Enzyme operational stability

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). 

Enzymatic Reactors Adding free enzyme to a batch reactor is practical only when the value of the enzyme is relatively low. With expensive enzymes, reuse by retaining the enzyme with some type of support makes great economic sense. As some activity is usually lost in tethering the enzyme and the additional operations cost money, stabihty is very important. However, many enzymes are stabilized by immobilization thus, many reuses may be possible. 

As with organic solvents, proteins are not soluble in most of the ionic liquids when they are used as pure solvent. As a result, the enzyme is either applied in immobilized form, coupled to a support, or as a suspension in its native form. For production processes, the majority of enzymes are used as immobilized catalysts in order to facilitate handling and to improve their operational stability [24—26]. As support, either inorganic materials such as porous glass or different organic polymers are used [27]. These heterogeneous catalyst particles are subject to internal and external... 

The class I FruA isolated from rabbit muscle aldolase (RAMA) is the aldolase employed for preparative synthesis in the widest sense, owing to its commercial availability and useful specific activity of 20 U mg . Its operative stability in solution is limiting, but the more robust homologous enzyme from Staphylococcus carnosus has been cloned for overexpression [87], which offers unusual stability for synthetic purposes. Recently, it was shown that less polar substrates may be converted as highly concentrated water-in-oil emulsions [88]. 

As a general rule, the optimal immobilization method is found empirically by a process of trial and error, where the selectivity, activity, and operational stability of the enzyme after immobilization are taken into account. The immobilization process is very sensitive to different parameters and is treated as a kind of art [16]. 

Bommarius, A.S., Drauz, K., Klenk, H. and Wandrey, C. (1992) Operational stability of enzymes - acylase catalyzed resolution of /V-acetyl amino acids to enantiomerically pure L-amino acids. Annals of the New York Academy of Sciences, 672, 126-136. 

Tor [7] developed a new method for the preparation of thin, uniform, self-mounted enzyme membrane, directly coating the surface of glass pH electrodes. The enzyme was dissolved in a solution containing synthetic prepolymers. The electrode was dipped in the solution, dried, and drained carefully. The backbone polymer was then cross-linked under controlled conditions to generate a thin enzyme membrane. The method was demonstrated and characterized by the determination of acetylcholine by an acetylcholine esterase electrode, urea by a urease electrode, and penicillin G by a penicillinase electrode. Linear response in a wide range of substrate concentrations and high storage and operational stability were recorded for all the enzymes tested. 

Additives such as polyethylene glycol, cationic antibiotics, polymers, small uncharged molecules, and negatively charged proteins have been used extensively in order to avoid the denaturing of enzymes or to improve the sensitivity and operational stability of biosensors. DNA has been proposed as an additive to improve the response and stability of biosensors based on CP. The biomolecules studied, such as tyrosinase [93], peroxidase [94], cytochrome C [95], have been shown to improve its performance by using adsorbed DNA within CP as an additive. 

Biphasic systems consisting of ionic liquids and supercritical CO2 showed dramatic enhancement in the operational stability of both free and immobilized Candida antarctica lipase B (CALB) in the catalyzed kinetic resolution of rac- -phenylethanol with vinyl propionate at 10 MPa and temperatures between 120 and 150°C (Scheme 30) 275). Hydrophobic ionic liquids, [EMIM]Tf2N or [BMIM]Tf2N, were shown to be essential for the stability of the enzyme in the biotransformation. Notwithstanding the extreme conditions, both the free and isolated enzymes were able specifically to catalyze the synthesis of (J )-l-phenylethyl propionate. The maximum enantiomeric excess needed for satisfactory product purity (ee >99.9%) was maintained. The (S)-l-phenylethanol reactant was not esterified. The authors suggested that the ionic liquids provide protection against enzyme denaturation by CO2 and heat. When the free enzyme was used, [EMIM]Tf2N appeared to be the best ionic liquid to protect the enzyme, which... 

Azevedo AM, Vojinovic V, Cabral JMS, Gibson TD, Eonseca LP (2004) Operational stability of immobilised horseradish peroxidase in mini-packed bed bioreactors. J Mol Cat B Enzym 28(2-3) 121-128... 

In order to improve the usability of enzymes, immobilization matrices have been proposed with both environmental decontamination as well as personal detoxification in mind. Effective immobilization methods allow for the preparation of an immobilized enzyme that retains most of its native activity, maintains high operational stability as well as high storage stability. Recent advances in material synthesis using enzymes have allowed the preparation of a variety of bioplastics and enzyme-polymer composites, which involve the incorporation of the enzyme material directly into the polymer. Enzymes stabilized in this way maintain considerable stability under normally denaturing conditions [21]. A number of methods have been used to prepare bioplastic or enzyme-polymer composite materials with OP-degrading enzymes. Drevon Russel described the incorporation... 

Alternatively, the enzyme can be modified such that it dissolves in a hydrophobic ionic liquid with retention of activity. This approach was demonstrated with cyt c, which, when covalently modified with polyethylene glycol (PEG), dissolved in [EMIm][ Tf2N] with retention of activity. The best results were obtained when the molecular weight of the polymer chain was >2000 [88]. Similarly, a copolymer of PEG and maleic anhydride solubilized subtilisin in [EMIm][NTf2] and a range of similar ionic liquids with good retention of activity and operational stability [89, 90]. 

If biocatalysis is so attractive, why was it not widely used in the past The answer is that only recent advances in biotechnology have made it possible. First, the availability of numerous whole-genome sequences has dramatically increased the number of potentially available enzymes. Second, in vitro evolution has enabled the manipulation of enzymes such that they exhibit the desired properties substrate specificity, activity, stability, and pH profile [42]. Third, recombinant DNA techniques have made it, in principle, possible to produce virtually any enzyme for a commercially acceptable price. Fourth, the cost-effective techniques that have now been developed for the immobilization of enzymes afford improved operational stability and enable their facile recovery and recycling [43]. 

F. Ricci, A. Amine, C.S. Tuta, A.A. Ciucu, F. Lucarelli, G. Palleschi and D. Moscone, Prussian Blue and enzyme bulk-modified screen-printed electrodes for hydrogen peroxide and glucose determination with improved storage and operational stability, Anal. Chim. Acta, 485(1) (2003) 111-120. 

Unlike activity, stability of enzymes is often interpreted simplistically as thermal stability, i.e., a temperature beyond which the enzyme loses stability. Although this quantity is important, first every statement of stability at a certain temperature depends on exposure time and thus is often ambiguous and second, for biocatalytic process applications, a more important quantity is the process or operational stability, which is the long-term stability under specified conditions. 

The relevant parameter for studies of operating stability of enzymes is the product of active enzyme concentration [E]active and residence time T, [E]active T. In a continuously stirred tank reactor (CSTR) the quantities [E]active and T are linked by Eq. (2.28), where [S0] denotes the initial substrate concentration, x the degree of conversion and r(x) the conversion-dependent reaction rate (Wandrey, 1977 Bommarius, 1992). 

If the enzyme is utilized for catalysis of a desired reaction the operational stability is often a much more interesting parameter than the resting stability. Catalyzing a reaction can influence enzyme stability in both positive and negative fashions ... 

The numerical value of the operational stability is expressed as the number of units of enzyme which have to be expended to generate a defined amount of product. The following conditions have to be specified besides the obvious ones such as T, pH, and substrate concentration, these are the quantity [E] T (see below) and the degree of conversion x. 

The relevant parameter for the study of the operating stability of impure enzyme preparations is E t, the product of active enzyme concentration E = fnE/V [g L-1] and residence time T. The following derivation illustrates the usefulness of this parameter. 

With both methods, the amount of enzyme added is compared to the amount of product produced ([P] = % [S]) to obtain a measure of the operating stability. At the beginning of a process design the prices for a unit of enzyme as well as for a mole (or pound or kilo or ton) of product are known, so an assessment of the influence of catalyst cost is possible. 

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