Enzymatic kinetically-controlled

FIGURE 5.10. Kinetic control by the enzymatic reaction. Normalized catalytic waves. From right to left log [feC l/fc.z + l/fci,2 + 1/fciCj)] = — oo,0,1,2,3. Adapted from Figure 2 of reference 20a, with permission from Elsevier. 

As in the homogeneous case, expression of the plateau current in equation (5.20) gives a simple representation of the competition between substrate and cosubstrate in the kinetic control of the enzymatic reaction. Equation (5.19) suggests the construction of primary and secondary plots allowing the derivation of the kinetic constants, as will be shown in the next section. 

FIGURE 5.11. Kinetic control by the enzymatic reaction. Substrate calibration curve. Adapted from Figure 1 of reference 20a, with permission from Elsevier. 

FIGURE 5.12. Cyclic voltammetry. Passage from kinetic control by enzymatic reaction (1) to control by substrate diffusion. From left to right log(I%k /yfDsFv/TIT) =6,5,4,3, 2,1,0, — 1. Adapted from Figure 4 of reference 20a, with permission from Elsevier. 

A practical enzymatic procedure using alcalase as biocatalyst has been developed for the synthesis of hydrophilic peptides.Alcalase is an industrial alkaline protease from Bacillus licheniformis produced by Novozymes that has been used as a detergent and for silk degumming. The major enzyme component of alcalase is the serine protease subtilisin Carlsberg, which is one of the fully characterized bacterial proteases. Alcalase has better stability and activity in polar organic solvents, such as alcohols, acetonitrile, dimethylformamide, etc., than other proteases. In addition, alcalase has wide specificity and both l- and o-amino acids that are accepted as nucleophiles at the p-1 subsite. Therefore, alcalase is a suitable biocatalyst to catalyse peptide bond formation in organic solvents under kinetic control without any racemization of the amino acids (Scheme 5.1). 

Figure 5.11 Qualitative illustration of the kinetic control in chemical reactions. Ziegler-Natta stereospecifle polymerizations are examples of kinetic control. Most enzymatic reactions are kinetically controlled - those for instance which lead to...

Self-organization systems under kinetic control (biological systems with genomic, enzymatic and/or evolutionary control), such as protein biosynthesis, virus assembly, formation of beehive and anthill, swarm intelligence. 

When the thermodynamics are unfavorable, one possible approach is to perform enzymatic reactions in a kinetically controlled strategy, and the reaction needs to be stopped at the maximum conversion (Figure 12.2) [16, 40]. 

Kinetically controlled strategies have been used to obtain high yields in the enzymatic synthesis of penicillins and cephalosporins. In the kinetically controlled... 

The advantages of diffusion-control enzyme electrodes over devices controlled by the enzymatic reaction (kinetic-control) are that the linearity is increased above the and the response is no longer dominated by the enzyme reaction. This implies that the enzyme electrode is less sensitive to pH and tern-... 

The solubilizing capacity of the choline residue is so pronounced that even substrates combining two hydrophobic amino acids are homogeneously soluble in aqueous buffer without any additional cosolvent. These favorable physical properties were also used in the enzymatic formation of peptide bonds. The amino acid choline ester 38 acts as the carboxyl component in kinetically controlled peptide syntheses with the amino acid amides 39 and 40 [52] (Fig. 11). The fully protected peptides 41 and 42 were built up by means of chymotrypsin in good yields. Other proteases like papain accept choline esters as substrates also, and even butyrylcholine esterase itself is able to generate peptides from these electrophiles. 

Besides being used for the production of the precursors 6-APA and 7-ADCA, penicillin amidases are also able to couple 6-APA and 7-ADCA with D-phenyl-glycine ester or amide in a kinetically controlled enzymatic peptide synthesis forming ampicillin or cephalexin (38, 39). These reactions (Scheme 21) have a great potential for being commercialized in the near future [95]. 

Cellulase was found to be effective in the synthesis of artificial cellulose in a single-step reaction by polycondensation of /J-D-cellobiosyl fluoride (Scheme 13).123 The polymerization is a repetition of the transglycosylation reaction, which became predominant over the hydrolysis reaction when the enzymatic polycondensation was carried out in a mixed solvent of acetonitrile/acetate buffer (5 1, pH 5). This synthesis is therefore kinetically controlled as well as equilibrium controlled. The fi configuration of the Cl fluorine atom is necessary to form a reactive intermediate leading to a / (1—4) product via a double displacement mechanism .124 Thus, this method provided the first successful in vitro synthesis of cellulose, the most abundant biomacromolecules on the earth, the synthesis of which had been unsolved for one-half a century.123... 

The enzyme has also been used in the production of several natural amino acids such as L-serine from glycine and formaldehyde and L-tryptophan from glycine, formaldehyde, and indole [77-79], In addition, SHMT has also been used for the production of a precursor, 20, to the artificial sweetener aspartame (21) through a non-phenylalanine-requiring route (Scheme 14) [80-83]. Glycine methyl ester (22) is condensed with benzaldehyde under kinetically controlled conditions to form L-enY/ ra-p-phenylserine (23). This is then coupled enzymatically using thermolysin with Z-aspartic acid (24) to form A -carbobcnzyloxy-L-a-aspartyl-L-eryt/zro-p-phenylserine (20). and affords aspartame upon catalytic hydrogenation. 

Polyethylene glycol is used to make the enzymes soluble in organic solvents [88], and high reaction yields have been obtained with polyethylene-glycol-modified chymotrypsin [89], and papain in benzene [90], Enzymatic modification reactions with deacylation, via aminolysis, of an intermediate covalent acyl-enzyme also support the mechanism of transpeptidation in kinetically controlled peptide syn-... 

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