PEG-enzyme preparation

PEG-modified enzymes prepared by coupling proteins with PEG in aqueous buffer solution are soluble not only in aqueous solution but in hydrophobic environments. Table 2 summarizes the list of PEG-enzymes prepared by the authors for the application to biotechnological processes. 

It should be noted that full treatment of the waste could be accomplished with a retention time of approximately 5 min when only 0.8 U/mL of crude or pure enzyme were used in the presence of PEG. In contrast, an aerobic biological treatment system that was used to treat this particular foundry waste required a retention time of approximately 6 hr. This illustrates one of the most significant advantages of enzymatic treatment. On the other hand, the use of crude enzyme preparations and PEG contributes significant quantities of organic compounds to the waste, which ultimately must be removed in a subsequent treatment step. 

Enzyme working solution is prepared by diluting the stock solution of enzyme (6 xM) 1 600 into cold (on an ice bath) enzyme dilution buffer (100 mM MES at pH 5.5, 400 mM NaCl, 0.2% PEG-8000, prepared analogously to assay buffer). The working solution is stored on ice and should be used within 10 min of dilution of the stock. 

The activity of the native and modified SC was also studied for the reaction methanolysis of APEE, using the substrate, methanol, itself as a solvent. This was done for three different salt-enzyme preparations, consisting of 99% salt, 50% salt, and no added salt (except that present in buffer). It was found that the PEG modification resulted in a six- to sevenfold increase in the initial rates of conversion for the cases with no surfactant and with T 20 (Table 2). AOT was found to reduce the activity of the enzyme preparations in all experiments conducted in the solvent methanol. Previous reports on enzymatic conversion in organic solvents have shown the effect of the solvent dielectric constant on enzyme activity for salt-free enzymes [22], Relationship between activity of AOT and PEG-modified SC to the hydrophobicity coefficient of various solvents has also been studied [20], however, only for the enzymes without salt-lyophilization. The decrease in activity of enzymes in organic solvents is attributed to the decreased water availability in organic media. Additionally, as the dielectric constant increases, the potential for removal of the... 

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. 

Enzyme preparations utilizing PEG for porphyrin synthesis have been isolated from a variety of sources plant 89-92), animal 31, 76, 93-96), and bacterial 97). Bogorad and Granick 89) showed that a preparation from Chlorella formed TJRO-III (Fig. 12) from PEG, but after heating to GO , only URO-I (Fig. 12) was formed. Similar observations have been made with preparations from chicken red blood cells (94) and from a bacterium 97) R. sphercides. 

In 2010, for the first time, Baccaro and Marx reported the enzymatic synthesis of DNA copolymers, composed of a DNA backbone and an organic polymer as a side chain synthesized by PCR. First, analogues of a thymidine-bearing polymer moiety (PEG) were prepared and subsequently converted into triphosphates. In order to incorporate polymer-functionalized building blocks (TTP), primer and nucleotide templates (304 and 1,062 bp, respectively) containing one adenine (A) residue were used. Subsequently, the original TTP moieties were substituted by modified triphosphates under the activity of the thermophilic enzyme 9°Nm DNA polymerase [27]. 

Currently, a common form of activated mPEG used for preparation of therapeutic enzymes is mPEG-succinate-N-hydroxysuccinimide ester (SS-PEG) (11). It reacts with proteins in short periods of time under mild conditions, producing extensively modified conjugates with well preserved biological activity. However, the ester linkage between the polymer and the succinic acid residue has limited stability in aqueous media (5,12). 

For the preparation of lipase-encapsulated derivatives, a procedure was adopted similar to that described for Method 1, except, in this case, the lipase solution (2.70 g of enzyme diluted in 15 mL of ultrapure water) was added simultaneously with the hydrolysis solution (NH4OH). The encapsulation of CRL in the hydrophobic silica gels was performed in the absence and presence of PEG-1450, resulting in the EN1 and EN2 derivatives, respectively. 

It can be noted that the way in which the enzyme is prepared in the dry form for catalysis in organic solvent is responsible for striking differences (up to two orders of magnitude) in the enzyme-specific activity. Furthermore, it is worth mentioning that the transesterification activity of lipase from B. cepacia entrapped in sol gel (sol gel-AK-lipase BC) was 83% of the activity in water measured using tributyrin as a substrate [6]. Analogously, in the case of CALB lyophilized with methoxypoly(ethylene glycol) (CALB -i- PEG) the activity was 51% of the activity in water in the hydrolysis of vinyl acetate [7]. It is important to note that, for both... 

Uyama and Kobayashi et al. [115,116] were first to prepare nearly mono-disperse sub-micron polyphenol particles by the enzyme-catalyzed dispersion polymerization of phenol andp-phenylphenol in a mixture of 1,4-dioxane and phosphate buffer using a water-soluble polymer as stabilizer (such as PVME, PVA, and PEG). 

A powder of hpoprotein hpase (LPL) esterified an organic substrate in toluene at a rather poor reaction rate (Table 4), which was to some extent explained by adhesion of the sticky enzyme powder to the surface of the reaction vessel [7]. When polyethylene glycol (PEG) was bound covalently to LPL and this modified enzyme was dissolved in toluene, approximately 3.5 U mg of enzyme protein were assayed. After simple addition of PEG to the reaction mixture together with LPL powder, the same poor reaction rate of the enzyme powder alone was observed. On the other hand, when LPL powder was lyophilized together with PEG the resultant preparation had an activity of 1.8 U mg L In this case, the enzyme... 

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