Lyophilic substrates

However, in most cases enzymes show lower activity in organic media than in water. This behavior has been ascribed to different causes such as diffusional limitations, high saturating substrate concentrations, restricted protein flexibility, low stabilization of the enzyme-substrate intermediate, partial enzyme denaturation by lyophilization that becomes irreversible in anhydrous organic media, and, last but not least, nonoptimal hydration of the biocatalyst [12d]. Numerous methods have been developed to activate enzymes for optimal use in organic media [13]. 

The simplest way to prepare a biocatalyst for use in organic solvents and, at the same time, to adjust key parameters, such as pH, is its lyophilization or precipitation from aqueous solutions. These preparations, however, can undergo substrate diffusion limitations or prevent enzyme-substrate interaction because of protein-protein stacking. Enzyme lyophilization in the presence of lyoprotectants (polyethylene glycol, various sugars), ligands, and salts have often yielded preparations that are markedly more active than those obtained in the absence of additives [19]. Besides that, the addition of these ligands can also affect enzyme selectivity as follows. 

Basically, there are three ways to tune enzyme enantioselectivity by means of additives (i) the additives are placed in the reaction medium together with the organic solvent, the enzyme, and the reagents (ii) the additives are co-lyophilized with the biocatalyst before use in the organic solvent (iii) the additives are complexed with the substrates before their transformation in the organic medium. 

Traps with Bio-Sep beads amended with [ Cg]-benzene and [ C]-toluene were used to assess biodegradation in an aquifer (Geyer et al. 2005). Beads were lyophilized after exposure, lipids were extracted with chloroform-methanol, and the fatty acids and values analyzed. High enrichment of was observed in several fatty acids, which showed that the label from the substrates had been incorporated. In addition, there were differences in the abundance of the fatty acids in beads amended with benzene or toluene that suggested the existence of different microbial degradative populations. 

Each enzyme has a working name, a specific name in relation to the enzyme action and a code of four numbers the first indicates the type of catalysed reaction the second and third, the sub- and sub-subclass of reaction and the fourth indentifies the enzyme [18]. In all relevant studies, it is necessary to state the source of the enzyme, the physical state of drying (lyophilized or air-dried), the purity and the catalytic activity. The main parameter, from an analytical viewpoint is the catalytic activity which is expressed in the enzyme Unit (U) or in katal. One U corresponds to the amount of enzyme that catalyzes the conversion of one micromole of substrate per minute whereas one katal (SI unit) is the amount of enzyme that converts 1 mole of substrate per second. The activity of the enzyme toward a specific reaction is evaluated by the rate of the catalytic reaction using the Michaelis-Menten equation V0 = Vmax[S]/([S] + kM) where V0 is the initial rate of the reaction, defined as the activity Vmax is the maximum rate, [S] the concentration of substrate and KM the Michaelis constant which give the relative enzyme-substrate affinity. 

Toxi-Chromotest is a commercial toxicity assay that is based on the assessment of the inhibition of (3-galactosidase activity, measured using a chromogenic substrate and a colorimeter. A mutant strain of Escherichia coli is revitalized from a lyophilized state prior to the test [39]. The principle of the MetSoil test is similar to that of the Toxi-Chromotest . 

Iduronidase solution lyophilized lysosomal enzymes purified from bovine testis (Moscerdam Substrates) are reconstituted in 2.2 ml demineralized water and aliquots are stored at -80°C. 

Lyophilized [200 unit single-dose vials 400 unit singledose vial (an enzyme unit (U) is defined as the amount of enzyme that catalyzes the hydrolysis of 1 mM of the synthetic substrate para-nitrophenyl-(beta)-D-glucopyranoside (pNP-Glc) per min at 37° C)]... 

Two general classes of methods can be functionally defined for preparing concentrates of organic substances. Concentration methods involve the removal of water (e.g., lyophilization, freeze concentration, vacuum distillation, reverse osmosis [RO], and ultrafiltration) and result in a more highly concentrated aqueous solution of organic contaminants. Isolation methods are those methods in which the organic substances are physically removed from the aqueous solution, for example, adsorption onto a solid substrate followed by desorption (I). 

Crude Cellulases. The culture filtrate obtained from four-day-old cultures was concentrated by lyophilization. The protease inhibitors, phenyl methyl sulfonyl fluoride (PMSF, 2.3mM) and ethylenediamine-tetraacetic acid (EDTA, 0.2mM) were added to the culture solution and the salt concentration was adjusted to 0.5M with NaCl. After stirring at 4°C for at least 30 min to release cellulases from any enzyme-substrate complex which might have formed, the culture solution was centrifuged... 

Substrate one step TMB (Zymed) is a common choice see Note 4). Although many other substrates are available for HRP, TMB has high sensitivity with a quick development time. OD can be monitored at 650 nm as the color develops, then at 450 nm, when the reaction is stopped with H2S04. TMB may also be obtained in a lyophilized state and made up fresh with hydrogen peroxidase or as a preprepared one-step solution. TMB can vary considerably between different manufacturers, and this can affect the sensitivity and specificity of the ELISA. With one-step TMB, there is often large batch-to-batch variation as well. Each batch therefore needs to be tested before use. 

At the 4th day of the fermentation 2.5 g compactin substrate is added in sterile filtered aqueous solution. Calculated for the volume of the broth 0.5-1.0% glucose was added into the culture depending on the pH in the form of 50% solution sterilized at 121°C for 25 min in parallel with the substrate feeding. After 24 hours the compactin substrate is consumed from the culture (is detected by HPLC) and was converted to pravastatin. By lyophilization of the aqueous residue 1.3 g pravastatin was obtained. The chromatographically pure product was crystallized from a mixture of ethanol and ethyl acetate. Melting point 170-173°C (decomp.). 

The PJ.P. assay requires the use of lyophilized, viable M. Ivieus ATCC 4698 cells. This substrate is available at the Centre for Standards as a ElJVcon trolled reference product. It should be stored at -20 and used within a year. 

If the substrate is a synthetic peptide, a stock solution can be prepared by dissolving the lyophilized peptide in anhydrous, Ar-saturated dimethylsulfoxide (DMSO). The stock solution with a typical substrate concentration of 5 mM can be kept in aliquots at -20°C. 

For compounds that might be decomposed by sublimation into vacuum, such as DNA, two different techniques have been developed to produce thin biomolecular films on metal substrates. When multilayer films are required, the molecules are put in a solution from which a small aliquot is lyophilized on a tantalum substrate [18]. The sample preparation and manipulations are performed within a sealed glove box under a pure dry nitrogen atmosphere. The average film thickness is usually estimated from the amount of biomolecular material deposited and its density... 

Figure 19-2. Schematic overview of the type of apparatus used to investigate the desorption of ions and neutral species induced by electron impact on thin molecular and bio-organic films. In the case of thin DNA films, they are formed outside vacuum by lyophilization on a metal substrate or as a self-assembled layer. The films are placed on the multi-sample holder in the load-lock chamber. From there, they can be transferred one by one to the main chamber for analysis...

Its utility as an enzyme label is due in part to its relatively good stability characteristics (as a lyophilized powder it may be stored for years at 4°C and as a purified aqueous solution it can retain undiminished activity for over 12 months at 4 ° C). H RP has a high specific activity and broad substrate specificity. The iron atom within the active site has a single free molecular orbital available for substrate and oxidant interaction hence, activity can be reversibly inhibited by alternative ligands such as cyanide and sulfide at concentrations of about 10- M. 

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... 

Optimization of the biotransformation showed that the best conditions for achieving high rates and molar conversions included the use of n-hexane and -heptane as solvents, a temperature of 50 °C, an equimolar concentration of the substrates around 50 mM, and a concentration of the lyophilized biocatalyst around 25-30 g/1. As a matter of fact, Table 6.1 shows how the carbon source affects the expressed activity of A. oryzae MIM and R. oryzae CBS 112.07. 

Kinetic and thermodynamic studies of geranyl acetate production by direct geraniol acetylation with lyophilized cells of A. oryzae MIM were carried out in n-heptane [13-15]. Batch tests were performed varying the starting substrates equimolar level from 25 to 150 mM, cell concentration from 5 to 30g/l, and temperature from 30 to 95 °C. The initial rates at different initial substrate concentrations were measured and an apparent Michaelis constant KJ of 62mM and a fee value of 0.88 mmol/g/h were estimated by direct fitting of the initial rates against the initial substrate concentrations <75 mM [15]. 

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