Phenolics enzymatic preparation

Reverse micellar systems were used for the polymerization of phenol derivatives. HRP-catalyzed polymerization of p-ethylphenol in the ternary system composed of a bis(2-ethylhexyl) sodium sulfosuccinate (AOT)—water—isooctane system produced spherical polyphenol particles having 0.1—2 /tm diameters quantitatively.21 Similar particles were obtained by pouring the solution of enzymatically prepared polyphenol into a nonsolvent containing AOT.22... 

Some phenolic acids like caffeic acid, p-coumaric acid and ferulic acid can act as precursors of volatile phenols, which could contribute positively to wine aroma, when they are present at low concentrations associated descriptors are smoky, dove-like and leather (Table 1). Yeasts can conduct the decarboxylation of phenolic adds to volatile phenols, as well as esterase activities present in enzymatic preparations used in winemaking. During wine storage and ageing, volatile phenols may be further transformed. 

Enzymatic preparations should never contain cinnamate decarboxylase. This enzyme can lead to the formation of ethyl-phenols with a very disagreeable animal odor (Chapter 2). 

Future research will be required to determine the selective effect of this enzymatic extraction on the various phenolic compounds, according to conditions. Its effect, with respect to standard practices for regulating maceration, also needs to be explored. Regardless of the mechanisms involved, tasting has confirmed the interest of using enzymatic preparations for maceration during red winemaking. 

Since the imidazolide method proceeds almost quantitatively, it has been used for the synthesis of isotopically labeled esters (see also Section 3.2), and it is always useful for the esterification of sensitive carboxylic acids, alcohols, and phenols under mild conditions. This advantage has been utilized in biochemistry for the study of transacylating enzymes. A number of enzymatic transacylations (e.g., those catalyzed by oc-chymo-trypsin) have been shown to proceed in two steps an acyl group is first transferred from the substrate to the enzyme to form an acyl enzyme, which is then deacylated in a second step. In this context it has been shown[21] that oc-chymotrypsin is rapidly and quantitatively acylated by Af-fraw.s-cinnamoylimidazole to give /ra/w-cinnamoyl-a-chymotrypsin, which can be isolated in preparative quantities and retains its enzymatic activity (see also Chapter 6). 

Most of the C-diazeniumdiolates are not NO donors since they hydrolyze to produce nitrous oxide directly [174]. However, it has been found that carefully selected compounds can serve as NO donors under physiological conditions via alternative reaction pathways. Many cupferron analogs release NO via enzymatic oxidation [175] as do Oi-alkylated diazeniumdiolates [176]. Several novel types of NO-releasing N-hydroxy-N-nitrosamines have been prepared. These new preparative methods have been described in earlier sections. The precursors are enamines (Scheme 3.10), phenolates (Scheme 3.12), nitriles, and N-hydroxyguanidines (Scheme 3.9). 

The 3-(2-hydroxy-4,6-dimethylphenyl)-3-methylbutanoic acid shown in Fig. 8.23, as well as another phenylpropanoic derivative presented below, have been used as pro-moieties to prepare a number of prodrugs of therapeutic peptides [169] [238], Of interest here is that the pro-moiety is linked to the peptide by both amide and ester bonds to form a cyclic, double prodrug, the two-step activation of which is schematized in Fig. 8.24. Briefly, enzymatic hydrolysis of the ester bond unmasks a nucleophile (in this case, a phenol) that carries out an intramolecular attack on the amide bond, resulting in cy-clization of the pro-moiety and elimination of the peptide. [Leu5]enkephalin was one of the therapeutic peptides used to validate the concept, as illustrated in Fig. 8.25 [241],... 

Monosaccharide, aliphatic acids, furan derivatives, and phenolic compound recoveries after posthydrolysis were calculated as the ratio between the concentration determined in the reaction media and the concentration that resulted from the quantitative acid hydrolysis (29) of oligosaccharides into monosaccharides and other compounds. In enzymatic treatments, the concentrations obtained were corrected by subtracting the corresponding concentration in the respective control assays, since the commercial enzymes contain mono-, di-, and oligosaccharides. The dilution factor introduced by adding the dilute enzyme preparation or the different volumes of sulfuric acid in posthydrolysis were also accounted for. 

To analyze free-form phenolic acids in fmit or vegetable juice, the sample preparation is straightforward and simple. The juice can be directly injected into an HPLC system after it is filtered to remove any insoluble particles. However, for a sample with a solid fraction containing both free and bound phenolic acids, the sample preparation is not as simple. A mechanical method is needed to physically break down the sample and release the free phenolic acids, which are blocked in the inner core of the sample matrix. Chemical (acid or alkaline) or enzymatic hydrolysis must be applied to break down linkages in the bound phenolic acids to release free phenolic acids. However, the recovery of total phenolic acids is significantly affected by hydrolysis and other extraction conditions. An intensive hydrolysis condition may increase the release rate of bound phenolic acids to free phenolic acids however, it can also cause degradation of some phenolic acids and lower... 

In general, the sample preparation and extraction steps of phenolic acid analysis are very critical to the final result. Solvent or solution composition, extraction temperature, extraction technology, acid, alkaline, or enzymatic hydrolysis, extraction time, and cleanup conditions are all factors that affect the recovery and profile of phenolic acids. Poor sample preparation and extraction result in unreliable outcomes, regardless of the precision of the chromatography quantification method. Table 3.1 lists various sample extraction methods for different types of samples. 

Enzymatically synthesized polyphenol derivatives are expected to have great potential for electronic applications. The surface resistivity of poly(p-phe-nylphenol) doped with nitrosylhexafluorophosphate was around 105 Q.4a The iodine-labeled poly(catechol) showed low electrical conductivity in the range from 10 6 to 10 9 S/cm.48 The iodine-doped thin film of poly (phenol- co- tetradecyloxyphenol) showed a conductivity of 10 2 S/cm, which was much larger than that obtained in aqueous 1,4-dioxane.24a The third-order optical nonlinearity (%3) of this film was 10 9 esu. An order of magnitude increase in the third-order nonlinear optical properties was observed in comparison with that prepared in the aqueous organic solution. 

Phenol-formaldehyde resins using prepolymers such as novolaks and resols are widely used in industry. These resins show excellent toughness and thermal-resistant properties, but the general concern over the toxicity of formaldehyde has resulted in limitations on their preparation and use. Therefore, an alternative process for the synthesis of phenolic polymers that avoids the use of formaldehyde is strongly desired. The enzymatic synthesis of phenolic polymers is thus an alternative, interesting and sustainable route. 

Amsberry and Borchardt" " have applied Cain s cascade concept to prepare lipophilic polypeptide prodrugs. The amine functionality of the polypeptide is coupled to 2 -acylated derivatives of 3-(2, 5 -dihydroxy-4, 6 -dimeth-ylphenyl)-3,3-dimethylpropionic acid (Figure 36.23). Under simulated physiological conditions the parent amine is regenerated in a two-step process enzymatic hydrolysis of the phenolic ester, followed by a non-enzymatic intramolecular cyclization, leading to the release of the free amine (polypeptide) and a lactone. 

APases hydrolyze numerous phosphate esters, such as those of primary and secondary alcohols, phenols and amines (Levine, 1974). One unit of activity of APase corresponds to the hydrolysis of 1.0 pmole of p-nitrophenyl phosphate (p-NPP) per min (in 100 mM glycine, 1 mM ZnCb, 1 mM MgCl2 and 6 mM p-NPP, pH 10.4 or in 1 M diethanolamine, 0.5 mM MgCU and 15 mM p-NPP, pH 9.8). The bovine enzyme generally has a specific activity of 1000 and 2000 U/mg in these two buffers, respectively, at 37°C. At 25°C, activity is reduced to about half. This demonstrates that buffers may have a marked influence on the enzymatic activity of APases which explains the great differences in activity given for commercial preparations. Assays with p-NPP above 30°C suffer from the spontaneous hydrolysis of this substrate, with serious consequences for the enzyme kinetics (see below). The bacterial enzyme has lower activity than the bovine intestinal enzyme. 

Enzymatic polymerization and oligomerization can be used to make polyesters, polypeptides, polysaccharides, polymers from phenols, polymers from anilines, and many others. This approach could lead to fewer side reactions, higher regio- and stereoselectivity, under milder conditions. Oligomeric polyesters can be prepared from lactones. Caprolactone can be polymerized in bulk with lipases (9.43) to polymers with molecular weights of 7000.305... 

---