Activation of crosslinked lipases in mesoporous silica via lid opening for recyclable biodiesel production.

Lipases catalyze a wide range of industrially important reactions, including the transesterification of triglycerides with alcohols for biodiesel production, and the stabilization of lipases are critical to achieve their recycled uses. Here, nanoscale enzyme reactor (NER) of lipase from Rhizopus oryzae (LP) was prepared via a simple two-step process, comprising of enzyme adsorption into magnetically-separable mesoporous silica and follow-up crosslinking of adsorbed enzymes. In aqueous phase, the specific hydrolysis activity of NER-LP was 4.7 times lower than that of free LP. On the other hand, however, the specific transesterification activity of NER-LP (130.4 µmol/min/mg LP) in organic phase for biodiesel production was 50 times higher than that of free LP (2.6 µmol/min/mg LP). These results reveal that the enzyme crosslinking for the preparation of NER does not interfere with the interfacial activation of LP molecules, opening the lid of LP active site under an optimal hydrophobic environment provided by the combination of organic solvent and mesoporous silica. Magnetic separation and optimized washing protocol facilitated the recycled uses of NER-LP. Highly stable and active NER-LP in magnetically-separable mesoporous silica has demonstrated its great potentials as an environmentally-friendly nanobiocatalyst for various lipase applications, including plasticizers, biosurfactants, functional fatty acids, as well as recyclable biodiesel production.

Controlled growth of redox polymer network on single enzyme molecule for stable and sensitive enzyme electrode.

The electrochemical applications of enzymes are often hampered by poor enzyme stability and low electron conductivity. In this work, a novel enzyme nanogel based on atom transfer radical polymerization (ATRP) has been developed for highly sensitive detection of glucose based on ferrocene (Fc) embedded in crosslinked polymer network nanogel. Enzyme surfaces are successively modified with Br initiator, and then in situ atom transfer radical polymerization (ATRP) was performed to build up crosslinked polyacrylamide network. The resulting single enzyme nanogel (ATRP-SEG) is uniform in size fairly. ATRP-SEG reveals bi-phasic inactivation, and the half-life of stable ATRP-SEG after 18-day incubation at 50 °C is 47 days, which is 197 times longer than that of free Gox (5.7 h). By introducing a ferrocene (Fc) containing redox polymer, poly(acrylamide-co-vinylferrocene), the half-life of Fc-ATRP-SEG after 18-day incubation at 50 °C is 49 days. Fc-ATRP-SEG is used for preparation of glucose-sensing electrode, and the sensitivity of Fc-ATRP-SEG electrode is 111 µA cm-2 mM-1, which is 366 and 1270 times higher than those of free GOx (0.303 µA cm-2 mM-1) and ATRP-SEG (0.0874 µA cm-2 mM-1), respectively. Fc-ATRP-SEG electrode maintained 90% of initial current density under 4 °C storage condition and repetitive usages every day for 7 days. Even the electrode repeatedly used in continuous harsh condition (250 rpm, room temperature), the current density maintained 96% after 12 h incubation at operational condition.

Effect of Surface and Bulk Properties of Mesoporous Carbons on the Electrochemical Behavior of GOx-Nanocomposites.

Biofuel cell (BFC) electrodes are typically manufactured by combining enzymes that act as catalysts with conductive carbon nanomaterials in a form of enzyme-nanocomposite. However, a little attention has been paid to effects of the carbon nanomaterials' structural properties on the electrochemical performances of the enzyme-nanocomposites. This work aims at studying the effects of surface and bulk properties of carbon nanomaterials with different degrees of graphitization on the electrochemical performances of glucose oxidase (GOx)-nanocomposites produced by immobilizing GOx within a network of carbon nanopaticles. Two types of carbon nanomaterials were used: graphitized mesoporous carbon (GMC) and purified mesoporous carbon (PMC). Graphitization index, surface functional groups, hydrophobic properties, and rate of aggregation were measured for as-received and acid-treated GMC and PMC samples by using Raman spectrometry, X-ray photoelectron spectroscopy (XPS), contact angle measurement, and dynamic light scattering (DLS), respectively. In addition to these physical property characterizations, the enzyme loading and electrochemical performances of the GOx-nanocomposites were studied via elemental analysis and cyclic voltammetry tests, respectively. We also fabricated BFCs using our GOx-nanocomposite materials as the enzyme anodes, and tested their performances by obtaining current-voltage (IV) plots. Our findings suggest that the electrochemical performance of GOx-nanocomposite material is determined by the combined effects of graphitization index, electrical conductivity and surface chemistry of carbon nanomaterials.

Tyrosinase-immobilized CNT based biosensor for highly-sensitive detection of phenolic compounds.

Highly sensitive phenol biosensor was developed by using well-dispersed carbon nanotubes (CNTs) in enzyme solution and adding CNTs in enzyme electrodes. First, the intact CNTs were dispersed in aqueous tyrosinase (TYR) solution, and TYR molecules were precipitated and crosslinked to prepare the sample of enzyme adsorption, precipitation and crosslinking (EAPC). EAPC exhibited 10.5- and 5.4-fold higher TYR activity per mg of CNTs as compared to enzyme adsorption (EA) and enzyme adsorption/crosslinking (EAC), respectively. EAPC retained 29% of its initial activity after incubation at 40 °C for 128 h, while EA and EAC showed no residual activities, respectively. In biosensing a model phenolic compound of catechol, the sensitivities of EA, EAC and EAPC electrodes on glassy carbon electrode (GCE) were 34, 281 and 675 µA/mM/cm2, respectively. When 90 w/w% CNTs were added to the enzyme electrodes, the sensitivities of EA, EAC, and EAPC electrodes were 146, 427, and 1160 µA/mM/cm2, respectively, and the EAPC electrode showed a 2.3-fold increase in sensitivity upon CNT addition. Catechol and phenol could also be detected by EAPC on the screen-printed electrode (SPE), with sensitivities of 1340 and 1170 µA/mM/cm2, respectively. The sensitivity of EAPC-SPE for phenol detection in the effluent from real municipal wastewater treatment plant was 1100 µA/mM/cm2. The sensitivity of EAPC-SPE retained 74% of its initial sensitivity after incubation at 40 °C for 12 h. The combination of EAPC immobilization and CNT addition has great potential for application in the development of sensitive enzyme biosensors for various analytes and phenols in water environments.

Fabrication of enzyme-based coatings on intact multi-walled carbon nanotubes as highly effective electrodes in biofuel cells.

CNTs need to be dispersed in aqueous solution for their successful use, and most methods to disperse CNTs rely on tedious and time-consuming acid-based oxidation. Here, we report the simple dispersion of intact multi-walled carbon nanotubes (CNTs) by adding them directly into an aqueous solution of glucose oxidase (GOx), resulting in simultaneous CNT dispersion and facile enzyme immobilization through sequential enzyme adsorption, precipitation, and crosslinking (EAPC). The EAPC achieved high enzyme loading and stability because of crosslinked enzyme coatings on intact CNTs, while obviating the chemical pretreatment that can seriously damage the electron conductivity of CNTs. EAPC-driven GOx activity was 4.5- and 11-times higher than those of covalently-attached GOx (CA) on acid-treated CNTs and simply-adsorbed GOx (ADS) on intact CNTs, respectively. EAPC showed no decrease of GOx activity for 270 days. EAPC was employed to prepare the enzyme anodes for biofuel cells, and the EAPC anode produced 7.5-times higher power output than the CA anode. Even with a higher amount of bound non-conductive enzymes, the EAPC anode showed 1.7-fold higher electron transfer rate than the CA anode. The EAPC on intact CNTs can improve enzyme loading and stability with key routes of improved electron transfer in various biosensing and bioelectronics devices.

Enzyme precipitate coating of pyranose oxidase on carbon nanotubes and their electrochemical applications.

Pyranose oxidase (POx), which doesn't have electrically non-conductive glycosylation moiety, was immobilized on carbon nanotubes (CNTs) via three different preparation methods: covalent attachment (CA), enzyme coating (EC) and enzyme precipitate coating (EPC). CA, EC and EPC of POx on CNTs were used to fabricate enzymatic electrodes for enzyme-based biosensors and biofuel cells. Improved enzyme loading of EPC resulted in 6.5 and 4.5 times higher activity per weight of CNTs than those of CA and EC, respectively. After 34 days at room temperature, EPC retained 65% of initial activity, while CA and EC maintained 9.2% and 26% of their initial activities, respectively. These results indicate that precipitation and crosslinking steps of EPC have an important role in maintaining enzyme activity. To demonstrate the feasibility of POx-based biosensors and biofuel cells, the enzyme electrodes were prepared using CA, EC, and EPC samples. In the case of biosensor, the sensitivities of the CA, EC, and EPC electrodes without BQ were measured to be 0.27, 0.76 and 3.7mA/M/cm2, while CA, EC and EPC electrode with BQ showed 25, 25, and 60mA/M/cm2 of sensitivities, respectively. The maximum power densities of biofuel cells using CA, EC and EPC electrodes without BQ were 41, 47 and 53µW/cm2, while CA, EC and EPC electrodes with BQ showed 260, 330 and 500µW/cm2, respectively. The POx immobilization and stabilization via the EPC approach can lead us to develop continuous glucose monitoring biosensors and high performing biofuel cells.