Enzymatic method

Enzymatic methods are very sensitive but are rather expensive, especially for a small number of samples. 

Lactose is first hydrolysed by j -galactosidase to glucose and galactose. The glucose may be quantified using  

glucose oxidase using a platinum electrode, or the H2O2 generated may be quantified by using a peroxidase and a suitable dye acceptor or 

The concentration of NADPH produced may be quantified by measuring the increase in absorbance at 334, 340 or 365 nm. 

Alternatively, the galactose produced may be quantified using galactose dehydrogenase (Gal-DH)  

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] 

After methionine is N-acetylated, the (L)-enantiomer is enzymatically hydrolysed in a stereoselective manner. The economy of the process depends on whether the (D)-enantiomer can then be racemised and recycled, for example by heating with acetic anhydride. 

The work-up of batch processes, run in stirred vessels, had often faced the challenge to efficiently separate and recover the enzyme used. Meanwhile, there is abundant know-how available to immobilise enzymes on different carriers, though some issues need always to be considered maintained activity of the enzyme, its stability towards solvents and the operating temperature used in a reaction. Enzyme immobilisation allows for continuous reactions carried out in columns or in a sequence of continuous stirred-tank reactors. Certain advantages are offered by Degussa s enzyme-membrane-reactor (EMR), where the enzyme is surrounded by a hoUow-fibre membrane, that is permeable to substrate and product. 

In this way, (L)-valine, (L)-alanine, (L)-phenylalanine and (L)-tryptophan, but also rare amino acids like (L)-propargylglycine, (L)-p-fluorophenylalanine or (L)-3-(r-naphthyl)alanine are prepared. (D)-acylases are used to obtain (D)-propargylglycine, (D)-tryptophan or (D)-p-chlorophenylalanine. [62] 

DSM has developed an industrial process for the preparation of (D)- and (L)-amino acids, which is based on the enantioselective hydrolysis of racemic amino acid amides using amidases, for example from Pseudomonasputida. It is often not necessary to isolate the pure enzyme standardised whole-cell or crude enzyme preparations can be used instead. It is noteworthy that in some cases the enzyme activity can be increased up to ten-fold by the addition of magnesium salts. The enzymes accommodate a broad spectrmn of substrates with considerable selectivity. Typical products are (L)-phenylalanine and (L)-homophenyl-alanine. 

Elaboration of new electrophoretic methods for PolyP separation is continuing. For example, capillary electrophoretic separations of sodium PolyPs with chain lengths of 5 to 44 has been reported. In this work, a buffer containing pyromellitic acid, triethanolamine and hexamethonium hydroxide gives high-resolution separation of linear and cyclic PolyPs (Stover, 1997 Wang and Li, 1998). 

Because of its efficiency, the electrophoretic method is now widely used in studies of PolyPs. It should be noted that for electrophoretic evaluation, the PolyPs must be extracted from biological material, while nucleic acids, proteins and other interfering compounds must be eliminated. 

The greatest advantage of enzymatic methods for PolyP determination is their high specificity to PolyPs. Their wide application in recent years results from the development of adequate methods of obtaining PolyP-dependent enzymes in sufficient quantities. 

The first method of enzymatic PolyP assay was proposed by Clark et al. (1986). In this technique, PolyPs were determined by polyphosphate glucokinase obtained from Pro-pionibacterium shermanii. Glucose-6-phosphate dehydrogenase reduced NADP through utilization of the formed glucose-6-phosphate, and the increase in NADPH concentration was measured. 

At the present time, many methods using PolyP-dependent enzymes for the assay of their substrates have now been developed. Polyphosphate kinase (PPK) catalyses the reversible 

Oilier oxidations catalyzed by enzymes are known (see Chapter 13). As our understanding of the processes coupled with schemes to alleviate the need for cofactors develop, we will no doubt see more examples of the use of enzymes to achieve oxidations. 

Methodology has been found that allows for the asymmetric oxidation of alkenes and allyl alcohol to the corresponding epoxides or diols. Although the methodologies have found widespread application at a laboratory scale for the preparation of a wide variety of compounds, examples of their use at scale are rare. With the potential of the approach, this will surely change. 

Asymmetric Synthetic Methodology, CRC Press Boca Raton, 1995. 

Sheldon, R. A. Chriotechnology Industrial Synthesis of Optically Active Compounds, Mareel Dekker New York, 1993. 

Takeuehi, A., Kageyama, H, Suzuki, M. Tetrahedron Lett. 1988, 29, 5423. 

However, the thermodynamic (equilibrium approach) generally does not provide yields in excess of 15%. 

Glycosidases may also be used in kinetically controlled glycosylations. These reactions depend on trapping by a glycosyl acceptor of a reactive intermediate. Suitable donors for such glycosylations include aryl glycosides, glycosyl fluorides 

although these transfers generally produce (l- 6) linkages, control of selectivity can be achieved by selection of the appropriate combination of donor and acceptor.92,93 

Both chemical and enzymatic syntheses have been reported of nucleoside sugar phosphates.88 An enzymatic synthesis of UDP-d-G1cNAc is shown next 95 

The driving force in the step producing UDP-G1cNH2 is provided by inorganic pyrophosphatase. 

The possibility of grafting synthetic polymer to chitosan has attracted much attention in the last years as a new way to modify the polysaccharide and develop practically useful derivatives. Graft copolymerization reactions introduce side chains and lead to the formation of novel types of tailored hybrid materials composed of natural and synthetic polymers. Grafting chitosan is a common way to improve chitosan properties such as formation of inclusion complexes [99], bacteriostatic effect [100], or to enhance adsorption properties [101, 102]. Although the grafting of chitosan modifies its properties, it is possible to retain some interesting characteristics such as mucoadhesivity [103], biocompatibility [104,105] and biodegradability [106]. 

Grafted chitosans have great utility in controlled drug release [115], tissue engineering [116], wound-healing [117] and cardiovascular applications [118-119]. Moreover, there are several reports regarding the use of enzymes in polymer s)m-thesis and modification [120-121]. In fact, enz)mies offer the potential advantage 

TABLE 6.3 Periodate oxidation of linked hexopyranose residues 

2-linkage Inner residues Termineil reducing residue One periodate consumed. Three periodate consumed/one formaldehyde (C ) and two formic add (C and C ) formed. 

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Guilbault, G. G. Enzymatic Methods of Analysis, Pergamon New York, 1970. 

Enzymatic fat splitting Enzymatic hydrolysis Enzymatic methods Enzymatic oxidation Enzymatic resolutions... 

Determination of DNA Sequence Information. Almost all DNA sequence is determined by enzymatic methods (12) which exploit the properties of the enzyme DNA polymerase. Whereas a chemical method for DNA sequencing exists, its use has been supplanted for the most part in the initial deterrnination of a sequence. Chemical or Maxam-Gilbett sequencing (13) is mote often used for mapping functional sites on DNA fragments of known sequence. 

In many cases only the racemic mixtures of a-amino acids can be obtained through chemical synthesis. Therefore, optical resolution (42) is indispensable to get the optically active L- or D-forms in the production of expensive or uncommon amino acids. The optical resolution of amino acids can be done in two general ways physical or chemical methods which apply the stereospecific properties of amino acids, and biological or enzymatic methods which are based on the characteristic behavior of amino acids in living cells in the presence of enzymes. 

Enzymatic Method. L-Amino acids can be produced by the enzymatic hydrolysis of chemically synthesized DL-amino acids or derivatives such as esters, hydantoins, carbamates, amides, and acylates (24). The enzyme which hydrolyzes the L-isomer specifically has been found in microbial sources. The resulting L-amino acid is isolated through routine chemical or physical processes. The D-isomer which remains unchanged is racemized chemically or enzymatically and the process is recycled. Conversely, enzymes which act specifically on D-isomers have been found. Thus various D-amino acids have been... 

An enzymatic method (45), which is specific for the citrate moiety, can be used as a combined assay and identification test for citric acid and its common salts down to 20 ppm. 

A new kinetic enzymatic method for the routine determination of urea in semm has been evaluated. This method is based upon an enzymatic reaction and formation of a coloured complex. The method is based on a modified Berthelot reaction. The reaction was monitored specRophotomebically at 700 nm (t = 25 0.1 °C). The optimal pH value, chosen for the investigation of complex, is 7.8. 

ACOCH2CF3, porcine pancreatic lipase, THF, 60 h, 77% yield. This enzymatic method was used to acetylate selectively the primary hydroxyl group of a variety of carbohydrates. 

Two new sections on the protection of phosphates and the alkyne-CH are included. All other sections of the book have been expanded, some more than others. The section on the protection of alcohols has increased substantially, reflecting the trend of the nineties to synthesize acetate- and propionate-derived natural products. An effort was made to include many more enzymatic methods of protection and deprotection. Most of these are associated with the protection of alcohols as esters and the protection of carboxylic acids. Here we have not attempted to be exhaustive, but hopefully, a sufficient number of cases are provided that illustrate the true power of this technology, so that the reader will examine some of the excellent monographs and review articles cited in the references. The Reactivity Charts in Chapter 10 are identical to those in the first edition. The chart number appears beside the name of each protective group when it is first introduced. No attempt was made to update these Charts, not only because of the sheer magnitude of the task, but because it is nearly impossible in... 

Highly selective cross-coupling benzoin condensations have been achieved via the use of enzymatic methods. ... 

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). 

Industrial production of amino acids by fermentation and chemo-enzymatic methods... 

In this chapter we consider amino acid production by fermentation and by chemo-enzymatic methods. We first consider the stereochemistry of amino adds and the importance of chirality in chemical synthesis. General approaches to amino add fermentation and recovery of amino adds from fermentation broths are then dealt with, followed by a detailed consideration of the production of L-phenylalanine by direct fermentation. Later in this chapter, chemo-enzymatic methods of amino acid... 

Two appendices are included at the end of this chapter. The first is intended to serve as a reminder, for those of you who might need it, of the nomendature and representation of stereoisomers. The second appendix contains descriptions of various chemo-enzymatic methods of amino acid production. This appendix has been constructed largely from the recent primary literature and includes many new advances in the field. It is not necessary for you to consult the appendix to satisfy the learning objectives of the chapter, rather the information is provided to illustrate the extensive range of methodology assodated with chemo-enzymatic approaches to amino add production. It is therefore available for those of you who may wish to extend your knowledge in this area. Where available, data derived from die literature are used to illustrate methods and to discuss economic aspects of large-scale production. 

Figure 8.6 Reaction schemes for the production of L-phenylalanine by enzymatic methods.

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