Oriented Enzyme Immobilization Research Articles

Oriented Enzyme Immobilization at the Oil/Water Interface Enhances Catalytic Activity and Recyclability in a Pickering Emulsion.

Enzyme-loaded water-in-oil Pickering emulsion is a promising system for biphasic catalytic reactions. In this paper, we report on oriented enzyme immobilization at the oil/water interface in a Pickering emulsion, in which CHO-Janus silica nanoparticles (CHO-JNPs) are utilized as a stabilizer of the emulsion and support for the enzyme to enhance both catalytic activity and recyclability. The catalytic performance of this immobilized enzyme (lipase from Candida sp.) was evaluated by esterification of hexanoic acid and 1-hexanol in a water/heptane biphasic medium. The results show that the specific catalytic activity of the immobilized enzyme (33.2 U mL-1) was 6.5 and 1.4 times higher than that of free enzyme (5.1 U mL-1) and encapsulated enzyme in the liquid core (23.3 U mL-1), respectively. Moreover, the immobilized enzyme demonstrated good stability and recyclability, retaining 75% of its activity after 9 cycles. We expect that oriented enzyme immobilization at the oil/water interface will be an important strategy for enhancing catalytic performance in Pickering emulsions.

Theoretical and experimental study of enzyme kinetics in a microreactor system with surface-immobilized biocatalyst

A mathematical model comprising transport phenomena and enzyme-catalyzed reaction performed on the inner walls of the continuously operated microreactor with surface-immobilized-transaminase was developed. Oriented enzyme immobilization enabling unhindered accessibility of enzyme active sites was obtained by using fusion protein N-SBM-ATA-wt consisting of selected ω-transaminase ATA-wt and the positively charged Zbasic2 tag, which established ionic interactions with silicon/glass microchannel surface. Enzyme-catalyzed transamination of (S)-(−)-α-methylbenzylamine and pyruvate to acetophenone and l-alanine was described by surface kinetics based on a ping-pong bi-bi mechanism. Reaction kinetic parameters were preliminarily defined in a batch system using various initial substrates concentrations and further applied in the surface reaction description. Based on the prevailing kinetic and convection/diffusion phenomena, the developed model could be reduced to the one-dimensional model which enabled immobilized enzyme concentration estimation and showed good agreement with experimental data from the outlet of the microreactor at various flow rates and inlet substrates concentrations. Moreover, the model successfully predicted performance of two consecutively connected microreactors coated with N-SBM-ATA-wt and could be further used to design and optimize efficient and sustainable processes of chiral amine syntheses catalyzed with surface immobilized enzymes.

Oriented immobilization and characterization of a poly-lysine-tagged cephalosporin C acylase on glyoxyl agarose support.

Cephalosporin C acylase (CCA), an important industrial enzyme for the production of 7-aminocephalosporanic acid, has very limited and scattered surface lysine residues. A mutant of cephalosporin C acylase (mCCA) has been designed to fuse a poly-lysine tag to the C-terminal of the β-subunit, which is far away from the active site. The free mCCA showed a near equal specific activity with the wild-type CCA, while a much higher activity recovery was obtained for the mCCA than its wild-type counterpart after immobilization on glyoxyl agarose support (73.3 versus 53.3 %). The mCCA's oriented immobilization enables it to obtain a higher substrate affinity and even a higher thermal stability than the wild-type enzyme. The improvement of stability might be attributed to the multipoint covalent attachment by the oriented enzyme immobilization via the adhered poly-lysine tag, which prevents the dissociation of the β-subunit of CCA from the support.

Biohydrogen for a New Generation of H2/O2 Biofuel Cells: A Sustainable Energy Perspective

AbstractAmong sustainable alternatives to fossil fuel, proton exchange membrane fuel cells are promising devices that deliver electricity from hydrogen and oxygen, producing only water. The transformation of the fuel and oxidant however relies on rare‐metal catalysts. H2/O2 enzymatic biofuel cells thus emerge as sustainable biotechnological devices in which chemical catalysts are replaced by hydrogenases at the anode and multicopper oxidases at the cathode. This review discusses the recent breakthroughs and the limitations in all the components of H2/O2 biofuel cells: 1) Catalytic mechanisms involved in multicopper oxidases and hydrogenases, in addition to the new potential of enzymes from the biodiversity pool, which exhibit outstanding properties. 2) The molecular basis for oriented enzyme immobilization on the electrochemical interfaces as a prerequisite for a fast direct electron‐transfer rate. 3) The requirement for 3D networks to enhance current densities. 4) The production of H2 from biomass. 5) The history of H2/O2 biofuel cells and recent devices, which also highlights the remaining issues that will allow the use of such devices in low‐power applications.

Oxygen Reduction by Enzyme-Carbon Nanotube Composites for the Development of Biofuel Cells

Glucose biofuel cells are promising candidates to replace lithium batteries in implanted devices one day. In fact, the possibility to convert the chemical energy of glucose and oxygen into electric energy is particularly interesting for in vivo applications because glucose and oxygen are present in body fluids at relatively low but constant concentration [1].Carbon nanotubes (CNTs) have great advantages as electrode material in biofuel cell design due to their high conductivity and shape which allow enhanced DET or MET with the biocatalysts. Furthermore, CNTs can be shaped to pellets, fibers, or films (Buckypapers, BPs) that makes them easy to process as mesoporous pure CNT bioelectrodes in biofuel cell designs. Moreover, the possibility to add appropriate functionalities via organic functionalization enabled optimal tuning of such nanostructured electrodes by attaching specific anchoring sites for enzymes.In this context, we report the non-covalent functionalization of multiwalled carbon nanotubes (MWCNT) with pyrene monomers bearing a boronic acid function [2]. This elegant technique consists in the formation of boronic acid ester through the reaction of a boronic acid function on the electrode surface with the sugar residues of glycosylated proteins like glucose oxidase (GOx) or horseradish peroxidase (HRP). By attaching at the same time GOx, a bi-enzymatic system was elaborated and showed efficient bioelectrocatalytic oxygen reduction in physiological conditions. To address the efficiency of this covalent binding, we also performed the same experiments using 1-pyrenebutyric acid N-hydroxysuccinimide ester.We also report the functionalization of MWCNT electrode by oxidative electropolymerization of pyrrole monomers bearing pyrene and N-hydroxysuccinimide groups [3]. Both polymers were applied to the immobilization and electrical wiring of laccase. A “pseudo” host-guest interaction of polymerized pyrene with a hydrophobic cavity of laccase was used for the oriented enzyme immobilization on MWCNT electrodes. The latter leads to higher catalytic current for oxygen reduction (1.85mA cm-2).Finally, we present here an efficient and easy way of designing free standing redox buckypaper electrodes via the elegant combination of MWCNT and a bis-pyrene derivative. This bis-pyrene 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (bis-Pyr-ABTS) that acts as a cross-linker between nanotubes, assures the formation of mechanically reinforced buckypaper obtained by a classical filtration technique of MWCNT suspension in presence of bis-Pyr-ABTS. In addition, the ABTS derivative assumes the mediated electron transfer to laccase. The buckypaper electrodes were applied to the mediated electron transfer using laccase in solution. For this setup, the developed mediator electrodes demonstrate high performances with maximum currents up to 2 mA ± 70 µA [4].Taking into account that carbon nanotubes became highly appropriate candidates for the elaboration of biofuel cells and electrochemical supercapacitors, we report here the original combination of these two devices. The possibility to recharge supercapacitors with an internal energy source could thus represent a significant improvement for the performance of biofuel cells. In this context, we propose an original hybrid battery-capacitor system using a compression of enzymes-carbon nanotubes as supercapacitors and electrodes for a biofuel cell setup. This hybrid supercapacitor/biofuel cell enables high power discharge cycles, the carbon nanotube disks being continuously recharged through the biocatalytic energy conversion under physiological conditions [5].ReferencesS. Cosnier, A. Le Goff, M. Holzinger. Electrochem. Commun, 38 (2014) 19-23.B. Reuillard, A. Le Goff, M. Holzinger,, S. Cosnier. J. Mat. Chem. B, in press.N. Lalaoui, K. Elouarzaki, A. Le Goff, M. Holzinger, S. Cosnier, Chem. Commun, 49 (2013) 9281-9283.M. Bourourou, K. Elouarzaki, M. Holzinger, C. Agnès, A. Le Goff, N. Bruas-Reverdy, D. Chaussy, M. Party, A. Maaref, S. Cosnier. Chem. Sci., in press.C. Agnès, M. Holzinger, A. Le Goff, B. Reuillard, K. Elouarzaki, S. Tingry, S. Cosnier, Energy Environ.Sci., in press

Addressable self‐immobilization of lactate dehydrogenase across multiple length scales

Successful nanobiotechnology implementation largely depends on control over the interfaces between inorganic materials and biological molecules. Controlling the orientations of biomolecules and their spatial arrangements on the surface may transform many technologies including sensors, to energy. Here, we demonstrate the self-organization of L-lactate dehydrogenase (LDH), which exhibits enhanced enzymatic activity and stability on a variety of gold surfaces ranging from nanoparticles to electrodes, by incorporating a gold-binding peptide tag (AuBP2) as the fusion partner for Bacillus stearothermophilus LDH (bsLDH). Binding kinetics and enzymatic assays verified orientation control of the enzyme on the gold surface through the genetically incorporated peptide tag. Finally, redox catalysis efficiency of the immobilized enzyme was detected using cyclic voltammetry analysis in enzyme-based biosensors for lactate detection as well as in biofuel cell energy systems as the anodic counterpart. Our results demonstrate that the LDH enzyme can be self-immobilized onto different gold substrates using the short peptide tag under a biologically friendly environment. Depending on the desired inorganic surface, the proposed peptide-mediated path could be extended to any surface to achieve single-step oriented enzyme immobilization for a wide range of applications.

Efficient direct oxygen reduction by laccases attached and oriented on pyrene-functionalized polypyrrole/carbon nanotube electrodes

We report the functionalization of multi-walled carbon nanotube (MWCNT) electrodes by oxidative electropolymerization of pyrrole monomers bearing pyrene and N-hydroxysuccinimide groups. Both polymers were applied to the immobilization and electrical wiring of Trametes versicolor laccase via chemical grafting or non-covalent binding. A "pseudo" host-guest interaction of polymerized pyrene with a hydrophobic cavity of laccase was used for the oriented enzyme immobilization on MWCNT electrodes. The latter leads to higher catalytic current for oxygen reduction (1.85 mA cm(-2)) and higher electroenzymatic stability (50% after one month).

Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self‐assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site‐specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N‐terminus of AP (nGBP1‐AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi‐functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple‐repeat constructs, 5GBP1‐AP displayed the best bi‐functional activity and, therefore, was chosen for self‐immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1‐AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self‐immobilization of the bi‐functional enzyme on micro‐patterned substrates where genetically linked 5GBP1‐AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site‐specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic‐binding peptides has a potential utility in a wide range of biosensing and bioconversion processes. Biotechnol. Bioeng. 2009;103: 696–705. © 2009 Wiley Periodicals, Inc.

Direct electron transfer: an approach for electrochemical biosensors with higher selectivity and sensitivity

The most promising approach for the development of electrochemical biosensors is to establish a direct electrical communication between the biomolecules and the electrode surface. This review focuses on advances, directions and strategies in the development of third generation electrochemical biosensors. Subjects covered include a brief description of the fundamentals of the electron transfer phenomenon and amperometric biosensor development (different types and new oriented enzyme immobilization techniques). Special attention is given to different redox enzymes and proteins capable of electrocatalyzing reactions via direct electron transfer. The analytical applications and future trends for third generation biosensors are also presented and discussed.