Enzyme biocatalysis plays a very relevant role in the development of many chemical industries, e.g., energy, food or fine chemistry. To achieve this goal, enzyme immobilization is a usual pre-requisite as a solution to get reusable biocatalysts and thus decrease the price of this relatively expensive compound. However, a proper immobilization technique may permit far more than to get a reusable enzyme; it may be used to improve enzyme performance by improving some enzyme limitations: enzyme purity, stability (including the possibility of enzyme reactivation), activity, specificity, selectivity, or inhibitions. Among the diverse immobilization techniques, the use of pre-existing supports to immobilize enzymes (via covalent or physical coupling) and the immobilization without supports [enzyme crosslinked aggregates (CLEAs) or crystals (CLECs)] are the most used or promising ones. This paper intends to give the advantages and disadvantages of the different existing immobilization strategies to solve the different aforementioned enzyme limitations. Moreover, the use of nanoparticles as immobilization supports is achieving an increasing importance, as the nanoparticles versatility increases and becomes more accessible to the researchers. We will also discuss here some of the advantages and drawbacks of these non porous supports compared to conventional porous supports. Although there are no universal optimal solutions for all cases, we will try to give some advice to select the optimal strategy for each particular enzyme and process, considering the enzyme properties, nature of the process and of the substrate. In some occasions the selection will be compulsory, for example due to the nature of the substrate. In other cases the optimal biocatalyst may depend on the company requirements (e.g., volumetric activity, enzyme stability, etc).

Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance

Enzyme inhibition-based biosensors for food safety and environmental monitoring

Biomolecular immobilization on conducting polymers for biosensing applications.

A new, slack, and uniformly porous TiO2 material is synthesized by a simple, carbon nanotube (CNT) template-assisted hydrothermal method and is further explored for protein immobilization and biosensing. Results demonstrate that the material has a large specific surface area and a unique nanostructure with a uniform pore-size distribution. Glucose oxidase (GOD) immobilized on the material exhibits facile, direct electrochemistry and good electrocatalytic performance without any electron mediator. The fabricated glucose oxidase sensor shows good stability and high sensitivity, which indicates that the slack porous TiO2 is an attractive material for use in the fabrication of biosensors, particularly enzymatic sensors, because of its direct electrochemistry, high specific surface area, and unique nanostructure for efficient immobilization of biomolecules.

New Nanostructured TiO2 for Direct Electrochemistry and Glucose Sensor Applications

Enzyme-based chemical biosensors are based on biological recognition. In order to operate, the enzymes must be available to catalyze a specific biochemical reaction and be stable under the normal operating conditions of the biosensor. Design of biosensors is based on knowledge about the target analyte, as well as the complexity of the matrix in which the analyte has to be quantified. This article reviews the problems resulting from the interaction of enzyme-based amperometric biosensors with complex biological matrices containing the target analyte(s). One of the most challenging disadvantages of amperometric enzyme-based biosensor detection is signal reduction from fouling agents and interference from chemicals present in the sample matrix. This article, therefore, investigates the principles of functioning of enzymatic biosensors, their analytical performance over time and the strategies used to optimize their performance. Moreover, the composition of biological fluids as a function of their interaction with biosensing will be presented.

Enzyme Biosensors for Biomedical Applications: Strategies for Safeguarding Analytical Performances in Biological Fluids

Enhancement of operational stability of an enzyme biosensor for glucose and sucrose using protein based stabilizing agents.

Reactivation of immobilized acetyl cholinesterase in an amperometric biosensor for organophosphorus pesticide.

Ascorbate oxidase based amperometric biosensor for organophosphorous pesticide monitoring.

A simple amperometric biosensor was used to study the stability of glucose oxidase (GOD) immobilized with different protein based
stabilizing agents (PBSA) against temperature, pH and urea concentrations. Lysozyme as PBSA showed signi®cant increase in the stability
of soluble as well as immobilized GOD compared to BSA and gelatin. At pH 6.0, the transition temperature (Tm) of the GOD immobilized
with lysozyme was found to be 74.9 �C whereas that of GOD immobilized with gelatin and BSA were 66.1 �C and 63.8 �C, respectively.
GOD immobilized with lysozyme retained more than 50% activity in 8M urea solution whereas with BSA and gelatin most of the activity
was lost. More than 3-fold increase in the stability of soluble GOD was observed in presence of lysozyme in 50mM phosphate buffer, pH
6.0 and at 60 �C. A strong adverse in¯uence on stabilization of soluble GOD in presence of 0.2M NaCl indicates that ionic interaction
between GOD and lysozyme may be playing a crucial role in the stabilization.

M.D. Gouda

Papers

Enhancement of operational stability of an enzyme biosensor for glucose and sucrose using protein based stabilizing agents.

Reactivation of immobilized acetyl cholinesterase in an amperometric biosensor for organophosphorus pesticide.

Ascorbate oxidase based amperometric biosensor for organophosphorous pesticide monitoring.

Stability Studies on Immobilized Glucose Oxidase Usingan Amperometric Biosensor – Effect of Protein Based Stabilizing Agents

Enhancement of stability of immobilized glucose oxidase by modification of free thiols generated by reducing disulfide bonds and using additives.