Antisolvent precipitation for the synergistic preparation of ultrafine particles of nobiletin under ultrasonication-homogenization and evaluation of the inhibitory effects of α-glucosidase and porcine pancreatic lipase in vitro.

To further enhance the application of nobiletin (an important active ingredient in Citrus fruits), we used ultrasonic homogenization-assisted antisolvent precipitation to create ultrafine particles of nobiletin (UPN). DMSO was used as the solvent, and deionized water was used as the antisolvent. When ultrasonication (670 W) and homogenization (16000 r/min) were synergistic, the solution concentration was 57 mg/mL, and the minimum particle size of UPN was 521.02 nm. The UPN samples outperformed the RN samples in terms of the inhibition of porcine pancreatic lipase, which was inhibited (by 500 mg/mL) by 68.41 % in the raw sample, 90.34 % in the ultrafine sample, and 83.59 % in the positive control, according to the data. Fourier transform infrared spectroscopy analysis revealed no chemical changes in the samples before or after preparation. However, the crystallinity of the processed ultrafine nobiletin particles decreased. Thus, this work offers significant relevance for applications in the realm of food chemistry and indirectly illustrates the expanded application potential of nobiletin.

Sterehirsutynes A - C: three new acetylenic aromatic metabolites from Stereum hirsutum.

Three new acetylenic aromatic compounds, sterehirsutynes A-C (1-3), along with three known congeners, frustulosinol (4), vibrayne (5), and sterehirsutinol (6), were isolated from the culture broth of Stereum hirsutum. Their structures were determined by detailed analyses of NMR and high-resolution mass. Chiral column analysis showed that compounds 2 and 3 were racemic mixtures. These new compounds were evaluated for porcine pancreatic lipase (PPL) inhibitory activities, and compounds 1 and (±) 3 showed moderate inhibitory activity against PPL, with IC50 values of 23.2 ± 1.04 and 21.8 ± 2.15 µM, respectively.

Biochemical and Structural Characterization of a novel thermophilic esterase EstD11 provide catalytic insights for the HSL family.

A novel esterase, EstD11, has been discovered in a hot spring metagenomic library. It is a thermophilic and thermostable esterase with an optimum temperature of 60°C. A detailed substrate preference analysis of EstD11 was done using a library of chromogenic ester substrate that revealed the broad substrate specificity of EstD11 with significant measurable activity against 16 substrates with varied chain length, steric hindrance, aromaticity and flexibility of the linker between the carboxyl and the alcohol moiety of the ester. The tridimensional structures of EstD11 and the inactive mutant have been determined at atomic resolutions. Structural and bioinformatic analysis, confirm that EstD11 belongs to the family IV, the hormone-sensitive lipase (HSL) family, from the α/ß-hydrolase superfamily. The canonical α/ß-hydrolase domain is completed by a cap domain, composed by two subdomains that can unmask of the active site to allow the substrate to enter. Eight crystallographic complexes were solved with different substrates and reaction products that allowed identification of the hot-spots in the active site underlying the specificity of the protein. Crystallization and/or incubation of EstD11 at high temperature provided unique information on cap dynamics and a first glimpse of enzymatic activity in vivo. Very interestingly, we have discovered a unique Met zipper lining the active site and the cap domains that could be essential in pivotal aspects as thermo-stability and substrate promiscuity in EstD11.

Janus Surface Micelles on Silica Particles: Synthesis and Application in Enzyme Immobilization.

In these years, synthesis and applications of Janus structures have aroused great interest for large-scale applications in chemistry and materials science. Up to now, Janus particles with different morphologies and different functionalities have been synthesized in solutions, but the synthesis of Janus particles on solid surfaces has not been touched. In this research, Janus surface micelles (JSMs) are fabricated on the surfaces of silica particles by polymerization induced surface self-assembly (PISSA) approach, and the JSMs are used for enzyme immobilization. Usually, enzyme immobilization should be able to optimize the performance of the immobilized enzymes, and an ideal immobilization system must offer protection to the immobilized enzyme with retained bioactivity. Herein, it is demonstrated that JSMs on silica particles can be used as an ideal platform for the immobilization of enzymes. To prepare JSMs, poly(2-(dimethylamino) ethyl methacrylate) macro chain transfer agent (PDMAEMA-CTA) brushes on silica particles and poly(di(ethylene glycol) methyl ether methacrylate) macro CTA (PDEGMA-CTA) are employed in reversible addition-fragmentation chain transfer dispersion polymerization of styrene. After polymerization, JSMs with polystyrene cores and PDMAEMA/PDEGMA patches on the surfaces are prepared on silica particles. After quaternization reaction, the quaternized PDMAEMA patches are used for the immobilization of enzymes. Experimental results turn out that enhanced bioactivities of the immobilized enzymes are achieved and the enzyme molecules are well protected by surface Janus structures.

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 ± 0.12 mM) and V max (13.01 ± 0.64 µmol/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 3 h. However, it merits further investigations to find out about large-scale application of the as-synthesized biocatalyst for esterification.

Chemical Constituents from the Whole Plant of Cuscuta reflexa.

Two new 2H-pyran-2-one glucosides, cuscutarosides A (1) and B (2), and one new steroidal glucoside, 7ß-methoxy-ß-sitosterol 3-O-ß-glucopyranoside (3), together with 12 known compounds (4-15) were isolated from the whole plant of Cuscuta reflexa (Convolvulaceae) collected from Myanmar. The chemical structures of these new compounds were elucidated based on extensive spectroscopic analysis. The antiobesity activity of these isolates was evaluated using porcine pancreatic lipase (PPL), and the antiplatelet aggregation activity was screened using rabbit platelets induced by thrombin, platelet-activating factor (PAF), arachidonate (AA), or collagen. 7ß-Methoxy-ß-sitosterol 3-O-ß-glucopyranoside (3) showed weak PPL inhibitory activity. Cuscutaroside A (1), its acetylated derivative (1a), and scrophenoside B (8) showed weak inhibitory activity against rabbit platelet aggregation induced by collagen. Compound 1a also showed inhibitory activity against rabbit platelet aggregation induced by AA.

Molecular Dynamic Simulation of the Porcine Pancreatic Lipase in Non-aqueous Organic Solvents.

This paper investigates the conformational stability of porcine pancreatic lipase (PPL) in three non-aqueous organic solvents, including dimethyl sulfoxide (DMSO), propylene glycol (PRG), and ethanol (EtOH) through molecular dynamic (MD) simulation. The root mean square deviations (RMSDs), radius of gyration (Rg), solution accessible surface area (SASA), radial distribution function (RDF), hydrogen bond (H-bond), Ramachandran plot analysis, secondary structure, and enzyme substrate affinity of the PPL in the various organic solvents were comparatively investigated. The results showed that the backbone and active pocket RMSD, and hydrophilic ASA of PPL in three solvents increase with the increase in the solvent LogP, while the Rg, hydrophobic ASA, and H-bond between the solvent and PPL decrease. Among the three organic solvents, DMSO acts as a better solvent, in which the PPL can be loose and extended, and retains its native backbone in DMSO compared to PRG and EtOH. Moreover, Ramachandran plot analysis indicated that the PPL structure quality in DMSO was higher than that in PRG and EtOH. Also, the molecular docking results showed that PPL in DMSO exhibited the highest enzyme-substrate affinity.

Structurally diverse vibralactones produced by the fungus Stereum hirsutum.

Eight unexpected vibralactone homodimers, bisvibralactones A-H (1-8), and three new vibralactone monomers, hirsutumins A-C (9-11), were isolated from the culture of Stereum hirsutum. Their structures and absolute configurations were determined by detailed analyses of NMR, optical rotations, ECD, and high-resolution mass spectra as well as chemical transformation. Compounds 1-8 are unusual vibralactone dimers formed by the esterification of two vibralactone monomers. The absolute configurations of compounds 1 and 5 were determined by chemical conversions. All of the isolated compounds were evaluated for Porcine Pancreatic Lipase (PPL) inhibitory activities, and compound 10 showed significant inhibitory activity against PPL, with an IC50 value of 8.31 ± 1.04 µM.

Effective degradation of the mycotoxin patulin in pear juice by porcine pancreatic lipase.

Porcine pancreatic lipase (PPL) was used to degrade the mycotoxin patulin (PAT) in pear juice. The dosage of PPL, the initial concentration of PAT, reaction temperature and time were investigated by batch experiments to study the optimal degradation condition. The concentration of PAT in pear juice was determined by high performance liquid chromatography with an ultraviolet detector (HPLC-UV). The results showed that the optimal condition was 0.02 g PPL/mL pear juice at 40 °C for 24 h. The content of organic acids, volatile flavor components, polyphenols, ascorbic acid and the degree of browning reaction in pear juice, relating to the quality of juice, changed insignificantly. Although the initial PAT concentration was very high, the degradation product was confirmed nontoxic by cytotoxicity test of Caco-2 cells. It suggested that PPL could be further considered to be applied in the degradation of PAT in pear juice.

Synthesis of functional ionic liquid modified magnetic chitosan nanoparticles for porcine pancreatic lipase immobilization.

We developed magnetic chitosan nanoparticles (CS­Fe3O4) with mean diameter of 15-20 nm. Subsequently, these inorganic-organic composite nanoparticles were modified using an imidazole-based functional ionic liquid (IL). The prepared support (IL­CS­Fe3O4), which was used to immobilize porcine pancreatic lipase (PPL), was characterized using Fourier transform infrared (FTIR) spectroscopy, vibrating sample magnetometry (VSM), thermogravimetry (TG), transmission electron microscopy (TEM) and X-ray diffraction (XRD). Circular dichroism (CD) was used to analyze the secondary structure of immobilized PPL. The immobilized PPL (PPL­IL­CS­Fe3O4) exhibited 1.93-fold higher specific activity than PPL­CS-Fe3O4 when triacetin was used as the substrate, and showed 95 mg/g of lipase immobilization capacity and 382% of activity recovery. The residual activity of PPL­IL­CS­Fe3O4 was above 60% of the initial activity after incubation at 50 °C for 6 h, as was higher than that of PPL­CS­Fe3O4 which showed 40% of the initial activity. In addition, PPL­IL­CS­Fe3O4 retained 84.6% of the initial activity after 10 cycles, whereas PPL­CS­Fe3O4 retained only 75.5% activity. Furthermore, the kinetic parameters, apparent Km and Vmax of PPL­IL­CS­Fe3O4 were 2.51 mg/mL and 1.395 U/mg respectively, these results indicated that the immobilized PPL had better affinity towards the substrate, especially when the nanoparticles were modified by functional IL. Besides, the magnetic chitosan nanoparticles loaded with PPL were easily recovered. A novel, efficient, and practical method for enzyme immobilization was developed.

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.

Effective Biodegradation of Mycotoxin Patulin by Porcine Pancreatic Lipase.

Patulin is a common contaminant in fruits and vegetables, which is difficult to remove. In this study, the biodegradation of patulin using porcine pancreatic lipase (PPL) was investigated. The method of HPLC was used to analyze the concentration of patulin. Batch degradation experiments were performed to illustrate the effect of PPL amount, pH, temperature, contact time, and initial concentration. Besides, the degradation product of patulin was characterized by full wavelength scanning and MS technologies. The results showed that the optimum degradation conditions of PPL for patulin was observed at pH 7.5, 40°C for 48 h. The percentage of degradation could reach above 90%. The structure of degradable product of patulin was inferred by the molecular weight 159.0594, named C7H11O4+. It indicated that PPL was effective for the degradation of patulin in fruits and vegetables juice.

Effect of Organic Solvents on Porcine Pancreatic Lipase Thermal Aggregation.

BACKGROUND: Proteins tend to form inactive aggregates under harsh conditions used in industrial processes. Lipases are enzymes that hydrolyse triglycerides to glycerol and free fatty acids, but are able to catalyse various other transformations in the presence of organic solvents. OBJECTIVES: The main objective of this study was to investigate lipases behavior at high temperature and in presence of organic solvents. METHODS: Heat-induced aggregation of porcine pancreatic lipase (PPL) was followed by UV-visible spectroscopy at 400 nm wavelength for 600 seconds, at the isoelectric point (pH 5, phosphate solution) and 50°C, and in presence or absence of various percentages of dimethyl sulfoxide (DMSO), propanol, isopropanol, acetone and trifluoroethanol (TFE). Possible positioning of each organic solvent molecule relative to PPL was investigated using docking method. RESULTS: Native enzyme aggregated under aforementioned conditions and amorphous aggregates formed which were visible to the naked eye. From the tested solvents, DMSO reduced protein aggregation in a concentration-dependent manner. On the other hand, protein aggregation intensified by adding any of propanol, isopropanol, acetone or TFE. This effect was more pronounced in TFE and propanol compared to isopropanol and acetone. CONCLUSION: Solvents with lower polarity led to aggregation, while solvent with higher polarity inhibited PPL aggregation, and DMSO could be effectively used to counteract lipase aggregation.

Enzyme-polymer hybrid nanogels fabricated by thiol-disulfide exchange reaction.

In this paper a novel method for the fabrication of hybrid nanogels based on thiol-disulfide exchange reaction is reported. Poly(oligo(ethylene glycol) monomethyl ether methacrylate-co-di(ethylene glycol) methyl ether methacrylate-co-2-(2-pyridyldisulfide) ethyl methacrylate) (POEGMA-co-PDEGMA-co-PDSMA) was synthesized by reversible addition-fragmentation chain transfer polymerization. Pyridyl disulfide functionalized porcine pancreatic lipase (PPL-S-S-Py) was prepared by treatment of PPL with Traut's reagent (2-iminothiolane) and 2,2'-dithiodipyridine. Upon addition of meso-2,3-dimercaptosuccinic acid into aqueous solutions of PPL-S-S-Py and POEGMA-co-PDEGMA-co-PDSMA, enzyme-polymer hybrid nanogels were prepared. The hybrid nanogels show thermal responsiveness. With an increase in the content of PPL in the nanogels, the lower critical solution temperature (LCST) shifts to the higher temperature. At a temperature below LCST, PPL molecules are in the shells of the nanogels, and at a temperature above LCST, PPL molecules are embedded inside the nanosized structures. The immobilized PPL show enhanced heat resistance and good reusability.

Kinetic and structural characterization of triacylglycerol lipases possessing phospholipase A1 activity.

The pancreatic lipase gene family displays various substrate selectivities for triglycerides and phospholipids. The structural basis for this difference in substrate specificity has not been definitively established. Based on a kinetic comparative study between various pancreatic lipase family members, we showed here that porcine pancreatic lipase (PPL), which was so far classified as "classical lipase", was able to hydrolyze phosphatidylcholine (PC). Amino acid sequence alignments revealed that Val260 residue in PPL lid could be critical for the interaction with lipid substrate. Molecular dynamics was applied to investigate PC binding modes within the catalytic cavity of PPL and human pancreatic lipase (HPL), aiming to explain the difference of specificity of these enzymes towards phospholipids. Results showed that with HPL, the oxyanion hole was not able to accommodate the PC molecule, suggesting that no activity could be obtained. With PPL, the formation of a large pocket involving Val260 allowed the PC molecule to come near the catalytic residues, suggesting that it could be hydrolyzed. One more interesting finding is that human pancreatic lipase related protein 2 could hydrolyze phospholipids through its PLA1 and PLA2 activities. Overall, our study shed the light on new structural features of the phospholipase activity of pancreatic lipase family members.

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.

Inhibition of pancreatic lipase, α-glucosidase, α-amylase, and hypolipidemic effects of the total flavonoids from Nelumbo nucifera leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Nelumbo nucifera Gaertn. leaves have been used as medicinal herbs in the past 1300 years, specifically utilized to cure hyperlipidemia, hyperglycemia, and obesity. It has been recorded in the most famous medicinal book in China for more than 400 years. The present study aims to identify the potential therapeutic activities of the flavonoids isolated from Nelumbo nucifera leaves. MATERIALS AND METHODS: Nelumbo nucifera leaf flavonoids (NLF) were tested for the inhibition of lipase, α-glucosidase, and α-amylase activities in vitro. A single dose of NLF was administered by oral gavage in mice for acute toxicity. Wistar rats with high-fat diet-induced hyperlipidemia and two other animal models were used to evaluate the hypolipidemic effects of NLF. RESULTS: Our in vitro biochemistry tests revealed that the NLF showed high inhibitory activity against porcine pancreatic lipase, α-amylase, and α-glucosidase with IC50 values of 0.38 ± 0.022, 2.20 ± 0.18, and 1.86 ± 0.018 mg/mL, respectively. Furthermore, the NLF significantly lowered the lipid components, such as the total cholesterol, triglycerides, low-density lipoprotein cholesterol, and malondialdehyde, in various established in vivo systems and raised the high-density lipoprotein cholesterol. Moreover, the NLF alleviated high-fat diet-induced lipid accumulation in the liver. CONCLUSIONS: The results demonstrate that NLFs can effectively ameliorate hyperlipidemia and inhibit the key enzymes related to type 2 diabetes mellitus. Our findings may provide new pharmacological basis for the treatment of hyperlipidemia, hyperglycemia, and obesity using NLFs.