Esterases acetyl esterase

Esterases. Acetyl esterase (EC 3.1.1.6) removes acetyl esters from acetylated xylose and short-chain xylo-oligomers. It s polymeracting counterpart, acetyl xylan esterase (EC 3.1.1.72), has a similar activity, but prefers polymeric xylan.244 In addition to acetate-specific enzyme detection kits, HPLC or GC analysis of acetate release from native extracted xylan and chemically acetylated xylan, colorimetric substrates, such as p-nitrophenol acetate and /3-napthyl acetate, or the fluorometric substrate, 4-methylumbelliferyl acetate are also used to assay acetyl esterases.244,253 The third esterase, ferulic acid esterase (EC 3.1.1.73), hydrolyzes the ester bond between ferulic acid or coumaric acid and the arabinose side chain of arabinoxylan. Assays for this activity are usually carried out using starch-free wheat bran or cellulase-treated gramineous biomass as a substrate and monitoring ferulic or coumaric acid released by HPLC or TLC. When preparing enzyme-treated substrates, care must be taken to employ phenolic-acid-esterase-free cellulases.244 Other substrates include methyl and ethyl esters of the phenolic acids, as well as finely ground plant biomass.240,254,255... 

Preparation of PhAcOZ amino acids proceeds from the chloroformate, and cleavage is accomplished enzymatically with penicillin G acylase (pH 7 phosphate buffer, 25°, NaHS03, 40-88% yield). In a related approach, the 4-ace-toxy derivative is used, but in this case deprotection is achieved using the lipase, acetyl esterase, from oranges (pH 7, NaCl buffer, 45°, 57-70% yield). 

Another important enzymatic process in the production of 7-ADCA, for use in the production of semi-synthetic cephalosporins, is the hydrolysis of 7-aminocephalosporanic add (7-ACA) by the enzyme acetyl esterase. This process, again using immobilisation techniques, is illustrated in Figure 6.16. Hie deacylated product can be used, for example, as an intermediate in the production of the important oral cephalosporin cefuroxime. We will return to cephalosporin antibiotics later in this chapter. 

Figure 6.16 Production of 7-aminodeacetyicephalosporanic acid from 7-ACA using an immobilised acetyl esterase. Following enzymatic removal of the acetyl group from 7-ACA, a 3-hydroxymethyl cephalosporin is obtained that can serve as intermediate in the production of cefuroxime.

As an accessory enzyme for the RGases, rhamnogalacturonan acetyl esterase (RGAE) was discovered in the same A. aculeatus preparation. This enzyme appeared to be specific for the de-acetylation of MHR and essential for the degradation of MHR by RGases A and B. 

The same enzyme (RGAE) could be purified from A. niger, together with two other esterases a feruloyl esterase (FAE) and an acetyl esterase (PAE) specific for the removal of one type of acetyl group present in the smooth regions of sugar-beet pectin. 

Making use of the chromatographic methods mentioned above, two different acetyl esterases could be purified from AEI. These esterases were named PAE and FAE. A third esterase was purified from AEII and this esterase was named RGAE, since it appeared to be similar to the... 

Table 5. Acetyl release (nmol/ml) from acetylated substrates by acetyl esterases at several stages of purification. Conditions protein concentration 2 pg/ml in 20 mM piperazine buffer pH 6.0, 20 h incubation. Solid and dotted lines indicate from which fraction the esterase has...

Isolation, characterization and inununo localization of orange fruit acetyl esterase. 

Acetyl esterase (AE) has been purified to homogeneity from orange peels. The purification steps included cation exchange chromatography and gel filtration. The enzyme has affinity for triacetin and sugar beet pectin with K, of 39 mM and of 26 mg/ml, respectively. AE has a MW of 42 kD and is a monomer. The isoelectric point is at pH > 9. 

The present work reports the purification and characterization of acetyl esterase from orange fruit as well as the in situ localization of the enzyme by immuno histology. 

Immuno localizations of AE in sections of orange fruits are shown in Fig. 3. The most intensive depositions of acetyl esterase were found in the outermost parts of the peel (exocarp or outermost albedo and the flavedo) and in the segments (juice vesicles), although quite high levels of acetyl esterase were found in most other tissues as well. The acetyl esterase depositions were all intracellular. 

In the peel strong immunological depositions of acetyl esterase were found in epidermis, the small cells of the exocarp and in the oil cavities (Fig. 3 A,B,C). In the mesocarp and endocarp the immunological depositions were more moderate (Fig. 3 D), but strong immunological depositions were found in the vascular bundles, especially in xylem. The immunological depositions in the peel seem to be correlated with cell size or cell age. The small cytoplasma rich cells have a higher content of acetyl esterase. 

In the segments strong immunological deposition was found throughout the tissue. Again the results indicate a slight correlation of cell size and the amount of acetyl esterase. In the small cells in the periphery of the juice vesicles, acetyl esterase is clearly intracellular (Fig 3 D,E), whereas the acetyl esterase was found on the cell walls of the large inner juice cells. This... 

Figure 3 Immuno localization of acetyl esterase. Sections were incubated with antibodies raised against the acetyl esterase, followed by visualization with alkaline phosphatase conjugated secondary antibodies and staining with Fast Red.

A Overview of the acetyl esterase immuno localizations in the peel (40x) (Ex exocarp, M mesocarp, OC oil cavity). B Immuno localizations of acetyl esterase in the exocarp (Ex) and oil cavity (OC) (294x). The most intensive acetyl esterase depositions are found in the small sized exocarp cells and in the oil cavity. C Immuno control with preimmune serum on the following section used in B (294x). D Immuno localization of acetyl esterase in endocarp (En) and juice vesicle (JV) (94x). Acetyl esterase depositions in the juice vesicles are more intensive than those observed in the endocarp. No acetyl esterase was detected in the innermost cell layer of the endocarp (see arrows). E Immuno localization of acetyl esterase in lamella (L) and juice vesicle (JV) (294x). Acetyl esterase depositions in the juice vesicles are more intensive than in lamella. Acetyl esterase was absent from the outermost cell layer of lamella (see arrows). F Immuno localization of acetyl esterase in core, where intensive acetyl esterase deposition was found in the xylem (94x). 

In lamella and core the strongest immunological depositions were found in the vascular bundles (Fig. 3 F), whereas acetyl esterase was present in moderate amounts in all other cells. No acetyl esterase was found in the outermost parts of the tissues, cuticula of epidermis, innermost cell layer of endocarp, outer walls of juice vesicles and outer cell layer of lamella. 

Acetyl esterases of Aspergillus niger puriHcation and mode of action on pectins... 

Acetyl esterases with different specificity occur in one Aspergillus niger preparation. Three acetyl esterases were purified and characterised pectin acetyl esterase (PAE), feruloyl acetyl esterase (FAE) and rhamnogalacturonan acetyl esterase (RGAE). 

Only PAE, a novel acetyl esterase, could remove acetyl from beet pectin, to a maximum of 30%. This was shown to be one specific acetyl group in theJiomogalacturonan chain of pectin (smooth region) by NMR spectroscopy. PAE activity was influenced by buffer salts and the addition of bivalent cations. PAE worked cooperatively with pectolytic enzymes. 

Enzymes can be used to specifically modify the pectins. Pectin methyl esterase is already widely used to adjust the gelling properties of commercially available pectins. The acetyl esters also strongly affect the gelation [2,3] and removal is important for the upgrading of sugar beet pectin, extractable from a by-product of the sugar industry. 

This study deals with the purification and characterisation of acetyl esterases from A.niger with different specificity. 

Enzyme purification and characterisation Acetyl esterases were isolated from a Rapidase C-80 preparation according to the scheme shown in Figure 1. The purified acetyl esterases were devoid of relevant side activities, and showed great differences in their specificity towards the different acetylated substrates. 

This novel enzyme was the only esterase able to release acetyl from sugar beet pectin and removed about 30% of the total acetyl groups present. It also caused the release of acetyl groups from a range of other acetylated substrates, either synthetic or extracted from plants, in small amounts. PAE had an apparent molecular weight of 60 kDa and showed optimal activity at pH 5.5 and a temperature of 50 C. The enzyme is sensitive to buffer composition and requires a bivalent cation for optimal activity and stability. In purified form this enzyme proved unstable, especially in phosphate buffers. 

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