Cross-linked Lipase Aggregates Research Articles

Enzyme-bimetallic hybrid catalyst for one-pot chemoenzymatic reactions

To overcome the intrinsic incompatibility between enzyme and chemical catalysts in one-pot chemoenzymatic cascade reactions, an enzyme-bimetallic hybrid catalyst was constructed by in situ reduction of ultrafine PdCu naonoclusters on the cross-linked lipase aggregates. The PdCu nanoalloy showed excellent activity in the Sonogashira coupling reaction because of the strong bimetallic synergistic effect between Pd and Cu within PdCu/CALB CLEAs. The activity of 3.4Pd1.7Cu/CALB CLEAs was 2.6-fold of Pd/CALB CLEAs in the synthesis of (R)-N-[1-(4-(phenylethynyl)phenyl)ethyl] acetamide. The highly active PdCu/CALB CLEAs rendered the one-pot chemoenzymatic reaction to perform at a lower temperature.

Magnetic cross-linked lipase aggregates coupled with ultrasonic pretreatment for efficient synthesis of phytosterol oleate

In this study, coupling with substrate ultrasonic pretreatment technology, magnetic cross-linked enzyme aggregates (CRL-FIL-CLEAs@Fe3O4) was used as novel biocatalyst for efficient preparation of phytosterol oleate in solvent-free reaction system. The CRL loading was 0.16 mg protein/mg CRL-FIL- CLEAs@Fe3O4. The effects of ultrasonic parameters, catalyst dosage, substrate molar ratio, and reaction temperature on the synthesis of phytosterol esters were studied. The results showed that the catalytical properties of CRL-FIL-CLEAs@Fe3O4 (7.09±0.11 µmol phytosterol/mg protein. hour) was significantly improved compared with natural lipase from Candida rugosa (CRL, 5.13±0.21 µmol phytosterol/mg protein. hour). Simultaneously, ultrasonic pretreatment greatly enhanced substrate mass transfer, further reducing the esterification time. The max conversion rate 94.72% of phytosterol was obtained after 9 h reaction under optimum condition (the molar ratio of oleic acid to phytosterol was 6:1, the concentration of CRL-FIL-CLEAs@Fe3O4 was 10 wt% of the phytosterol mass). After five recycles, the activity of CRL-FIL-CLEAs@Fe3O4 (80.02%) was still 2.68 times higher than that of natural lipase system (29.91%). Meanwhile, the novel immobilized lipase (CRL-FIL-CLEAs@Fe3O4) before and after reaction was characterized by SEM, AFM, VSM, TEM, and TGA. The results showed that CRL-FIL-CLEAs@Fe3O4 was a robust and recyclable biocatalyst for phytosterol esterification.

PEI-crosslinked lipase on the surface of magnetic microspheres and its characteristics

Here, PEI@PMMA microspheres were prepared by grafting polyethyleneimine (PEI) on poly(methyl methacrylate) (PMMA) magnetic microspheres and successfully used to immobilize lipase. The results showed that PEI@PMMA microspheres had strongly adsorbed lipase (49.1 mg/g microsphere) via electrostatic attraction. To prevent lipase shedding, the adsorbed lipase was further crosslinked with PEI on microspheres using glutaraldehyde as crosslinker. Consequently, PEI-crosslinked lipase (2.14 U/mg) exhibited 2.6 times and 1.4 times higher activity respectively than the directly covalent lipase (0.82 U/mg) and the crosslinked lipase aggregates (1.57 U/mg), which was close to the activity of adsorbed lipase (2.20 U/mg). Conformational analysis from FTIR spectroscopy showed that PEI-crosslinked lipase retained its natural structure well. And the α-helix structure seemed to play a key role in enhancing lipase activity. Furthermore, the effects of various parameters on crosslinking reaction were investigated. Also, PEI-crosslinked lipase revealed higher pH and thermal stability. The Michaelis constant (Km) was increased and the optimum temperature of lipase was widened observably after crosslinking with PEI on PEI@PMMA magnetic microspheres.

Kinetic resolution of racemic naproxen methyl ester by magnetic and non-magnetic cross-linked lipase aggregates

In this study, the non-magnetic and the magnetic cross-linked enzyme aggregates (CLEAs) from Candida rugosa lipase were synthesized to catalyze the kinetic resolution reaction of naproxen methyl ester (NME). Magnetic iron oxide nanoparticles (MIONPs) were produced through co-precipitation method and their surfaces were modified by silanization reaction. The MIONPs were used as a platform to synthesize the magnetic CLEAs (M-CLEAs). The biocatalysts and MIONPs synthesized were characterized by FTIR spectroscopy and SEM analysis. The kinetic resolution of racemic NME was studied in aqueous buffer solution/isooctane biphasic system to compare the performance of M-CLEAs and CLEAs. The effects of reaction parameters such as temperature, pH, stirring rate on the enantiomeric excess of the substrate (ees%) were investigated in a batch reactor system. The activity recovery of CRL enzyme in CLEAs was higher than M-CLEAs. Compared with M-CLEAs, CLEAs biocatalysts had previously reached ees% values. Although both biocatalysts showed similar cavity structure from SEM analysis, the lower performance of M-CLEAs may be due to the different microenvironments of M-CLEAs from CLEAs. However, the reusability performance of M-CLEAs was higher than that of CLEAs. The optimal reaction conditions for M-CLEAs and CLEAs were found to be 37 °C, pH 7.5, and 300 rpm.

Improved Performance of Magnetic Cross-Linked Lipase Aggregates by Interfacial Activation: A Robust and Magnetically Recyclable Biocatalyst for Transesterification of Jatropha Oil.

Lipases are the most widely employed enzymes in commercial industries. The catalytic mechanism of most lipases involves a step called “interfacial activation”. As interfacial activation can lead to a significant increase in catalytic activity, it is of profound importance in developing lipase immobilization methods. To obtain a potential biocatalyst for industrial biodiesel production, an effective strategy for enhancement of catalytic activity and stability of immobilized lipase was developed. This was performed through the combination of interfacial activation with hybrid magnetic cross-linked lipase aggregates. This biocatalyst was investigated for the immobilization of lipase from Rhizomucor miehei (RML). Under the optimal conditions, the activity recovery of the surfactant-activated magnetic RML cross-linked enzyme aggregates (CLEAs) was as high as 2058%, with a 20-fold improvement over the free RML. Moreover, the immobilized RML showed excellent catalytic performance for the biodiesel reaction at a yield of 93%, and more importantly, could be easily separated from the reaction mixture by simple magnetic decantation, and retained more than 84% of its initial activities after five instances of reuse. This study provides a new and versatile approach for designing and fabricating immobilized lipase with high activation and stability.

Preparation of spherical cross-linked lipase aggregates with improved activity, stability and reusability characteristic in water-in-ionic liquid microemulsion

BACKGROUND Cross-linking enzyme aggregates (CLEAs) seem to be a promising immobilization method due to mild processing conditions and short preparation time. However, the CLEAs prepared in aqueous solution exhibit amorphous large clusters that cause significant mass-transfer limitations, leading to low catalytic efficiency. In this work, novel spherical CLEAs of lipase from bovine pancreas (spherical CLEAs) were prepared within droplets dispersed in a water-in-hydrophobic ionic liquid microemulsion rather than a conventional aqueous solution. The properties of the spherical CLEAs were determined. RESULTS The resultant CLEAs exhibited spherical structure with good monodispersity instead of the amorphous clusters of conventional CLEAs, and showed higher activity recovery (84.6%) than that of conventional CLEAs (52.8%). The high activity recovery of spherical CLEAs could be attributed to both interfacial activation of the microemulsion system and the well-defined structure of the spherical CLEAs, which not only activated lipases in an active form, but also decreased mass-transfer limitations. Moreover, the thermostability, storage stability, tolerance against hydrophilic solvents, and mechanical stability of the spherical CLEAs were enhanced significantly. The spherical CLEAs evidenced excellent reusability in comparison with conventional CLEAs. CONCLUSIONS The novel strategy in this study might open an attractive way towards effectively improving the catalytic properties of traditional CLEAs. © 2016 Society of Chemical Industry

Hybrid Cross-Linked Lipase Aggregates with Magnetic Nanoparticles: A Robust and Recyclable Biocatalysis for the Epoxidation of Oleic Acid.

Highly stable and easily recyclable hybrid magnetic cross-linked lipase aggregates (HM-CSL-CLEAs) were prepared by coaggregation of lipase aggregates with nonfunctionalized magnetic nanoparticles and subsequent chemical cross-linking with glutaraldehyde. Analysis by SEM and CLSM indicated that the CLEAs were embedded in nanoparticle aggregates instead of covalently immobilized. The resulting HM-CSL-CLEAs exhibited higher thermostability, storage stability, and reusability than standard CLEAs. For example, HM-CSL-CLEAs maintained >60% of their initial activity after 40 min of incubation at 60 °C, whereas standard CLEAs lost most of their activities. The HM-CSL-CLEAs can be easily recovered from the reaction mixture by an external magnetic field. Moreover, the H2O2 tolerance of the lipase in HM-CSL-CLEAs was also enhanced, which could relieve the inhibitory effect on lipase activity. A high conversion yield (55%) for the epoxidation of oleic acid using H2O2 as oxidizing agent was achieved by HM-CSL-CLEAs.

Biocatalytic epoxidation of α-pinene to oxy-derivatives over cross-linked lipase aggregates

Lipase-based cross-linked aggregates were investigated for a non-specific reaction, i.e. the epoxidation of α-pinene to its oxygenated derivatives. The activity of the biocatalysts has been evaluated in a green context, i.e. ethyl acetate as both acetate-supplier and organic solvent with H2O2/UHP/TBHP as oxidant. Screening of the lipase sources indicated Aspergillus niger lipase as the most efficient biocatalyst for this reaction. Different immobilization protocols ((i) cross-linked enzyme aggregates (CLEA), (ii) cross-linked enzyme aggregates onto magnetic particles (CLEMPA) and (iii) covalent immobilized enzyme (CIE) onto magnetic particles (MP)) were evaluated considering the activity as main parameter. Thus, CLEA and CLEMPA afforded better epoxidation yields of α-pinene towards CIE. The investigated biocatalytic systems allowed to transform α-pinene into oxigenated derivatives with industrial and commercial applications (e.g. α-pinene oxide, camphene, pinanediol and camphonelic aldehyde). FTIR investigations on the biocatalysts revealed the effects of the immobilization protocol on the enzyme secondary-structure. Additionally, textural characterizations were performed by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) analysis.

Enhancing the resolution of (R,S)-2-octanol catalyzed by magnetic cross-linked lipase aggregates using an alternating magnetic field

Magnetic cross-linked lipase aggregates (MCLEAs) were used as a biocatalyst to catalyze the resolution of (R,S)-2-octanol under an alternating magnetic field. The magnetic field resulted in enhanced apparent enzymatic activity. The effects of varying the magnetic field parameters showed that the magnetic field frequency played a more dominant role than field strength on the resolution of (R,S)-2-octanol. Using the optimal conditions, the MCLEAs were highly stable under the alternating magnetic field and were suitable for multiple reuse cycles for the resolution of (R,S)-2-octanol.

Novel magnetic cross-linked lipase aggregates for improving the resolution of (R, S)-2-octanol.

Novel magnetic cross-linked lipase aggregates were fabricated by immobilizing the cross-linked lipase aggregates onto magnetic particles with a high number of -NH2 terminal groups using p-benzoquinone as the cross-linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross-linked lipase aggregates were achieved. The magnetic cross-linked lipase aggregates were able to efficiently resolve (R, S)-2-octanol, and retained 100% activity and 100% enantioselectivity after 10 cycles of reuse, whereas the cross-linked lipase aggregates only retained about 50% activity and 70% enantioselectivity due to insufficient cross-linking. These results provide a great potential for industrial applications of the magnetic cross-linked lipase aggregates.

Synthesis of crosslinked lipase aggregates and their use in the synthesis of aspirin

Biocatalytic synthesis of drugs is imperative to avoid the hazardous environmental effects of the pharmaceutical production processes. In view of this, biocatalytic synthesis of aspirin is discussed in the present work. A crude lipase extracted from Geobacillus sp. was crosslinked using N,N-methylenebisacrylamide (N,N-MBAAm) and two different initiators: ammonium persulphate (APS) and γ-irradiation. The crude lipase was crosslinked as such bypassing the separation procedures using a biocompatible crosslinker. The crosslinked lipase was characterized by various techniques and evaluated for its activity profile as a function of time, temperature, pH and substrate concentration and results were compared to its free form. The activity profile of the crosslinked lipase was observed to be only marginally lower than the free lipase. The crosslinked lipases were used as catalysts in the green synthesis of acetylsalicylic acid, aspirin, directly from the acetic acid. The reusability studies of the crosslinked lipases were carried out up to 10 cycles.

Designing cross-linked lipase aggregates for optimum performance as biocatalysts

Cross-linked enzyme aggregates (CLEAs) are prepared by precipitation of an enzyme and then chemical cross-linking the precipitate. Three CLEAs of lipase with glutaraldehyde concentrations of 10 mM (CLEA A), 40 mM (CLEA B) and 60 mM (CLEA C) were prepared. Studies show that there is a trade-off between thermal stability vs transesterification/hydrolysis rate vs enantioselectivity. The initial rates for transesterification of β-citronellol for the uncross-linked enzyme and CLEAs A, B and C were 243, 167, 102 and 40 µmol mg−1 h−1, respectively. Their thermal stabilities in aqueous media, as reflected by their half-life values at 55°C, were 6, 9, 13 and 16 h, respectively. The enantioselectivity, E values (for kinetic resolution of β-citronellol by transesterification) were 19, 74, 11 and 6, respectively. These results show that CLEA C was the most thermostable; the uncross-linked enzyme was best at obtaining the highest transesterification rate; and CLEA A was best suited for the enantioselective synthesis. Scanning electron microscopy (SEM) showed that the morphology of CLEA was dependent upon the extent of cross-linking.

Immobilization of cross-linked lipase aggregates within microporous polymeric membranes

A new approach is proposed to immobilize lipase into microporous polymeric membrane via embedding cross-linked enzyme aggregates (CLEA) within membrane pores. Preparation of biocatalytic membranes was implemented with hydrophilic cellulose and hydrophobic PTFE membranes. Formation of lipase CLEAs is initiated by solvent exchange between aqueous solution of the enzyme filling membrane pores and organic media containing cross-linking agent. The influence of organic media composition and matrix hydrophilicity/hydrophobicity on membrane biocatalytic properties has been studied. Highly effective biocatalytic membranes with embedded lipase CLEA have been obtained on the base of both hydrophilic and hydrophobic membranes by varying the content of glutaric dialdehyde used as a cross-linking agent. Bio-imprinting has been tested as a mean to improve efficiency of the biocatalytic membranes. Biocatalytic esterification of oleic acid with n-butanol has been studied in cross-flow membrane bioreactor with membrane-supported lipase CLEA using hexane and iso-octane as reaction solvents of different hydrophobicity.