Enzymatic method procedure

Many procedures have been suggested to achieve efficient cofactor recycling, including enzymatic and non-enzymatic methods. However, the practical problems associated with the commercial application of coenzyme dependent biocatalysts have not yet been generally solved. Figure A8.18 illustrates the continuous production of L-amino adds in a multi-enzyme-membrane-reactor, where the enzymes together with NAD covalently bound to water soluble polyethylene glycol 20,000 (PEG-20,000-NAD) are retained by means of an ultrafiltration membrane. 

Schneider, A. J. Willis, H. J. Sources of variation in a standardized and semi-micro procedure for serum glutamic oxalacetic transaminase. Clin. Chem. (1958), 4, 392-408. Atwood, J. G. DiCesare, J. L. Making enzymatic methods optimum for measuring compounds with a kinetic analyzer. Clin. Chem. (1975), n, 1263-1269. 

The enzymatic method (5,6) is probably the method of choice as a starting point for most tissue types because of its ability not only to release a large number of cells, but also to preserve cellular integrity and viability (7). Last, chemical dissociation is commonly used in conjunction with mechanical or enzymatic procedures. Chemical methods are designed to omit or sequester the Ca + and Mg + ions needed for maintenance of the intercellular matrix and cell-surface integrity. Ethylenediaminoacetate (EDTA) or citrate ion is commonly used to remove these cations, but does not adequately dissociate all types of tissue. 

Enzymes play an important role in biochemical analysis. In biological material—e. g in body fluids—even tiny quantities of an enzyme can be detected by measuring its catalytic activity. However, enzymes are also used as reagents to determine the concentrations of metabolites—e.g., the blood glucose level (C). Most enzymatic analysis procedures use the method of spectrophotometry (A). 

In summary, for aU of the above-mentioned applications, the synthesis of carbohydrates and derivatives thereof is necessary to foster the field of glyco-engineering. Many procedures have been established to produce synthetic (neo)glycoconjugates by chemical or enzymatic methods and these are summarized in excellent reviews [12, 27, 28]. 

Most of the other posttranslational modifications involving the N- or C-terminus (Table 1) as well as the side-chain functionalities (Table 2) of the polypeptide chains occur under the control of enzymes that also dictate the regioselectivity of such chemical transformations. This regioselectivity is difficult to attain by synthetic procedures. Sophisticated protection schemes are required when additional chemistry must be performed on preassembled peptides, unless enzymatic methods can be used to supplement the synthetic strategies. As a consequence, the use of suitably modified amino acids as synthons is generally the preferred approach as will be discussed in the following sections. 

Another interesting example of resolution through formation of diastereo-mers is the isolation of four stereoisomers of 3-amino-2-methyl-3-trifluoro-methyl butanoic acid [55]. In this process, the chemical-enzymatic method by the combination of chemical and enzymatic reaction is a very convenient. At first, -phenylacetyl derivatives 61a and 61b were prepared in excellent isolated yields via the Schotten-Baumann procedure. After these materials were hydrolysed with penicillin acylase (EC 3.5.1.11) from Escherichia coli until attainment of 50% conversion, enzymatically unconverted -phenylacetyl derivatives 62 a and 62 b (organic layer) and amino acids 63 b and 63 d (aqueous layer) were separated. Acidic hydrolysis of unconverted materials produced other stereoisomers 63 a and 63 c in high optical pure form. 

The COOH-terminal amino acid of a peptide or protein may be analyzed by either chemical or enzymatic methods. The chemical methods are similar to the procedures for NH2-terminal analysis. COOH-terminal amino acids are identified by hydrazinolysis or are reduced to amino alcohols by lithium borohydride. The modified amino acids are released by acid hydrolysis and identified by chromatography. Both of these chemical methods are difficult, and clear-cut results are not readily obtained. The method of choice is peptide hydrolysis catalyzed by carboxypeptidases A and B. These two enzymes catalyze the hydrolysis of amide bonds at the COOH-terminal end of a peptide (Equation E2.3), since carboxypeptidase action requires the presence of a free a-carboxyl group in the substrate. 

Pentz et al. (P5) estimate taurine with fluorodinitrobenzene in urine passed through Dowex 50 H+ columns, but there are doubts as to whether this procedure is really specific for taurine (B38). Dent et al. have compared results obtained for the estimation of sulfur-containing amino acids in urine of cystinuric patients, by polarographic and microbiological methods (D18, D19). Hier (H12) and Schreier and Pliickthun (S10, Sll) have published data on amino acid excretion as determined microbiologically. Enzymatic methods have been used with success in the case of histidine in urine with specific decarboxylase preparations (S23). 

All residual lignins isolated by the enzymatic method are contaminated with enzymes to varying extents. This is especially true for the residual lignins isolated from semi-bleached pulps (Jiang et al. 1987). The contamination can be largely removed by the following procedure ... 

Analysis is an indispensable element of any biochemical investigation analytical methods will need to be used whether one needs such simple information as a protein concentration, or an enzyme activity, or the results of more complex studies like the sequence of a nucleic acid or the affinity of a protein for a specific ligand. From the diverse range of analytical techniques that can be exploited in biochemistry, we focus here on core methods for investigating the concentration, structure and stability of proteins and nudeic adds, and the essentials of enzymatic analytical procedures. 

The hydrolysis of proteins by chemical and enzymatic methods is discussed by R. L. Hill in the second chapter. The chapter should serve as a standard source for information on the many operational techniques with which only the experienced specialist is likely to be familiar. Dr. Hill covers not only the partial hydrolysis of proteins by general and specific procedures but also tbe total enzymatic hydrolysis of proteins for the purpose of amino acid analysis. 

Due to the absence of any sufficiently selective chenoical deprotection procedures, anoides generally represent irreversible carboxy protection. An enzymatic method for the hydrolysis of a-anoides using highly selective peptide anoidases has been proposed. ... 

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