Two enzyme system

Improved sensitivity and scope can be achieved by coupling two (or more) enzymatic reactions hi a chain, cycling, or catalytic mechanism (9). For example, a considerable enhancement of the sensitivity of enzyme electrodes can be achieved by enzymatic recycling of the analyte in two-enzyme systems. Such an amplification... 

Asano et al. have developed an approach for the synthesis of D-amino acids through DKR using a two-enzyme system [55]. They had previously reported the discovery of new D-stereospecific hydrolases that can be applied to KR of racemic amino acid amides to yield D-amino acids. Combination of a D-stereospedfic hydrolase with an amino acid amide racemase allows performing DKR of i-amino acid amides yielding enantiomerically pure D-amino acids in excellent yields (Figure 4.29). 

The synthesis of long-chain fatty acids (lipogenesis) is carried out by two enzyme systems acetyl-CoA carboxylase and fatty acid synthase. 

Witschel M, S Nagel, T Egli (1997) Identification and characterization of the two-enzyme system catalyzing the oxidation of EDTA in the EDTA-degrading bacterial strain DSM 9103. J Bacterial 179 6937-6943. 

Sutherland TD, I Home, RJ Russell, JG Oakeshott (2002) Gene cloning and molecular characterization of a two-enzyme system catalyzing the oxidative detoxification of 3-endosulfan. Appl Environ Microbiol 68 6237-6245. 

FIG. 5 Rate of hydroperoxide production in (a, ) lipoxygenation in pure aqueous medium, (b, ) lipoxygenation in biphasic system, (c, x) two-enzyme (lipase-lipoxygenase) system in two-phase medium, determined experimentally, and (d, ) modeled kinetic of the two enzyme system. (From Ref 63.)... 

C. The Kinetic Behavior of a Two-Enzyme System in Biphasic Media ... 

Recently [63], we studied the behavior of two-enzyme system catalyzing two consecutive reactions in a macroheterogeneous medium (modified Lewis cell). The system consisted of lipase-catalyzed hydrolysis of trilinolein and subsequent lipoxygenation of liberated fatty acids (Fig. 3). Our approach compared the kinetic behavior of coupled enzymes in the Lewis cell with the sequential study of separated phenomena presented before ... 

Kinetic behavior of the two-enzyme system (lipase-lipoxygenase) in biphasic media (curve c in Fig. 5) is compared with kinetics of lipoxygenase in the same biphasic medium (b) and in an aqueous medium (a). These curves demonstrated that the configuration of the media influences the production rate of HP. As previously stated, lipoxygenase in biphasic media has an apparent kinetic behavior different from that in aqueous media (see difference between curves a and b in Fig. 5). 

In conventional synthetic transformations, enzymes are normally used in aqueous or organic solvent at moderate temperatures to preserve the activity of enzymes. Consequently, some of these reactions require longer reaction times. In view of the newer developments wherein enzymes can be immobilized on solid supports [183], they are amenable to relatively higher temperature reaction with adequate pH control. The application of MW irradiation has been explored with two enzyme systems namely Pseudomonas lipase dispersed in Hyflo Super Cell and commercially available SP 435 Novozym (Candida antarctica lipase grafted on an acrylic resin). 

The two enzyme systems responsible for the transulfuration process are thiosulfate-cyanide sul-furtransferase — also known as rhodanese — and beta-mercaptopyruvate cyanide sulfurtransferase. 

The majority of microbial hydrogen production is driven by the anaerobic metabolism of pyruvate, formed during the catabolism of various substrates. The breakdown of pyruvate is catalyzed by one of two enzyme systems ... 

The same group has exploited this interesting dehydrogenase for the synthesis of cyclic amino acids from linear precursors by developing a one-pot, two-enzyme system (Scheme 2.16). t-Lysine oxidase or L- or d-AAO were initially used to... 

Ethanol is metabolized primarily in the liver by at least two enzyme systems. The best-studied and most important enzyme is zinc dependent alcohol dehydrogenase. Salient features of the reaction can be seen in Fig. 35.1. The rate of metabolism catalyzed by alcohol dehydrogenase is generally linear with time except at low ethanol concentrations and is relatively independent of the ethanol concentration (i.e., zero-order kinetics). The rate of metabolism after ingestion of different amounts of ethanol is illustrated in Fig. 35.2. 

Biological Decay. The two major enzyme systems that are used to control intracellular H202 concentrations in organisms are catalases and peroxidases (47). The different overall reactions for H202 decomposition mediated by these two enzyme systems can be illustrated as follows for catalase ... 

The two major enzyme systems that lead to the decomposition of H202 are catalases and the peroxidases. By using 1802, Moffett and Zafiriou (1) showed that catalase is responsible for 65-80% of the decomposition of H202 and that peroxidase-mediated decay accounts for 20-35% of the loss. These experiments have not been extended to freshwater systems, and therefore the relative contribution of the two enzyme systems has not been established. 

The two enzyme systems most frequently mentioned in connection with biomineralization are (1) carbonic anhydrase and (2) alkaline phosphatase. Little information, however, has been presented on their specific role in the deposition of minerals. Recent advances in the field of biochemistry may shed new light on this intriguing problem. 

The food industry is a fertile area for biocatalysis applications high-fructose corn syrup (HFCS) from glucose with glucose isomerase, the thermolysin-catalyzed synthesis of the artificial sweetener Aspartame , hydrolysis of lactose for lactose-intolerant consumers, and the synthesis of the nutraceutical i-camitine in a two-enzyme system from "ybutyrobetaine all serve as examples. 

Two enzyme systems that act on alcohols formed in the above fashion have been documented to date. By far the most common is the action of an acetyl transferase which yields acetates. Acetyl transferase activity can be invoked to explain the production of acetate pheromone components by most tortricid moths (17), and we... 

FMO levels may, however, vary with nutrition, diurnal rhythms, gender, pregnancy, and corticosteroids, although the effects appear to be both species- and tissue-dependent. Such alterations in the relative contributions of the two enzyme systems may assume toxicological importance when the products from the two enzymes differ, particularly when one metabolite is more toxic than the others. Thus prior exposure of animals to environmental agents can have a significant effect on activation/detoxication pathways and the toxicity of other xenobiotics. 

In DERA reactions, where acetaldehyde is the donor, products are also themselves aldehydes. In certain cases a second aldol reaction will proceed until a product has been formed that can cyclize to a stable hemiacetal.71 For example, when a-substituted aldehydes were used, containing functionality that could not cyclize to a hemiacetal after the first aldol reaction, these products reacted with a second molecule of acetaldehyde to form 2,4-dideoxyhexoses, which then cyclized to a hemiacetal, preventing further reaction. Oxidation of these materials to the corresponding lactone, provided a rapid entry to the mevinic acids and compactins (Scheme 5.43). Similar sequential aldol reactions have been studied, where two enzyme systems have been employed72 (Scheme 5.44). The synthesis of 5-deoxy ketoses with three substitutents in the axial position was accomplished by the application of DERA and RAMA in one-pot (Scheme 5.44). The long reaction time required for the formation of these thermodynamically less stable products, results in some breakdown of the normally observed stereoselectivity of the DERA and FDP aldolases. In a two-pot procedure, DERA and NeuAc aldolase have... 

A reaction mixture containing IMP, aspartic acid, and GTP was prepared, and the reaction was started by the addition of purified sAMP synthetase. As the incubation proceeded, samples were removed and analyzed by HPLC. As shown in Figure 10.9, the chromatograms reveal the presence of the substrates and the formation of sAMP. After incubation for 20 minutes, the multienzyme system was reconstituted by the addition of a sample of sAMP lyase to the reaction. The reconstituted system was incubated for an additional 10 minutes, and a sample removed at that time was analyzed by HPLC. The profile (Fig. 10.9) illustrates a decline in the level of sAMP and the appearance of a new peak, AMP, confirming the successful reconstitution of this two-enzyme system. 

Many other deracemizahon methods based on a two-enzyme system or in the successive enzymatic resolution/base-catalyzed racemization have been reported. They are often regarded as having a reduced environmental impact, not requiring a transition metal catalyst in the racemization step [4]. 

Deracemization of Hydroxy Acids by DKR with a Two-enzyme System... 

In order to extend the two-enzyme system to other 2-hydroxy acids, a racemase with a broader activity was found in Lactobacillus paracasei. This was exploited for deracemization of 2-hydroxy-4-phenylbutanoic acid and 3-phenyllactic acid, which are important synthetic intermediates. In addition, in this case the procedure requires a kinetic resolution step and a successive racemization step. O-Acetyl derivatives of the absolute (S)-configuration can be obtained in two successive repeating cycles. Yields are around 60%. Of course the 0-acetyl derivatives of opposite configuration can be obtained when the lipase-catalyzed reaction is apphed in the hydrolysis direction. Obtaining the O-acetyl derivatives of the absolute (R)-configuration requires an additional acetylation step of the initially resolved and racemized (S)-hydroxy acid [12]. 

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