 0.3% and 80.1 ± 0.6, respectively) and the presence of enzyme was confirmed by EDS method. The attained results of the reusability of PPL@mDE revealed that 57% of the initial activity was retained after 11 cycles of biocatalyst application. PPL@mDE demonstrated higher storage stability than the free enzyme at 4°C, 25°C, and 37°C. The apparent K m (2.35 ± 0.12mM) and V max (13.01 ± 0.64µmol/min) values for the immobilized enzyme were considerably altered compared to those of the free enzyme (p > 0.05). PPL@mDE was subsequently employed for the synthesis of banana flavor (isoamyl acetate) in n-hexane, which yields an esterification percentage of 100 at 37°C after 3h. However, it merits further investigations to find out about large-scale application of the as-synthesized biocatalyst for esterification.

Immobilization of porcine pancreatic lipase onto a metal-organic framework, PPL@MOF: A new platform for efficient ligand discovery from natural herbs

In terms of ligand fishing, the amount and the relative activity recovery of enzymes immobilized on magnetic particles and nanoparticles are not preeminent. Therefore, the metal-organic framework (MOF) UiO-66-NH2 was synthesized to immobilize the porcine pancreatic lipase (PPL) via precipitation-cross-linking, and the resulting novel biological matrices named PPL@MOF manifested high PPL loading capacity (98.31 mg/g) and relative activity recovery (104.4%). Moreover, the novel enzyme-MOF composite was applied to screen lipase inhibitors from Prunella vulgaris L. to enrich and improve the techniques of ligand fishing. As a result, 13 lipase ligands were obtained, and 12 compounds were determined by HPLC-Q-TOF-MS/MS. All of these ligands were further confirmed to be potential inhibitors through the verification of the activity assay and molecular docking. The proposed approach based on PPL@MOF was superior in terms of abundant protein loading capacity, high enzyme catalytic activity and easy preparation process. Taken together, our newly developed method provided a new platform for efficient discovering bioactive molecules from natural herbs.

Utilization of Nano-SiO₂ as a Supporting Material for Immobilization of Porcine Pancreatic Lipase.

Nano-SiO2 is an inexpensive material for enzyme immobilization and has been frequently utilized for this purpose. In addition to its low cost, Nano-SiO2 has several advantages when used as a supporting material, including its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, Porcine Pancreatic lipase was non-covalently immobilized on Nano-SiO2. The properties of immobilized enzyme were then defined. The optimum pH and temperature for the immobilized PPL for hydrolysis of olive oil were determined as 7.6 and 40-50 °C, respectively. The immobilized enzyme possessed high stability in batch operation; with 73.5% of the initial activity still remaining after 8 times of repeated operation of the immobilized lipase. The immobilized enzyme was more stable than free enzyme and t1/2 was 25 d, while free lipase activity was lost 50% in 2 days. The apparent Km for the immobilized enzyme was significantly smaller than that of the free one.

Enhancement of catalytic performance of porcine pancreatic lipase immobilized on functional ionic liquid modified Fe3O4-Chitosan nanocomposites

Magnetic chitosan nanocomposites were designed and fabricated by combining the magnetic nanoparticles Fe3O4 and chitosan covalently modified by imidazole-based ionic liquids with various functional groups, to be utilized as a support matrix for immobilization of porcine pancreatic lipase (PPL). Ionic liquids modified chitosan was characterized with nuclear magnetic resonance (NMR) and the nanocomposites were characterized with Fourier transform infrared spectroscopy (FTIR), vibrating-sample magnetometer (VSM), thermogravimetry analysis (TGA), Transmission electron microscope (TEM) and Scanning electron microscopy (SEM). The enzymatic properties of PPL were significantly improved by immobilization onto all thus prepared nanocomposites, and among the supports, modified with ionic liquids bearing hydroxyl group exhibited relatively enhanced performance. Particularly, the MCS-IL(8C)-OH-PPL exhibited the highest specific activity which was 6.68 times that of free PPL, and the residual activity of MCS-IL(8C)-OH-PPL remarkably maintained 91.5% of its initial activity even after 10 repeated cycles. The residual activity of MCS-IL(8C)-OH-PPL retained 55.8% after incubated in 6 M urea solution for 1 h and maintained the noticeable initial activity of 75.5% after incubated in phosphate buffer at 50 °C with pH 7.5 for 6 h. In conclusion, the study demonstrated that Fe3O4@Chitosan nanocomposites modified with functional ionic liquids could be utilized as a novel nanosupport for enzyme immobilization.

Polyoxometalate-metal organic framework-lipase: An efficient green catalyst for synthesis of benzyl cinnamate by enzymatic esterification of cinnamic acid

Iron-carboxylate (MIL-100(Fe)) and HKUST-1 (Cu3(BTC)2, BTC=1,3,5-benzenetricarboxylic acid) as nanoporous metal organic framework supports were compared for immobilization of porcine pancreatic lipase (PPL). These immobilizations improved thermal, pH and operational stability of PPL compared to the soluble enzyme. Stability of MIL-100(Fe) was better than HKUST-1 as support. MIL-100(Fe) encapsulated Keggin phosphotungstic acid H3PW12O40 (PW) (PW@MIL-100(Fe)) was synthesized to develop novel enzyme immobilized system and characterized by Fourier transform infrared spectroscopy (FT-IR), field emission scanning electron microscopy (FE-SEM), Brunauer–Emmett–Teller (BET), X-ray diffraction (XRD) and Barrett Joyner Halenda (BJH) analysis. Relative activity for immobilized lipase on PW@MIL-100(Fe) was more than MIL-100(Fe) in pH range of 3–9. At the elevated temperature of 70°C, the PW@MIL-100(Fe) was the most stable one. PW@MIL-100(Fe)/PPL substrate exhibited the higher stability at 4°C and 25°C, along with other supports. Moreover, PW@MIL-100(Fe) was chosen as the best support for immobilization of PPL and was also applied for the synthesis of benzyl cinnamate by enzymatic esterification of cinnamic acid. The immobilized enzyme retained 90.4% of its initial activity during synthesis of benzyl cinnamate after 5 successive catalytic rounds and reached 80.0% yield after 8 reuses.

Porcine pancreatic lipase hydrophobically adsorbed on octyl-silica: A robust biocatalyst for syntheses of xylose fatty acid esters

This work describes the immobilization of porcine pancreatic lipase (PPL), obtained from crude extract, on silica coated with octyl groups (OS) by interfacial adsorption, a simple and low-cost immobilization protocol. The biocatalyst PPL-OS was employed to the enzymatic preparation of fatty acid esters of d-xylose, a product used especially in the field of cosmetics and pharmacy, especially dermatology, improving the functionality of epidermal cells. The yields of the immobilization in terms of enzymatic activity and protein concentration (98% and 75%, respectively) suggested that PPL present in the crude extract was selectively immobilized on the octyl-silica support, which allowed the hyperactivation of the biocatalyst (recovered activity, 144%), a phenomenon that may be attributed to the interfacial activation of the enzyme on hydrophobic surfaces. The biocatalyst PPL-OS showed to be very robust in organic medium and at high temperature, which is an extremely important characteristic to produce sugar fatty acid esters from the industrial point of view. The syntheses of xylose fatty esters (oleate, caprylate and butyrate) yielded conversions around 70% after short reaction period (2 h) at 60 °C in tert-butyl alcohol. The biocatalyst, even after incubation at 60 °C for 24 h, could be reused in four esterification cycles of 2-h reaction at 60 °C, maintaining 100% of its catalytic activity.

Graphene oxide-based Fe3O4 nanoparticles as a novel scaffold for the immobilization of porcine pancreatic lipase

A chloropropyl-functionalized graphene oxide decorated with Fe3O4 nanoparticles, CPS/GO-Fe3O4@MCM-41, was made, and porcine pancreas lipase (PPL) was immobilized onto the graphene oxide based magnetic nanoparticles via covalent bonding.

Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: Characterization and application for enzymatic inhibition assays

Using carboxyl functionalized silica-coated magnetic nanoparticles (MNPs) as carrier, a novel immobilized porcine pancreatic lipase (PPL) was prepared through the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. Transmission electron microscopic images showed that the synthesized nanoparticles (Fe3O4–SiO2) possessed three dimensional core–shell structures with an average diameter of ~20nm. The effective enzyme immobilization onto the nanocomposite was confirmed by atomic force microscopic (AFM) analysis. Results from Fourier-transform infrared spectroscopy (FT-IR), Bradford protein assay, and thermo-gravimetric analysis (TGA) indicated that PPL was covalently attached to the surface of magnetic nanoparticles with a PPL immobilization yield of 50mg enzyme/g MNPs. Vibrating sample magnetometer (VSM) analysis revealed that the MNPs-PPL nanocomposite had a high saturation magnetization of 42.25emu·g−1. The properties of the immobilized PPL were investigated in comparison with the free enzyme counterpart. Enzymatic activity, reusability, thermo-stability, and storage stability of the immobilized PPL were found significantly superior to those of the free one. The Km and the Vmax values (0.02mM, 6.40U·mg−1 enzyme) indicated the enhanced activity of the immobilized PPL compared to those of the free enzyme (0.29mM, 3.16U·mg−1 enzyme). Furthermore, at an elevated temperature of 70°C, immobilized PPL retained 60% of its initial activity. The PPL-MNPs nanocomposite was applied in the enzyme inhibition assays using orlistat, and two natural products isolated from oolong tea (i.e., EGCG and EGC) as the test compounds.

Immobilization of porcine pancreatic lipase on poly-hydroxybutyrate particles for the production of ethyl esters from macaw palm oils and pineapple flavor

Poly-hydroxybutyrate particles (PHB) were used as support to immobilize porcine pancreatic lipase (PPL). The biocatalysts prepared were tested in the synthesis of pineapple flavor by esterification of butanol and butyric acid in heptane medium, and in the synthesis of ethyl esters by transesterification of macaw palm pulp (MPPO) and macaw palm kernel (MPKO) oils with ethanol in solvent-free systems. The effect of protein loading on the biocatalyst activity was assessed in olive oil hydrolysis. Maximum hydrolytic activity of 292.8±8.60IU/g was observed. Langmuir isotherm model was applicable to the adsorption of PPL on PHB particles. Maximum immobilized protein amount was 24.3±1.70mg/g. The optimal pH and temperature in hydrolysis reaction for the immobilized PPL were at pH 8.5 and 50°C, while for the crude PPL extract were at pH 8.0 and 45°C. Immobilized PPL exhibited full hydrolytic activity after 2h of incubation in non-polar solvents. In esterification reaction, optimal conversion was around 93% after 2h of reaction. After six esterification cycles, the biocatalyst retained 63% of its initial activity. The biocatalyst prepared attained transesterification yield of 50% after 48h of reaction for MPKO and 35% after 96h of reaction for MPPO.

Sonication assisted assemblage of exotic polymer supported nanostructured bio-hybrid system and prospective application

This work was focused on sonication mediated immobilization of porcine pancreatic lipase (PPL) onto poly(ethylene glycol) supported silver-iron oxide hybrid nanoparticles (PEG-Ag/IONPs). Selected process parameters of sonication were optimized using response surface methodology. Sonication assisted assemblage of spherical PEG-Ag/IONPs and consequent evolution of nanorods post PPL immobilization were documented. The efficacy of the reported immobilization strategy was attested by the increased thermostability, storage stability and enhanced activity of the biocatalyst, suggestive of plausible structural modulations post immobilization. The commercial prospect of the antibacterial and magnetically recyclable system was vouched by its excellent compatibility with some commercial detergents for oil de-staining.

Immobilization of Porcine pancreatic Lipase on Vesicle-Like Silica Material with Large Pore

Vesicle-like silica (VS) was prepared by utilizing cationic surfactant cetyltrimethylammonium bromide (CTAB) and anionic surfactant sodium dodecyl sulfate (SDS) as a dual-template. The mesostructures of VS were characterized by N2adsorption-desorption isotherms, field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM). Results showed that mean mesopore size of unilayer and multilayer VS was about 14 nm and shell thickness was nearly 6 nm. The VS was chosen as support for immobilization of Porcine pancreatic lipase (PPL) by physical adsorption. The immobilized PPL possessed excellent thermal stability and reusability in the hydrolysis reaction of triacetin in phosphate buffer solution.

Immobilization and catalytic activity of lipase on mesoporous silica prepared from biocompatible gelatin organic template

Mesoporous silica (MPS) with high surface area (518.8m2/g), tunable pore size (3.6–13.7nm) was synthesized using inexpensive sodium silicate as the source of silica and the biodegradable gelatin as the structure directing agent. The amorphous mesostructures of the materials were characterized by N2 adsorption–desorption isotherms, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The synthesized MPS was then utilized as supports for immobilization of Porcine pancreatic lipase (PPL) by physical adsorption. The measurements of Fourier transform infrared spectroscopy (FT-IR), N2 adsorption–desorption isotherms and energy-dispersive spectroscopy (EDS) demonstrated that PPL had been adsorbed into the channels of the MPS. The immobilized PPL was used for catalyzing hydrolysis reaction of triacetin, and its correlative catalytic activity was studied. Compared with free PPL, the immobilized PPL possessed superior adaptability in alkaline range (pH=7.0–9.0), good thermal stability and excellent reusability in phosphate buffer solution in hydrolysis reactions of triacetin. The results also indicated that the immobilized PPL maintained 45% of its initial activity even after being reused six times.

Mesoporous Silica Materials Templated with an Anionic Surfactant/Natural Polymer System for Enzyme Immobilization

Mesoporous silica (MPS) has been synthesized by using biodegradable gelatin and anionic surfactant sodium dodecylsulphate (SDS) as a dual-template. The mesostructures of MPS were characterized by N2 adsorption-desorption isotherms and transmission electron microscopy (TEM). The MPS was chosen as support for immobilization of porcine pancreatic lipase (PPL) by physical adsorption. The immobilizedPPL possessed good thermal stability and excellent reusability solution in hydrolysis reactions of triacetin in phosphate buffer. The results also indicated that the immobilized PPL maintained about 40% of its initial activity even after being reused six times.

Immobilization of porcine pancreatic lipase in zeolite MCM 22 with different Si/Al ratios

The use of zeolites as support for immobilization of enzymes has been a matter of great interest recently due to the potential properties of these materials. In this work, the MCM 22 zeolite with different Si/Al ratios (15, 25 and 50) was used as support for immobilization of porcine pancreatic lipase, assessing the effect of its structure on the immobilization yield and enzyme activity. Results showed that the material composition influenced significantly the immobilization process. Higher yields of immobilization and enzymatic activities were achieved when MCM 22 with Si/Al ratio of 25 was used as support. In general, results demonstrate the potential of MCM 22 as support for porcine pancreatic lipase immobilization.