Shi-Lin Cao

Preparation and Characterization of Immobilized Lipase from Pseudomonas Cepacia onto Magnetic Cellulose Nanocrystals

Magnetic cellulose nanocrystals (MCNCs) were prepared and used as an enzyme support for immobilization of Pseudomonas cepacialipase (PCL). PCL was successfully immobilized onto MCNCs (PCL@MCNC) by a precipitation-cross-linking method. The resulting PCL@MCNC with a nanoscale size had high enzyme loading (82.2 mg enzyme/g) and activity recovery (95.9%). Compared with free PCL, PCL@MCNC exhibited significantly enhanced stability and solvent tolerance, due to the increase of enzyme structure rigidity. The observable optimum pH and temperature for PCL@MCNC were higher than those of free PCL. PCL@MCNC manifested relatively higher enzyme-substrate affinity and catalytic efficiency. Moreover, PCL@MCNC was capable of effectively catalyzing asymmetric hydrolysis of ketoprofenethyl ester with high yield of 43.4% and product e.e. of 83.5%. Besides, immobilization allowed PCL@MCNC reuse for at least 6 consecutive cycles retaining over 66% of its initial activity. PCL@MCNC was readily recycled by magnetic forces. Remarkably, the as-prepared nanobiocatalyst PCL@MCNC is promising for biocatalysis.

Preparation of a novel magnetic cellulose nanocrystal and its efficient use for enzyme immobilization

A novel biocompatible magnetic cellulose nanocrystal (MCNC) composite was in situ prepared via a simple co-precipitation-electrostatic-self-assembly technique and was structurally characterized. The results showed that the anionic cellulose nanocrystals (CNCs) were successfully composited with cationic chitosan-coated Fe3O4 by self-assembly technology. The electrostatic interaction between CNCs and chitosan, and that between chitosan and Fe3O4, were the key driving forces for the formation of the composite. Papain, a widely used protease, could be successfully immobilized on the activated MCNCs with formaldehyde. The immobilized papain exhibited higher thermal stability than the free enzyme, with the relative activity being higher than 80% after incubation at 40 °C for 7 h while that of free papain was less than 30%. Also, the pH stability of immobilized papain was superior to that of free papain. Moreover, the immobilized papain showed significantly better tolerance to the three solvents tested compared with its free counterpart. The optimum range of pH for immobilized papain (pH 5-10) was remarkably wider than that of free enzyme (pH 5-7). The relative activities of immobilized papain at 50-70 °C were more than 90%, which significantly surpassed those of free papain. The immobilized papain also manifested excellent storage stability, with relative activity being as high as 93.6% after 16 days of storage at 4 °C. Furthermore, the obtained kinetic constant values showed that papain immobilized on the MCNCs had relatively high catalytic efficiency. Additionally, the immobilized papain could be easily separated and recycled from the reaction system through magnetic forces. Obviously, the prepared MCNCs as novel supports are promising and competitive for enzyme immobilization.

Recent advances in immobilized enzymes on nanocarriers

Abstract Recent progress in nanotechnology has provided high-performance nanomaterials for enzyme immobilization. Nanobiocatalysts combining enzymes and nanocarriers are drawing increasing attention because of their high catalytic performance, enhanced stabilities, improved enzyme–substrate affinities, and reusabilities. Many studies have been performed to investigate the efficient use of cellulose nanocrystals, polydopamine-based nanomaterials, and synthetic polymer nanogels for enzyme immobilization. Various nanobiocatalysts are highlighted in this review, with the emphasis on the design, preparation, properties, and potential applications of nanoscale enzyme carriers and nanobiocatalysts.

A magnetic biocatalyst based on mussel-inspired polydopamine and its acylation of dihydromyricetin

A support made of mussel-inspired polydopamine-coated magnetic iron oxide nanoparticles (PD-MNPs) was prepared and characterized. The widely used Aspergillus niger lipase (ANL) was immobilized on the PD-MNPs (ANL@PD-MNPs) with a protein loading of 138 mg/g and an activity recovery of 83.6% under optimized conditions. For the immobilization, the pH and immobilization time were investigated. The pH and thermal and storage stability of the ANL@PD-MNPs significantly surpassed those of free ANL. The ANL@PD-MNPs had better solvent tolerance than free ANL. The secondary structure of free ANL and ANL@PD-MNPs was analyzed by infrared spectroscopy. A kinetic study demonstrated that the ANL@PD-MNPs had enhanced enzyme-substrate affinity and high catalytic efficiency. The ANL@PD-MNPs was applied as a biocatalyst for the regioselective acylation of dihydromyricetin (DMY) in DMSO and gave a conversion of 79.3%, which was higher than that of previous reports. The ANL@PD-MNPs retained over 55% of its initial activity after 10 cycles of reuse. The ANL@PD-MNPs were readily separated from the reaction system by a magnet. The PD-MNPs is an excellent support for ANL and the resulting ANL@PD-MNPs displayed good potential for the efficient synthesis of dihydromyricetin-3-acetate by enzymatic regioselective acylation.

Highly Efficient Enzymatic Acylation of Dihydromyricetin by the Immobilized Lipase with Deep Eutectic Solvents as Cosolvent

A novel deep eutectic solvent (DES)-DMSO cosolvent system has been, for the first time, successfully used as the reaction medium for the enzymatic acylation of dihydromyricetin (DMY) catalyzed by the immobilized lipase from Aspergillus niger (ANL). The cosolvent mixture, ChCl:Glycerol-DMSO (1:3, v/v) proved to be the optimal medium. With the newly developed cosolvent, the initial reaction rate of enzymatic acylation of DMY achieved 11.1 mM/h and the conversion of DMY was 91.6%. ANL@PD-MNPs is stable and recyclable in this cosolvent, offering 90% conversion rate after repeated use of 5 times. The lipid-solubility of DMY-16-acetate was 10 times higher than that of its raw materials DMY. The results showed that the DMY-16-acetate product exhibits good antioxidative activity. The present research illustrated that the use of DES-DMSO cosolvent may become a feasible alternative for the synthesis of DMY ester.

Preparation of a Nanobiocatalyst by Efficiently Immobilizing Aspergillus niger Lipase onto Magnetic Metal-Biomolecule Frameworks (BioMOF)

A biocompatible support made of ZnGlu‐coated magnetic iron oxide nanoparticles (ZnGlu–MNPs) was prepared by a simple electrostatic self‐assembly technique and structurally characterized. Aspergillus niger lipase (ANL) was immobilized onto the ZnGlu–MNPs with high protein loading (118.0 mg g−1) and high enzyme‐activity recovery (more than 82.0 %). The as‐prepared immobilized ANL exhibits high catalytic performance and a wide pH and temperature adaptability. Moreover, the stabilities and the solvent tolerance of ANL@ZnGlu‐MNPs were clearly superior to those of the free counterpart. The novel ANL@ZnGlu‐MNPs was easily recycled from the reaction medium by magnetic forces. Therefore, the as‐prepared ZnGlu‐MNPs is a promising novel carrier for immobilized enzymes.