Reaction mixture enzymes

The use of organic solvent in the medium is one stategy that has been proposed for biocatalysis [9-15]. In the organic phase, the reactant has a much greater solubility than in the aqueous phase. This could significantly reduce the volume of the reaction mixture. Enzymes and micro-organisms have been shown to be active in the presence of organic solvents [16-20]. 

Enzyme reactions are performed at a constant temperature and stopped by inactivation of enzymes in the reaction mixtures. Enzymes are easily inactivated at a low pH by the addition of an acid such as HCl to the reaction mixture. 

Mix 3 g. of starch well with loml. of water in a test-tube and pour the mixture into 90 ml. of boiling water contained in a 300 ml. conical flask, stirring at the same time. Cool to about 70 and then place in a water-bath maintained at 65-70 , but not higher. Now add 2-3 ml. of the malt extract prepared as above, mix well and allow the hydrolysis to proceed. Take a series of test -tubes and in each put 10 ml. of water and 2 drops of a 1 % iodine solution. At intervals of about 4 minutes (depending upon the activity of the enzyme solution), remove 1 ml. of the reaction mixture, cool and add it to one of the test-tubes and note the colour obtained. At the beginning of the experiment the colour will be blue due to the starch alone. As the reaction proceeds, the colour gradually becomes violet, reddish, yellowish and finally colourless. 

In this type of sequential reaction, all possible binary enzyme substrate complexes (AE, EB, QE, EP) are formed rapidly and reversibly when the enzyme is added to a reaction mixture containing A, B, P, and Q ... 

A very simple and elegant alternative to the use of ion-exchange columns or extraction to separate the mixture of D-amino add amide and the L-amino add has been elaborated. Addition of one equivalent of benzaldehyde (with respect to die D-amino add amide) to the enzymic hydrolysate results in the formation of a Schiff base with die D-amino add amide, which is insoluble in water and, therefore, can be easily separated. Add hydrolysis (H2SQ4, HX, HNO3, etc.) results in the formation of die D-amino add (without racemizadon). Alternatively the D-amino add amide can be hydrolysed by cell-preparations of Rhodococcus erythropolis. This biocatalyst lacks stereoselectivity. This option is very useful for amino adds which are highly soluble in die neutralised reaction mixture obtained after acid hydrolysis of the amide. 

In general, pyruvate decarboxylase (EC 4.1.1.1) catalyzes the decarboxylation of a 2-oxocar-boxylic acid to give the corresponding aldehyde6. Using pyruvic acid, the intermediately formed enzyme-substrate complex can add an acetyl unit to acetaldehyde already present in the reaction mixture, to give optically active acetoin (l-hydroxy-2-butanone)4 26. Although the formation of... 

A primary goal of this chapter is to learn how to achieve control over the pH of solutions of acids, bases, and their salts. The control of pH is crucial for the ability of organisms—including ourselves—to survive, because even minor drifts from the optimum value of the pH can cause enzymes to change their shape and cease to function. The information in this chapter is used in industry to control the pH of reaction mixtures and to purify water. In agriculture it is used to maintain the soil at an optimal pH. In the laboratory it is used to interpret the change in pH of a solution during a titration, one of the most common quantitative analytical technique. It also helps us appreciate the basis of qualitative analysis, the identification of the substances and ions present in a sample. 

Another consequence of application of organic solvents as a reaction medium is associated with the mechanism of enzyme-catalyzed transformations. According to the commonly accepted mechanism, the first product of the interaction of a hydrolytic (serine) enzyme with an ester is an O-acylenzyme (Scheme 5.4). When the reaction is performed in an aqueous solution, water acts as a nucleophile in the next step, to give acid B. If more nucleophilic hydrogen peroxide is present in the reaction mixture, peroxycarboxylic acids C are formed. However, in organic solvents the O-acylenzyme also reacts readily with other nucleophiles, such as... 

For most applications, enzymes are purified after isolation from various types of organisms and microorganisms. Unfortunately, for process application, they are then usually quite unstable and highly sensitive to reaction conditions, which results in their short operational hfetimes. Moreover, while used in chemical transformations performed in water, most enzymes operate under homogeneous catalysis conditions and, as a rule, cannot be recovered in the active form from reaction mixtures for reuse. A common approach to overcome these limitations is based on immobilization of enzymes on solid supports. As a result of such an operation, heterogeneous biocatalysts, both for the aqueous and nonaqueous procedures, are obtained. 

Analysis of reaction mixtures for 1-propanol and 2-propanol following incubation of NDPA with various rat liver fractions in the presence of an NADPH-generating system is shown in Table I ( ). Presence of microsomes leads to production of both alcohols, but there was no propanol formed with either the soluble enzyme fraction or with microsomes incubated with SKF-525A (an inhibitor of cytochrome P450-dependent oxidations). The combined yield of propanols from 280 ymoles of NDPA was 6.1 ymoles and 28.5 ymoles for the microsomal pellet and the 9000 g supernatant respectively. The difference in the ratio of 1- to 2-propanol in the two rat liver fractions may be due to differences in the chemical composition of the reaction mixtures (2) Subsequent experiments have shown that these ratios are quite reproducible. For comparison, Table I also shows formation of propanols following base catalyzed decomposition of N-propyl-N-nitrosourea. As expected (10,11), both propanol isomers were formed, the total yield in this case being almost quantitative. 

Typically the reaction was carried out as follows to a mixture of lipase in the IL were added this racemic alcohol and vinyl acetate as the acyl donor. The resulting mixture was stirred at 35°C and the reaction course was monitored by GC analysis. After the reaction, ether was added to the reaction mixture to form a biphasic layer, and product acetate and unreacted alcohol were extracted with ether quantitatively. The enzyme remained in the IL phase as expected (Fig. 2). Two months later, Kim and co-workers reported similar results and Lozano and Ibora " reported other examples of lipase-catalyzed reaction in June. Further Park and Kazlauskas reported full details of lipase-catalyzed reaction in an IL solvent... 

We succeeded in showing that recycling of the enzyme was indeed possible in our IL solvent system, though the reaction rate gradually dropped with repetition of the reaction process. Since vinyl acetate was used as acyl donor, acetaldehyde was produced hy the hpase-catalyzed transesterification. It is well known that acetaldehyde acts as an inhibitor of enzymes because it forms a Schiff base with amino residue in the enzyme. However, due to the very volatile nature of acetaldehyde, it easily escapes from the reaction mixture and therefore has no inhibitory action on the lipase. However, this drop in reactivity was assumed to be caused by the inhibitory action of acetaldehyde oligomer which had accumulated in the [bmim][PFg] solvent system. In fact, it was confirmed that the reaction was inhibited by addition of acetaldehyde trimer. =... 

The conjugation of catechin on poly(allylamine) using ML as catalyst was examined under air. During the conjugation, the reaction mixture turned brown and a new peak at 430 nm was observed in the UV-vis spectrum. At pH 7, the reaction rate was the highest. The conjugation hardly occurred in the absence of laccase, indicating that the reaction proceeded via enzyme catalysis. 

Our standard incorporation assays contained resuspended particulate enzyme, labelled UDP-Gal (0.1 mM) and (10 mM) in resuspension buffer (Tris, pH 7.5). After incubation, reaction mixtures were heated briefly to 100°C and soluble lupin galactan was added, to ensure the precipitation of small amounts of galactan formed in the en me reaction and dissolved during the heating step. Precipitation of macromolecular products was achieved by adding methanol to a final concentration of 70%. The pellet was freed of soluble labelled products, including residual UDP-Gal, by repeated extraction with hot 70% methanol and was then analysed for labelled (l- )-P-D-galactan. The supernatant was analysed for soluble labelled products. 

HPAEC analyses were carried out to determine the oligomeric products released from various pectic substrates after depolymerization by the PL isoenzymes. Action pattern analyses for the concerted action of PL isoenzymes utilized 68% esterified pectin as substrate. One-ml reaction mixtures in a buffer system as detailed in section 2.2. comprising 0.5% (w/v) substrate and 5 U of enzyme were incubated for 30 s to 18 h, and then thermoinactivated. Samples of 750 pi were applied to a Carbopac PA-1 (Dionex) column before the carbohydrates were eluted over a period of 70 min using a gradient of 0.2 M KOH, 0.05 M K-acetate to 0.2 M KOH, 0.7 M K-acetate. Detection employed a Pulsed Electrochemical Detector (PED, Dionex) in the integrated amperometry mode according to the manufacturer s recommendations. 

In liquid medium, the thiobarbuturic acid test was used to determine polygalacturonase and pectate lyase activity (Sherwood, 1965). 1 ml of the crude enzyme preparation was added to 2 ml of 0.5 N HCl in a test tube. 4 ml of 0.01 M thiobarbuturic acid, dissolved in distilled water, were added. The tubes were heated in a boiling water for Ih and centrifuged. The absorption of the supernatant was determined in the spectrophotometer over the range 480-580 nm. Reaction mixtures without enzyme, which showed no reaction with thiobarbuturic acid, were used as controls. 

Pectolytic activity was also studied in batch reactors, following the reaction progress in thermostated quartz cuvettes. The reaction medium (3 cm ) was prepared with 1.5 g/L pectin in the standard buffer and 0.063 mg of enzyme. The absorbance of the reaction mixture against the substrate blank was continuously recorded at the spectrophotometer (Perkin Elmer Lambda 2, USA). Typical reaction time was 15 minutes, but initial reaction rates were estimated considering only the absorbances recorded during the first 200 seconds, range of totally linear response. 

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