Enzymatic method principle

In section 6.6.1, we described how enzymatic methods have come to dominate the production of the important intermediates used in the manufacture of semi-synthetic -lactams. In principle, the hydrolytic penicillin acylases may be used in the reverse direction to add acyl groups to 6-APA. For example, a two-step enzymatic process has been described for the preparation of ampiciilin (D-(-)-a-aminobenzylpenidllin structure shown in Figure 6.17). 

Unmodified lysine is, in principle, biologically available and will be determined as such by animal assays (21) and enzymatic methods (22, 23,24), or determined as reactive lysine by the Carpenter method (23,24,25, 26) and by guanidina-tion (24, 27). 

The quantitation of enzymes and substrates has long been of critical importance in clinical chemistry, since metabolic levels of a variety of species are known to be associated with certain disease states. Enzymatic methods may be used in complex matrices, such as serum or urine, due to the high selectivity of enzymes for their natural substrates. Because of this selectivity, enzymatic assays are also used in chemical and biochemical research. This chapter considers quantitative experimental methods, the biochemical species that is being measured, how the measurement is made, and how experimental data relate to concentration. This chapter assumes familiarity with the principles of spectroscopic (absorbance, fluorescence, chemi-and bioluminescence, nephelometry, and turbidimetry), electrochemical (poten-tiometry and amperometry), calorimetry, and radiochemical methods. For an excellent coverage of these topics, the student is referred to Daniel C. Harris, Quantitative Chemical Analysis (6th ed.). In addition, statistical terms and methods, such as detection limit, signal-to-noise ratio (S/N), sensitivity, relative standard deviation (RSD), and linear regression are assumed familiar Chapter 16 in this volume discusses statistical parameters. 

Enzymatic methods offer in principle the possibility of a direct enantioselective synthesis of amino acids. Enzymes are often used for separation of racemic mixtures, as examplified in the case of methionine. Although racemic methionine is adequate for the animal feed sector, other applications require the enan-tiomerically pure (L)-form. For the resolution, (L)-acylases from Aspergillus sp. are often used, since they can accept a broad spectrum of substrates, are highly active, and very stable under the production conditions. [62]... 

This colour change is the principle on which the breath analyser operates. Instrumental methods of breath alcohol analysis have recently been reviewed [24]. Enzymatic methods are usually based on alcohol dehydrogenase which catalyses the oxidation of ethanol to acetaldehyde ... 

Urea is the most important end product of protein degradation in the body. Its concentration in blood depends on the protein catabolism and nutritive protein intake and is regulated by renal excretion. Thus the estimation of blood urea nitrogen is important in the assessment of kidney failure. The normal level of urea ranges from 3.6 mM to 8.9 mM. All enzymatic methods for urea determination are based on the principle of urea hydrolysis by urease ... 

The principles of CHO structure are explained in this chapter and applications of MS for structure determination of CHOs obtained by chemical or enzymatic methods as well as applications of MS for investigations of the kinetics and mechanisms of chitinolytic enzymes are reviewed. We will not consider stereoisomerism due to a- and p-anomeric configurations, investigations on V-acetylhexosaminidases, studies on V-glycans of glycoproteins containing a Af,Af -diacetylchitobiose core, detection of chitinous materials in biological samples by pyrolysis GC-MS, and use of MS for... 

Enzymatic methods rely on similar principles redox reactions involving the acids associated with either the oxidation of NADH or the reduction of NAD. The reaction is followed by the appearance or disappearance of NADH, which is measured spectrophotometrically at 340 nm. The primary reaction with the acid is often coupled to a second reaction in order to remove the product and thus drive the reaction equilibria to completion. Some of the possible reactions are summarized in the following schemes ... 

An important application area of ion chromatography in the characterization of fruit juices is the analysis of carbohydrates. Traditionally, sorbitol and the main sugars such as glucose, fructose, and sucrose were determined with enzymatic methods, which are usually very sensitive and specific. Interferences caused by the matrix are rare however, they are possible when determining sucrose. The principle of the sucrose determination is based on the determination of the glucose content prior to and after enzymatic hydrolysis of sucrose. Because fixiit juices often contain large amounts of glucose in the presence of small amounts of sucrose, precise quantification of small sucrose concentrations... 

The enzymatic method described above has two disadvantages (1) trapping of CO2 is a cumbersome procedure, and (2) the use of a radioactive substrate requires special precautions for use and disposal of reagents. Measurement of the primary amine formed by decarboxylation of the amino acid can also be exploited to monitor the PLP-dependent, enzyme-catalyzed reaction. This principle has been applied by Allenmark et al. (106), who used L-3,4-dihydroxyphenyl-alanine (L-DOPA) as substrate for tyrosine decarboxylase the dopamine produced by the decarboxylation reaction was determined by HPLC followed by amperometric detection. Both Hamfelt (107) and Lequeu et al. (108) utilized apo-tyrosine decarboxylase with tyrosine as substrate. The tyramine produced by the decarboxylation reaction was separated from the substrate (tyrosine) by HPLC and quantitated by either amperometric (108) or fluorometric (107) detection. The procedures discussed above are still subject to the main disadvantage of enzymatic methods possible interference by other materials present in the PLP containing extract which could either inhibit reconstitution of the holoenzyme or alter the reaction rate of enzyme catalysis. Moreover, HPLC with amperometric detection can hardly be described as less cumbersome than CO2 trapping difficulties in baseline-stabilization encountered with these detectors are well known. 

A second principle used widely for glucose analysis, is that of the oxidation of glucose enzymatically, mediated by the action of glucose oxidase with the formation of gluconic acid and hydrogen peroxide (22). In this procedure it is the hydrogen peroxide which is usually assayed for determination of glucose. This method suffers from the action of inhibitors which occur, particularly with patients in a diabetic coma and these need to be removed. 

When a colour layer of an artwork is analysed, a drop (5 10 pi) of this solution is localised on the sample surface. This method of enzymatic digestion can be, in principle, applied to all types of samples that occur in restoration practice fragments, cross-sections, microtome slices, etc. The samples are digested in closed microtubes to prevent evaporation of the solution. In the case of the cross-sections and microtome slices, it is essential to ensure the wetness of sample surface for the whole time of digestion. 

Recently, a method has been developed to microinject cell-polymer suspensions into 3-D collagen matrices in a 96-well plate format. This was used to perform a proof-of-principle screen of known signaling pathway inhibitors in murine breast cancer cells. The cells were visualized by staining of the actin cytoskeleton and effects of the compounds measured using multiparametric imaging. The system also proved amenable to the use of enzymatically... 

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