Enzyme immobilization procedure

Amperometric biosensors incorporating certain enzymes on the electrode for the determination of D- and L-amino acids were investigated. The parameters included enzyme immobilization procedure, composition of the immobilizing matrix, amount of enzyme,... 

In c, d, and e we have the typical case of a bioelectrocatalyst where, through a mediator, there is electron transfer between the electrode and the enzyme active centre where the substrate is in its turn activated and reacts. In c the components are in solution in d and e the mediator or the enzyme are immobilized on the electrode surface, the electron transfer reaction occurring between mediator and electrode. In case/we have the ideal situation direct electron exchange between the electrode and active centre of the enzyme, the mediator being eliminated. It is, nevertheless, very difficult to reconcile the enzyme characteristics and the electrochemical process, and it continues to be important to find adequate mediators and enzyme immobilization procedures. 

In this chapter we describe the applications of biological macromolecules in analytical chemistry and present an update on enzyme immobilization procedures (see Everse et td. (9)). 

The electrode preparation, the enzyme immobilization procedure was described as follows. About two mg of acid treated MWCNTs was ultrasonicated in 1 pL of N, N dimethylformamide (DMF) until a black suspension was obtained. About 15 pi of this MWCNTs suspension was casted on the working area of SPE surface and dried in an oven at 80 °C for 30 min. About 10 pi of AChE solution (0.132 U) was dropped on the MWCNTs modified electrode surface and dried at room temperature under a current of air and used. Hydrodynamic voltammetric studies results shows that significant response was observed at MWCNT-SPE towards 2mM thiocholine, whereas the response was poor at the unmodified electrode (Fig. 2). The linear response of the MWCNT-SPE modified sensor was found to be between 5 pM - 430 pM (r2 = 0.999) with a sensitivity of 6.018 mA/M. In contrast, the response of AChE/SPE modified electrode was only 5 % and thus this result further reveals the contribution of MWCNTs in improving the sensitivity. 

Other than AUNPs, Quantum dots have also been employed in the development of pesticide sensors. Li et al. have synthesized Poly (N-vinyl-2-pyrrolione) (PVP)-capped CdS quantum dots (QCdS-PVP) from CdC and Na2S in the presence of PVP [37], AChE was immobilized onto this QCdS-PVP matrix incorporated GCE surface. The resulting GCE/ QCdS-PVP/AChE sensor was used for the detection of OP pesticides, such as trichlorfon. The enzyme immobilization procedure was described as follows. About 3 ml of QCdS-PVP was deposited on the surface of the GCE and dried in air. Then 3 ml of 0.5 mg ml"1 AChE along with 2.5% GA was deposited on the surface of the QCdS-PVP modified GCE and dried for 1 h at room temperature. TEM results show that, the QCdS-PVP particles were homogeneously distributed and they possess an average size of 2-4 nm (Fig. 9). 

Nunes G. S., Jeanty G., and Marty J. L., Enzyme immobilization procedures on screen-printed electrodes used for the detection of anticholinesterase pesticides—Comparative study. Anal. Chim. Acta., 523(1), 107-115, 2004. 

Another case of heterogeneous systems refers to immobilized enzymes. The kinetic behavior of a bound enzyme can differ significantly from that of the same enzyme in free solution. The properties of an enzyme can be modified by suitable choice of the immobilization protocol, whereas the same method may have appreciably different effects on different enzymes. These changes may be due to conformational alterations within the enzyme, immobilization procedure and the presence and nature of the immobifization support. The advantages of immobifized enzymes are for instance in reusabifity and possibility to use continuous mode. 

In the last decades, many studies have focused on the development of low-cost screen-printed biosensors, well suited for mass production and portable devices. They have the additional advantage of allowing both real-time and in situ monitoring. Depending on the biosensor properties, which include the use of electrochemical mediators and the enzyme immobilization procedure, the electrochemical response can be suitable to detect low levels of several organic contaminants, such as pesticides. 

The immobilization procedure may alter the behavior of the enzyme (compared to its behavior in homogeneous solution). For example, the apparent parameters of an enzyme-catalyzed reaction (optimum temperature or pH, maximum velocity, etc.) may all be changed when an enzyme is immobilized. Improved stability may also accrue from the minimization of enzyme unfolding associated with the immobilization step. Overall, careful engineering of the enzyme microenvironment (on the surface) can be used to greatly enhance the sensor performance. More information on enzyme immobilization schemes can be found in several reviews (7,8). 

Enzymes are immobilized by a variety of methods. Two general types of immobilization procedures are used. The first-type procedures are based on weak interactions between the support and the enzyme and are classified as physical methods. The second-type procedures rest upon the formation of covalent bonds between the enzyme and the support and are classified as chemical methods. 

The first belief in the possibility of enzyme stabilization on a silica matrix was stated by Dickey in 1955, but he did not give experimental evidence, only mentioning that his experiments were unsuccessful [65]. A sol-gel procedure for enzyme immobilization in silica was first developed by Johnson and Whateley in 1971 [66]. The entrapped trypsin retained about 34 % of its tryptic activity observed in solution before the encapsulation. Furthermore, the enzyme was not released from the silica matrix by washing, demonstrating the increased stability and working pH range. Unfortunately, the article did not attract attention, although their method contained all the details that may be found in the present-day common approach. This was probably due to its publication in a colloid journal that was not read by biochemists. 

The immobilization procedure performed in two stages allows one to exclude the detrimental effect of acid on the entrapped proteins [44,71,86]. It was demonstrated [87] by the example of a set of oxidases that their activity was retained if the entrapment was carried out at a pH as close to their isoelectric point (pi) as possible. Because the pi value of most enzymes is in the neutral region, the two-stage procedure favors the retention of their functionality. Therefore, the pH shift to the optimal region provides a means of extending the sol-gel entrapment to a wide range of enzymes [45,71]. 

One of the most promising applications of enzyme-immobilized mesoporous materials is as microscopic reactors. Galameau et al. investigated the effect of mesoporous silica structures and their surface natures on the activity of immobilized lipases [199]. Too hydrophilic (pure silica) or too hydrophobic (butyl-grafted silica) supports are not appropriate for the development of high activity for lipases. An adequate hydrophobic/hydrophilic balance of the support, such as a supported-micelle, provides the best route to enhance lipase activity. They also encapsulated the lipases in sponge mesoporous silicates, a new procedure based on the addition of a mixture of lecithin and amines to a sol-gel synthesis to provide pore-size control. 

In this communication a study of the catalytic behavior of the immobilized Rhizomucor miehei lipase in the transesterification reaction to biodiesel production has been reported. The main drawbacks associated to the current biodiesel production by basic homogeneous catalysis could be overcome by using immobilized lipases. Immobilization by adsorption and entrapment have been used as methods to prepare the heterogeneous biocatalyst. Zeolites and related materials have been used as inorganic lipase supports. To promote the enzyme adsorption, the surface of the supports have been functionalized by synthesis procedures or by post-treatments. While, the enzyme entrapping procedure has been carried out by sol-gel method in order to obtain the biocatalyst protected by a mesoporous matrix and to reduce its leaching after several catalytic uses. 

Table 1 shows the enzyme immobilization results. By adsorption procedure, it is possible to observe that no enzyme was retained on the N-ITQ-6 material after... 

Moreover, the catalytic results clearly show that the lipase immobilization procedure strongly influences the final activity of the enzyme. Adsorption and entrapping procedures allow to preserve the open and active conformation of the enzyme whit respect to electrostatic ones. Using the entrapped lipase, the enzyme leaching can be avoided and the biodiesel total productivity strongly increases if compared with the one obtained by the free enzyme. 

Depending on the immobilization procedure the enzyme microenvironment can also be modified significantly and the biocatalyst properties such as selectivity, pH and temperature dependence may be altered for the better or the worse. Mass-transfer limitations should also be accounted for particularly when the increase in the local concentration of the reaction product can be harmful to the enzyme activity. For instance H2O2, the reaction product of the enzyme glucose oxidase, is able to deactivate it. Operationally, this problem can be overcome sometimes by co-immobilizing a second enzyme able to decompose such product (e.g. catalase to destroy H202). 

Among various enzyme immobilization protocols, entrapment in polymer membranes is a general one for a variety of transducers. Formation of a membrane from a solution of already synthesized polymer is simpler and reproducible compared to chemical polymerization. The simplicity of this immobilization procedure should provide reproducibility for the resulting biosensors the latter is strongly required for mass production. 

The methodology of the proposed immobilization approach [164, 179] is, however, quite different from non-aqueous enzymology . Though during an immobilization procedure, enzymes have to be exposed to organic solvents, their activity is required only in aqueous solution in which resulting biosensors are operated. Hence, it is only important that the enzymes are able to retain their catalytic properties after exposure to organic solvents. 

Chemical immobilization procedures of bioluminescent enzymes such as firefly luciferase and bacterial luciferase-NAD(P)H FMN oxidoreductase to glass beads or rods [174, 175], sepharose particles [176], and cellophane films [177] have produced active immobilized enzymes. Picomole-femtomole amounts of ATP or NAD(P)H could be detected using immobilized firefly luciferase or bacterial luciferase-oxidoreductase, respectively. 

For farther improvement of hydrogen enzyme electrode the commercial carbon filament materials were used as an electrode matrix. Such type of materials are accessible and well characterized, that provides the reproducibility of the results. A procedure for hydrogen enzyme electrode preparation included the pretreatment of electrode support with sulfuric acid followed by enzyme immobilization. This procedure is a critical step, since initially carbon filament material is completely hydrophobic [9]. 

The techniques developed in enzyme immobilization have facilitated the development of enzyme electrodes and of novel enzyme -based, automated, analytical methods (l6,17,l8). Enzyme electrodes have resulted from the combination of an enzyme membrane and an ion-selective electrode they were used successfully to assay directly appropriate substrates. Enzyme columns or enzyme tubes, prepared in a conventional manner, were used as a specific auxiliary component in the indirect assay of substrates in many of the novel automated analytical procedures. 

Even though, the immobilization procedure should be engineered to maximum retained activity of immobilized enzyme, it is difficult to measure the amount of active enzyme on the carrier without an active site titration. However, this has been done in the case of immobilized trypsin, although only covalent immobilized. (Daly and Shih, 1982)... 

Kennedy JF (1975) Data on techniques of enzyme immobilization and bioaffinity procedure. In Wiseman A (ed) Handbook of Enzyme Biotechnology. Wiley, New York, pp 147-201... 

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