Enzyme-immobilised membrane

Despite these improvements, there are other important biosensor limitations related to stability and reproducibility that have to be addressed. In this context, enzyme immobilisation is a critical factor for optimal biosensor design. Typical immobilisation methods are direct adsorption of the catalytic protein on the electrode surface, or covalent binding. The first method leads to unstable sensors, and the second one presents the drawback of reducing enzyme activity to a great extent. A commonly used procedure, due to its simplicity and easy implementation, is the immobilisation of the enzyme on a membrane. The simplest way is to sandwich the enzyme between the membrane and the electrode. Higher activity and greater stability can be achieved if the enzyme is previously cross-linked with a bi-functional reagent. 

The microbial lipoxygenase was immobilised on polymer membrane, which was done according to the procedure in the methodological part. Examination of the obtained conjugate for protein contents was performed spectrophotometricaly in the way it was done for the enzyme immobilised on polymer granules. The relative activity of the immobilised enzyme is 65% and the protein contents 5.4 mg protein/g dry carrier. 

The work-up of batch processes, run in stirred vessels, had often faced the challenge to efficiently separate and recover the enzyme used. Meanwhile, there is abundant know-how available to immobilise enzymes on different carriers, though some issues need always to be considered maintained activity of the enzyme, its stability towards solvents and the operating temperature used in a reaction. Enzyme immobilisation allows for continuous reactions carried out in columns or in a sequence of continuous stirred-tank reactors. Certain advantages are offered by Degussa s enzyme-membrane-reactor (EMR), where the enzyme is surrounded by a hoUow-fibre membrane, that is permeable to substrate and product. 

Uragami T (2011), Enzyme-immobilised polymer membranes for chemical reactions. In Membranes for Membrane Reactors Preparation, Optimization and Selection, Ed. by Basile, Gallucc, John Wiley Sons, pp. 567-589. 

A three-enzyme electrode system, such as needed for creatinine measurement, poses a more difficult enzyme-immobilisation problem, in that different enzymes have different immobilisation requirements and their microenvironmental interrelationships need to be optimised. For one creatine sensor, the requisite creatine amidinohydrolase and sarcosine oxidase were immobihsed in polyurethane pre-polymer and PEG-hnked creatinine amidohydrolase was attached via diisocyanate pre-polymer to create a polyurethane adduct [14]. The likelihood of enzyme inactivation with chemical immobih-sation is high, but provided an enzyme preparation survives this, long-term stability is feasible. In the case of these three particular enzymes, a loss of activity resulted from silver ions diffusing from the reference electrode the material solution was to protect the enzyme layer with a diffusion-resisting cellulose acetate membrane. 

Several L-amino acids are produced on a large scale by enzymatic resolution of N-acetyl-D,L-amino adds (Figure A8.4). Acylase immobilised on DEAE-Sephadex is for example employed in a continuous process while Degussa uses the free acylase retained in a membrane reactor. In the latter process the advantage of reuse of the enzyme and homogeneous catalysis are combined. 

Degussa AG uses immobilised acylase to produce a variety of L-amino adds, for example L-methionine (80,000 tonnes per annum). The prindples of the process are the same as those of the Tanabe-process, described above. Degussa uses a new type of reactor, an enzyme membrane reactor, on a pilot plant scale to produce L-methionine, L-phenylalanine and L-valine in an amount of 200 tonnes per annum. 

In this case study, an enzymatic hydrolysis reaction, the racemic ibuprofen ester, i.e. (R)-and (S)-ibuprofen esters in equimolar mixture, undergoes a kinetic resolution in a biphasic enzymatic membrane reactor (EMR). In kinetic resolution, the two enantiomers react at different rates lipase originated from Candida rugosa shows a greater stereopreference towards the (S)-enantiomer. The membrane module consisted of multiple bundles of polymeric hydrophilic hollow fibre. The membrane separated the two immiscible phases, i.e. organic in the shell side and aqueous in the lumen. Racemic substrate in the organic phase reacted with immobilised enzyme on the membrane where the hydrolysis reaction took place, and the product (S)-ibuprofen acid was extracted into the aqueous phase. 

The values of the Michaelis-Menten kinetic parameters, Vj3 and C,PP characterise the kinetic expression for the micro-environment within the porous structure. Kinetic analyses of the immobilised lipase in the membrane reactor were performed because the kinetic parameters cannot be assumed to be the same values as for die native enzymes. 

The inhibition analyses were examined differently for free lipase in a batch and immobilised lipase in membrane reactor system. Figure 5.14 shows the kinetics plot for substrate inhibition of the free lipase in the batch system, where [5] is the concentration of (S)-ibuprofen ester in isooctane, and v0 is the initial reaction rate for (S)-ester conversion. The data for immobilised lipase are shown in Figure 5.15 that is, the kinetics plot for substrate inhibition for immobilised lipase in the EMR system. The Hanes-Woolf plots in both systems show similar trends for substrate inhibition. The graphical presentation of rate curves for immobilised lipase shows higher values compared with free enzymes. The value for the... 

However, there are disadvantages to using immobilised cells. The cell may contain numerous catalytically active enzymes, which may catalyse unwanted side reactions. Also, the cell membrane itself may serve as a diffusion barrier, and may reduce productivity. The matrix may sharply reduce productivity if the microorganism is sensitive to product inhibition. One of the disadvantages of immobilised cell reactors is that the physiological state of the microorganism cannot be controlled. 

The reaction in a homogeneous solution with a polar organic solvent in which the enzymes and substrates are both soluble, occurs often at the expense of the enzyme stability [4, 5]. Besides immobilised enzymes in organic solvents [6], emulsion reactors, especially enzyme-membrane-reactors coupled with a product separation by membrane based extractive processes [7-9] and two-phase membrane reactors [10-12], are already established on a production scale. 

In some systems, for example, the immobilised enzyme is sandwiched between an external cellulose acetate membrane that blocks the passage of heavy molecules and an internal polycarbonate membrane that allows passage of the transformed products to the electrode. 

Membranes can be used as well as a supporting material for immobilisation (e.g. polycarbonate membranes with created amino groups on the surface that allow covalent binding with glutaraldehyde). Entrapment of enzymes on electrode surfaces can be carried out with polymeric membranes such as polyacrylamide and gelatine, or by electropolymerisation of small monomers (o-phenylenediamine). Enzyme encapsulation within a sol-gel matrix has also been reported. 

A wide variety of methods exist for the immobilisation of enzymes on a sensor surface. Screen-printed carbon electrodes are often the favourite base for these sensors due to their inexpensiveness and ease of mass production. Methods used for the construction of AChE-containing electrodes include simple adsorption from solution [22], entrapment within a photo-crosslinkable polymer [20,23], adsorption from solution onto microporous carbon and incorporation into a hydroxyethyl cellulose membrane [24], binding to a carbon electrode via Concanavalin A affinity [25,26] and entrapment within conducting electrodeposited polymers [27]. 

An important parameter in a number of fields is the study of inorganic phosphate. Recently, Kwan et al. [206,207] have reported on a screen-printed phosphate biosensor based on immobilised pyruvate oxidase (PyOD) for monitoring phosphate concentrations in a sequencing batch reactor system [206] and in human saliva [207]. The enzyme was immobilised by drop-coating a Nation solution onto the working electrode surface this was then covered by a poly(carbamoyl) sulfonate (PCS) hydrogel membrane. 

Phosphate is another anion which is of biological significance as well as being biologically active. Numerous phosphate biosensors have been developed, with one based on a multi-enzyme system immobilised on a cellulose membrane on a Pt electrode being able to detect levels down to 10 8 M [104]. Simpler methods, however, use enzymes such as polyphenol oxidase combined with alkaline phosphatase bound within an electropolymerised layer based on a substituted pyrrole [ 105]. This was reported to give a sensor with a detection... 

Thus, glucose oxidase can be randomly immobilised on the modified nylon mesh (I). The resultant enzyme membrane (II) when held tautly over a platinun anode disc provides a high performance, long life glucose electrode which can be housed in a Stelte cell adapted for flow injection analysis (4). 

New reactor designs and immobilisation methods have been used to extend the lifetime of lipases in scCC>2 (Lozano et al., 2004). Ceramic membranes have been coated with hydrophilic polymers and the enzyme covalently attached to these. In SCCO2, activities and selectivities were excellent and the half-life of the catalyst was enhanced. It is thought the hydrophilic layer of the membrane protected the enzyme. Operational stability of enzymes has also been increased by using ionic liquid/scC02 biphasic systems (Lozano et al., 2002 Reetz et al., 2003). 

Bodzek M, Bohdziewicz J, Kowalska M. Preparation of membrane-immobilised enzymes for phenol decomposition. J Chem Technol Biotechnol 1994 61 231-239. 

Gekas VC (1986) Artificial membranes as carriers for the immobilisation of biocatalysts. Enzyme Microb Technol 8 450 -61... 

Various Japanese researchers show the possibilities of immobilising an enzyme or yeast on/in a ceramic membrane [99-101]. In the first example [99], the enzyme is boimd by Nakajima et al. to the ceramic surface of the TOTO 50 run membrane by activating it first with a silane-glutaraldehyde technique. Inver-tase is then bound to this activated surface and converts 100% of the 10-50 wt% sucrose in the feed solution. Alternatively glucose-isomerase jdelds a fructose ratio of 42% in a 45 wt% glucose feed at a residence time of 1000 s. The productivity of such systems is 10-fold higher than in conventional columns in which the enzyme is immobilised in beads. 

The biosensors were constructed with optical sensor for oxygen based on optical fibre Avaspec -Oxy (Avantes, Holland). The membrane with immobilised enzyme was attached to the electrodes with a dialysis membrane and a pipette tip. 

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