Co-Immobilization of Tri-Enzymes for the Conversion of Hydroxymethylfurfural to 2,5-Diformylfuran.

Acting as a "green" manufacturing route, the enzyme toolbox made up of galactose oxidase, catalase, and horseradish peroxidase can achieve a satisfactory yield of 2,5-diformylfuran derived from 30 mM hydroxymethylfurfural. However, as the concentration of hydroxymethylfurfural increases, the substrate causes oxidative damage to the activity of the tri-enzyme system, and the accumulated hydrogen peroxide produced by galactose oxidase causes tri-enzyme inactivation. The cost of tri-enzymes is also very high. These problems prevent the utilization of this enzyme toolbox in practice. To address this, galactose oxidase, catalase, and horseradish peroxidase were co-immobilized into Cu3(PO4)2 nanoflowers in this study. The resulting co-immobilized tri-enzymes possessed better tolerance towards the oxidative damage caused by hydroxymethylfurfural at high concentrations, as compared to free tri-enzymes. Moreover, the 2,5-diformylfuran yield of co-immobilized tri-enzymes (95.7 ± 2.7%) was 1.06 times higher than that of separately immobilized enzymes (90.4 ± 1.9%). This result could be attributed to the boosted protective effect provided by catalase to the activity of galactose oxidase, owing to the physical proximity between them on the same support. After 30 recycles, co-immobilized tri-enzymes still achieves 86% of the initial yield. Moreover, co-immobilized tri-enzymes show enhanced thermal stability compared with free tri-enzymes. This work paves the way for the production of 2,5-diformylfuran from hydroxymethylfurfural via co-immobilized tri-enzymes.

Prevention of Bacterial Contamination of a Silica Matrix Containing Entrapped ß-Galactosidase through the Action of Covalently Bound Lysozymes.

ß-galactosidase was successfully encapsulated within an amino-functionalised silica matrix using a "fish-in-net" approach and molecular imprinting technique followed by covalent binding of lysozyme via a glutaraldehyde-based method. Transmission electron microscopy (TEM), X-ray diffraction (XRD), scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) spectroscopy were used to characterise the silica matrix hosting the two enzymes. Both encapsulated ß-galactosidase and bound lysozyme exhibited high enzymatic activities and outstanding operational stability in model reactions. Moreover, enzyme activities of the co-immobilised enzymes did not obviously change relative to enzymes immobilised separately. In antibacterial tests, bound lysozyme exhibited 95.5% and 89.6% growth inhibition of Staphylococcus aureus ATCC (American type culture collection) 653 and Escherichia coli ATCC 1122, respectively. In milk treated with co-immobilised enzymes, favourable results were obtained regarding reduction of cell viability and high lactose hydrolysis rate. In addition, when both co-immobilised enzymes were employed to treat milk, high operational and storage stabilities were observed. The results demonstrate that the use of co-immobilised enzymes holds promise as an industrial strategy for producing low lactose milk to benefit people with lactose intolerance.

Ultrasound-Assisted Enantioselective Esterification of Ibuprofen Catalyzed by a Flower-Like Nanobioreactor.

A flower-like nanobioreactor was prepared for resolution of ibuprofen in organic solvents. Ultrasound irradiation has been used to improve the enzyme performance of APE1547 (a thermophilic esterase from the archaeon Aeropyrum pernix K1) in the enantioselective esterification. Under optimum reaction conditions (ultrasound power, 225 W; temperature, 45 °C; water activity, 0.21), the immobilized APE1547 showed an excellent catalytic performance (enzyme activity, 13.26 µmol/h/mg; E value, 147.1). After ten repeated reaction batches, the nanobioreactor retained almost 100% of its initial enzyme activity and enantioselectivity. These results indicated that the combination of the immobilization method and ultrasound irradiation can enhance the enzyme performance dramatically.

Microwave-Assisted Resolution of α-Lipoic Acid Catalyzed by an Ionic Liquid Co-Lyophilized Lipase.

The combination of the ionic liquid co-lyophilized lipase and microwave irradiation was used to improve enzyme performance in enantioselective esterification of α-lipoic acid. Effects of various reaction conditions on enzyme activity and enantioselectivity were investigated. Under optimal condition, the highest enantioselectivity (E = 41.2) was observed with a high enzyme activity (178.1 µmol/h/mg) when using the ionic liquid co-lyophilized lipase with microwave assistance. Furthermore, the ionic liquid co-lyophilized lipase exhibited excellent reusability under low power microwave.

Improvement of the enzyme performance of trypsin via adsorption in mesoporous silica SBA-15: hydrolysis of BAPNA.

The enzymatic performance of trypsin in hydrolysis of N-α-benzoyl-DL-arginine-4-nitroanilide (BAPNA) was improved by adsorption on Santa Barbara Amorphous (SBA)-15 mesoporous silica. The optimal immobilization conditions were screened and the properties of immobilized enzyme have also been studied. Under the optimal conditions, the immobilized trypsin displays maximum specific activity (49.8 µmol/min/g). The results also indicate that the immobilized trypsin exhibits better storage stability.

Combining the physical adsorption approach and the covalent attachment method to prepare a bifunctional bioreactor.

Aminopropyl-functionalized SBA-15 mesoporous silica was used as a support to adsorb myoglobin. Then, in order to avoid the leakage of adsorbed myoglobin, lysozyme was covalently tethered to the internal and external surface of the mesoporous silica with glutaraldehyde as the coupling agent. The property of amino-functionalized mesoporous silica was characterized by N(2) adsorption-desorption and thermogravimetric (TG) analysis. The feature of the silica-based matrix before and after myoglobin adsorption was identified by fourier transform infrared (FTIR) and UV/VIS measurement. With o-dianisidine and H(2)O(2) as the substrate, the peroxidase activity of adsorbed myoglobin was determined. With Micrococus lysodeilicus as the substrate, the antibacterial activity of covalently tethered lysozyme was measured. Results demonstrated that the final product not only presented peroxidase activity of the myoglobin but yielded antibacterial activity of the lysozyme.

Xanthan gum/oil body-microgel emulsions with enhanced transdermal absorption for accelerating wound healing.

The oil body comprises lipid droplets surrounded by a surface embedded with oil body-related proteins. To form a drug delivery system, an oleosin can be fused with foreign proteins and bound to the oil body surface. Here, safflower oil bodies carrying oleosin-human epidermal growth factor (hEGF) were mixed with xanthan gum to form self-assembled polymers, referred as an oil body microgel emulsion (OBEME) without any chemical crosslinking agent. The physicochemical properties of OBEME were evaluated and compared with those of natural lipid droplets. The electrostatic interaction between xanthan gum and oil bodies prevents excessive cross-linking and forms a uniform network structure. The basic properties of OBEME were characterized by scanning electron microscopy, cryo-scanning electron microscopy, rheology, and thermogravimetric analysis. The OBEME is an interconnected network and presents a smooth surface without any pores; it remains stable at room temperature for 90 days, and is not affected by low-speed centrifugation and repeated freeze-thaw cycles as indicated by particle size, potential, and fluorescence microscopy analyses. The OBEME enlarges the skin tissue gap, enhances skin permeability, and shows a good slow-release effect in the transdermal absorption test in vivo. It demonstrates a wound healing effect; further, it regulates the inflammatory response of full-layer skin wounds in rats, as well as accelerate angiogenesis, and promote re-epithelialization and remodeling. The OBEME as a bioactive molecule-carbohydrate complex can effectively accelerate skin regeneration and has great translational potential to provide low-cost alternative wound care treatments.

The Fabrication of Amino Acid Incorporated Nanoflowers with Intrinsic Peroxidase-like Activity and Its Application for Efficiently Determining Glutathione with TMB Radical Cation as Indicator.

The assessment of glutathione (GSH) levels is associated with early diagnostics and pathological analysis for various disorders. Among all kinds of techniques for detecting GSH, the colorimetric assay relying on the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) catalyzed by many nanomaterials with peroxidase-like activity attracts increasing attention owing to its outstanding merits, such as high sensitivity and high selectivity. However, the aggregation between the nanomaterials severely hinders the entrance of TMB into the "active site" of these peroxidase mimics. To address this problem, the D-amino acid incorporated nanoflowers possessing peroxidase-like activity with a diameter of 10-15 µm, TMB and H2O2 were employed to establish the detection system for determining the level of glutathione. The larger diameter size of the hybrid nanoflowers substantially averts the aggregation between them. The results confirm that the hybrid nanoflowers detection system presents a low limit of detection, wide linear range, perfect selectivity, good storage stability and desired operational stability for the detection of GSH relying on the intrinsic peroxidase-like activity and favorable mechanical stability of the hybrid nanoflowers, indicating that the hybrid nanoflowers detection system has tremendous application potential in clinical diagnosis and treatment.

Nitroxide-Modified Protein-Incorporated Nanoflowers with Dual Enzyme-Like Activities.

PURPOSE: Combined superoxide dismutase (SOD)/catalase mimetics have attracted much attention because of their efficacy against reactive oxygen species-associated diseases; however, their application is often limited owing to their poor stability and the absence of favorable grafting sites. To address this, we developed a new class of SOD/catalase mimetics based on hybrid nanoflowers, which exhibit superior stability and possess the desired grafting sites for drugs and endogenous molecules. METHODS: In this work, for the first time, we used polynitroxylated human serum albumin (PNA) to mediate the formation of hybrid copper-based nanoflowers. H2O2 depletion and O2 evolution assays were first performed to determine the catalase-like activity of the hybrid nanoflowers. Next, the xanthine oxidase/cytochrome c method was used to assay the SOD-like activity of the nanoflowers. Further characteristics of the nanoflowers were evaluated using scanning electron microscopy (SEM), electron paramagnetic resonance (EPR), and Fourier-transform infrared spectroscopy (FTIR). Operational stability was assessed via the reusability assay. RESULTS: The H2O2 depletion and O2 evolution assays indicated that PNA-incorporated nanoflowers have genuine catalase-like activity. Kinetic analysis revealed that the reactions of the incorporated nanoflowers with H2O2 not only obey Michaelis-Menton kinetics, but that the nanoflowers also possess a higher affinity for H2O2 than that of native catalase. The FTIR spectra corroborated the presence of PNA in the hybrid nanoflowers, while the EPR spectra confirmed the intermolecular interaction of nitroxides bound to the human serum albumin incorporated into the nanoflowers. The remarkable operational reproducibility of the hybrid nanoflowers in catalase-like and SOD-like reactions was verified across successive batches. CONCLUSION: Herein, a comparison of Michaelis constants showed that the hybrid nanoflower, a catalase mimetics, outperforms the native catalase. Acting as a "better-than-nature" enzyme mimetics, the hybrid nanoflower with superior stability and desired ligand grafting sites will find widespread utilization in the medical sciences.

Ordered cubic mesoporous silicas with large pore sizes synthesized via high-temperature route.

Ordered cubic mesoporous silicas with large pore sizes (LP-SBA-16) have been successfully synthesized at high temperatures (180-220 degrees C) using polymer surfactant of F127 as a template. Compared with conventional SBA-16 with entrance size at 3.6 nm, LP-SBA-16 samples synthesized at high temperatures show large entrance sizes at 11.8-24.7 nm, confirmed by the pore size distribution and TEM images. The formation of these large pore sizes in the samples is attributed to the increase of surfactant hydrophobicity and the merging of mesopores at high temperatures. The results obtained from NMR and XRD techniques show that ordered mesostructure in LP-SBA-16 samples basically remained even if the surfactant (F127) is fully decomposed at high temperatures, indicating that F127 surfactant becomes unimportant when the mesoporous walls of silica have been condensed. Furthermore, we have compared the adsorption capacity of myoglobin over conventional SBA-16 and LP-SBA-16 samples, and it is found that LP-SBA-16 samples exhibit much higher adsorption capacity than conventional SBA-16, which is potentially important for immobilization of enzymes on ordered mesoporous materials.

Optimization of a dual-functional biocatalytic system for continuous hydrolysis of lactose in milk.

In this study, an amino-functionalized silica matrix encapsulating ß-galactosidase was first synthesized in situ, with subsequent covalent anchoring of lysozyme to the outer part of the amino-grafted matrix. Fourier transform infrared (FTIR) spectra verified that ß-galactosidase was successfully encapsulated. Meanwhile, the co-immobilized enzymes were demonstrated to retain suitable enzymatic activities and outstanding operational stability during successive reaction cycles. Furthermore, when used for lactose removal from skim milk, the packed-bed column system achieved both a high lactose hydrolysis rate and microbial inactivation ratio during 30 days of continuous operation. Notably, this system exhibited favorable stability during 60 days of continuous hydrolysis of lactose in solution and thus may be appropriate for further development for use in industrial lactose removal from milk.

Immobilization of Bacillus subtilis lipase on a Cu-BTC based hierarchically porous metal-organic framework material: a biocatalyst for esterification.

Bacillus subtilis lipase (BSL2) has been successfully immobilized into a Cu-BTC based hierarchically porous metal-organic framework material for the first time. The Cu-BTC hierarchically porous MOF material with large mesopore apertures is prepared conveniently by using a template-free strategy under mild conditions. The immobilized BSL2 presents high enzymatic activity and perfect reusability during the esterification reaction. After 10 cycles, the immobilized BSL2 still exhibits 90.7% of its initial enzymatic activity and 99.6% of its initial conversion.

Amino acids-incorporated nanoflowers with an intrinsic peroxidase-like activity.

Functional molecules synthesized by self-assembly between inorganic salts and amino acids have attracted much attention in recent years. A simple method is reported here for fabricating hybrid organic-inorganic nanoflowers using copper (II) ions as the inorganic component and natural amino acids as the organic component. The results indicate that the interactions between amino acid and copper ions cause the growth of the nanoflowers composed by C, N, Cu, P and O elements. The Cu ions and Cu(AA)n complexes containing Cu-O bond are present in the nanoflowers. The nanoflowers have flower-like porous structure dominated by the R groups of amino acids with high surface-to-volume ratios, which is beneficial for exerting its peroxidase-like activity depending on Fenton-like reaction mechanism with ABTS and Rhodamine B as the substrates. It is expected that the nanoflowers hold great promise as enzyme mimics for application in the field of biosensor, bioanalysis and biocatalysis.