Enzyme enzymatic conversions

Biochemistry resulted from the early elucidation of the pathway of enzymatic conversion of glucose to ethanol by yeasts and its relation to carbohydrate metaboHsm in animals. The word enzyme means "in yeast," and the earfler word ferment has an obvious connection. Partly because of the importance of wine and related products and partly because yeasts are relatively easily studied, yeasts and fermentation were important in early scientific development and stiU figure widely in studies of biochemical mechanisms, genetic control, cell characteristics, etc. Fermentation yeast was the first eukaryote to have its genome elucidated. 

The dopamine is then concentrated in storage vesicles via an ATP-dependent process. Here the rate-limiting step appears not to be precursor uptake, under normal conditions, but tyrosine hydroxylase activity. This is regulated by protein phosphorylation and by de novo enzyme synthesis. The enzyme requites oxygen, ferrous iron, and tetrahydrobiopterin (BH. The enzymatic conversion of the precursor to the active agent and its subsequent storage in a vesicle are energy-dependent processes. 

Figure 4B. Chromatogram illustrating the studies conducted on the enzymatic conversion of the PSP toxins to decarbamoyl metabolites (appended with an M in these figures). Conversion of a mixture of Cl and C2 by the clam enzymes was chromatographically distinct from conversion to GTX II and GTX III with weak acid.

A Box-Behnken design was employed to investigate statistically the main and interactive effects of four process variables (reaction time, enzyme to substrate ratio, surfactant addition, and substrate pretreatment) on enzymatic conversion of waste office paper to sugars. A response surface model relating sugar yield to the four variables was developed on the basis of the experimental results. The model could be successfully used to identify the most efficient combination of the four variables for maximizing the extent of sugar production. 

The high specificity required for the analysis of physiological fluids often necessitates the incorporation of permselective membranes between the sample and the sensor. A typical configuration is presented in Fig. 7, where the membrane system comprises three distinct layers. The outer membrane. A, which encounters the sample solution is indicated by the dashed lines. It most commonly serves to eliminate high molecular weight interferences, such as other enzymes and proteins. The substrate, S, and other small molecules are allowed to enter the enzyme layer, B, which typically consist of a gelatinous material or a porous solid support. The immobilized enzyme catalyzes the conversion of substrate, S, to product, P. The substrate, product or a cofactor may be the species detected electrochemically. In many cases the electrochemical sensor may be prone to interferences and a permselective membrane, C, is required. The response time and sensitivity of the enzyme electrode will depend on the rate of permeation through layers A, B and C the kinetics of enzymatic conversion as well as the charac-... 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

Enzyme electrodes. Guilbault52 was the first to introduce enzyme electrodes. The bulb of a glass electrode was covered with a homogeneous enzyme-containing gel-like layer (e.g., urease in polyacrylamide) and the layer was protected with nylon gauze or Cellophane foil when placed in a substrate solution (e.g., urea) an enzymatic conversion took place via diffusion of substrate into the enzymatic layer. 

Extending these ideas to enzymatic catalysis, Jiang et al. reported the use of protamine-silica hybrid microcapsules in combination with a host gel-like bead structure to encapsulate several enzymes individually in the enzymatic conversion of C02 to methanol [20]. They used a layer-by-layer (LbL) method where alternately charged layers were deposited on an enzyme-containing CaC03 core. The layers, however, were not polyelectrolytes, but protamine and silica (Scheme 5.6). 

Several hundred tons of L-methionine per year are produced by enzymatic conversion in an enzyme membrane reactor. An alternative approach is dynamic resolution, where the unconverted enantiomer is racemized in situ. Starting from racemic /V-acetyl-amino acid, the enantioselective L-acylase is used in combination with an TV-acyl-amino acid racemase to enable nearly total conversion of the substrate. 

As most enzymes function under compatible ambient conditions, bio-bio cascades had already been successfully developed by the 1970s. By far, most examples have been reported in the field of carbohydrates, using combinations of enzymatic conversions (up to eight enzymes in one-pot), as well as for the in situ cofactor regeneration of enzymatic redox reactions towards amino and hydroxy acids. 

Another interesting case is the one-pot four-enzyme cascade conversion of glycerol into a heptose sugar on gram scale [11], in which a pH switch method is applied to temporarily turn off one of the enzymes involved (Fig. 13.6). The four consecutive enzymatic conversion steps in one and the same reactor, without separation of intermediates, consist of ... 

An impressive one-pot six-step enzymatic synthesis of riboflavine from glucose on the laboratory scale has been reported with an overall yield of 35-50%. Six different enzymes are involved in the various synthesis steps, while two other enzymes take care for the in situ cofactor regenerations [12]. This example again shows that many more multi-enzyme cascade conversions will be developed in the near future, as a much greater variety of enzymes in sufficient amounts for organic synthetic purposes will become available through rapid developments in genomics and proteomics. 

The fact that enzymes also work in organic solvents [42], ranging from apolar alkanes up to the very polar N,N-dimethylformamide [65], has (Fig. 13.10) and will continue to open up new cascade opportunities for the integration of enzymatic conversions with chemocatalytic methods that require organic solvents. Notably, however, some enzyme classes, for instance the carbohydrate-converting enzymes, do not show activity in non-aqueous media [40, 41]. 

The general basis for success in the three above-mentioned areas is the development of chemical and chemo-catalytic procedures that are mutually compatible (temperature, pressure and solvent). In addition, to exploit fully the power of enzymatic conversions, this compatibility is best sought within the range of reaction conditions microorganisms and enzymes are able to work in. 

It is interesting to note that serine peptidases can, under special conditions in vitro, catalyze the reverse reaction, namely the formation of a peptide bond (Fig. 3.4). The overall mechanism of peptide-bond synthesis by peptidases is represented by the reverse sequence f-a in Fig. 3.3. The nucleophilic amino group of an amino acid residue competes with H20 and reacts with the acyl-enzyme intermediate to form a new peptide bond (Steps d-c in Fig. 3.3). This mechanism is not relevant to the in vivo biosynthesis of proteins but has proved useful for preparative peptide synthesis in vitro [17]. An interesting application of the peptidase-catalyzed peptide synthesis is the enzymatic conversion of porcine insulin to human insulin [18][19]. 

When lipases are used for enzymatic conversions, the enzyme is mainly active at a phase boundary, which can effectively be provided by a membrane. Additionally, for conversions requiring two phases (e.g. fat splitting [84—86] and esterifications [87]), the membrane also keeps the two liquid phases (an oil and an aqueous phase, respectively) separated. This is schematically depicted in Fig. 13.11. The equilibrium reactions involved are... 

More recently, Kutney and co-workers (220) have investigated whether the same dihydropyridinium intermediate 109 is involved in the enzymatic conversion of catharanthine (4) and vindoline (3) to anhydrovinblastine (8) as is involved in the chemical conversion. Use of a cell-free preparation from a 5-day culture of the AC3 cell line gave 18% of the bisindole alkaloids leurosine (11), Catharine (10), vinamidine (25), and hydroxy-vinamidine (110), with 10 predominating. When the incubations were carried out for only 5-10 min, the dihydropyridinium intermediate was detected followed by conversion to the other bisindole alkaloids, with FAD and MnClj required as cofactors. Clearly a multienzyme complex is present in the supernatant, but further purification led to substantial loss of enzymatic activity. The chemical formation of anhydrovinblastine (3) is carried out with catharanthine A-oxide (107), but when this compound was used in the enzyme preparation described, no condensation with vindoline (3) occurred to give bisindole alkaloids. This has led Kutney and co-workers to suggest (220) that the A-oxide 108 is not an intermediate in the biosynthetic pathway, but rather that a 7-hydroperoxyindolenine... 

The enzymatic conversion of many analogues of the naturally occurring purines directly to their biologically active form, the ribonucleotides, in vivo [5, 8, 10, 13, 39] underlines the importance of these enzymes to the drug action of this class of compounds. 2-Aminoadenine (2, 6-diaminopurine, I) [107], 2-fluoroadenine (II) [108], 4-aminopyrazolo [3, 4-d] pyrimidine (VIll) [109]. and 2- and 8-aza-adenine (IX and X) [ 110, 111] have all been shown to be substrates for the adenine phosphoribosyltransferase [J12, 113]. Extensive studies on the metabolism of 2-aminoadenine (I) in E. coli [114, 115], L cells [116], and mice [117] have also shown its conversion by this enzyme to the ribonucleotide. 

A synthetically derivatized substrate designed to undergo a change in absorption and/or fluorescence spectrum upon its enzymatic conversion to product. Chromo-genie substrates provide valuable assays for enzymes that otherwise fail to produce a spectral change, especially phosphotransferases, amide bond synthases, isomerases, and hydrolases. 

This procedure involves the enzymatic conversion of the chiral acetate (C(HDT)COO ), obtained from experiments involving chiral methyl groups, to labeled malate. The enzymes used in this procedure include acetate kinase, phosphotransacetylase, malate synthase, and fumarase. 

The sensing microzone of the flow-through sensor depicted in Fig. 5.9.B1 integrates gas-diffusion and detection with two analytical reactions [28], viz. (a) the urease-catalysed formation of ammonium ion by hydrolysis of urea (the analyte), which takes places on a hydrophilic enzyme membrane in contact with the sample-donor stream, which contains a gel where the enzyme is covalently bound and (b) an acid-b reaction that takes place at the microzone on the other side of the diffusion membrane and involves Bromothymol Blue as indicator. This is a sandwich-type sensor including a hydrophilic and a hydrophobic membrane across which the sample stream is circulated —whence it is formally similar to some enzyme electrodes. Since the enzymatic conversion of the analyte must be as efficient as possible, deteetion (based on fibre optics) is performed after the donor and acceptor streams have passed through the sensor. Unlike the previous sensor (Fig. 5.9.A), this does not rely on the wall-jet approach in addition, each stream has its own outlet and the system includes two sensing microzones... 

Coumaroyl-CoA is produced from the amino acid phenylalanine by what has been termed the general phenylpropanoid pathway, through three enzymatic conversions catalyzed by phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate CoA ligase (4CL). Malonyl-CoA is formed from acetyl-CoA by acetyl-CoA carboxylase (ACC) (Figure 3.2). Acetyl-CoA may be produced in mitochondria, plastids, peroxisomes, and the cytosol by a variety of routes. It is the cytosolic acetyl-CoA that is used for flavonoid biosynthesis, and it is produced by the multiple subunit enzyme ATP-citrate lyase that converts citrate, ATP, and Co-A to acetyl-CoA, oxaloacetate, ADP, and inorganic phosphate. ... 

Prior to fermentation, the wort is then cooled to temperatures below 85°F (30°C), and the pH is adjusted to about 5. Yeast such as Saccharomyces cerevisiae, Saccharomyces carlsbergensis or Candida brassicae are added and fermentation proceeds for 2 to 3 days under batch processing conditions. Yeast produces the enzymes maltase, zymase, and invertase. Maltase converts maltose to glucose. Zymase converts glucose to ethanol. Invertase converts any sucrose present to fermentable sugar. The following equations illustrate the enzymatic conversion of starch to ethanol ... 

Figure 9.1 Schematic presentation of an enzymatic conversion in a two-phase system. S= substrate, P=product and E=enzyme.

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