Acetyl esterase

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. 

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. 

The A. niger preparation investigated in this study contains at least three different acetyl esterases, each with its own specificity. The activities of RGAE and FAE are comparable to those of similar enzymes isolated previously from A. aculeatus and a different A. niger preparation. PAE appears to be a new enzyme, with an activity specific towards one type of acetyl ester in the homogalacturonan chain of beet pectin. 

Christensen TMIE, Nielsen JE, Mikkelsen JD. Isolation, characterization and immunolocalization of orange fruit acetyl esterase, in Progress in Biotechnology 14 Pectins and Pectinases (Visser J, Voragen AGJ, eds.), Elsevier, Amsterdam, The Netherlands, 1996, pp. 723-730. 

The p-acetoxybenzyloxycarbonyl (AcOZ) group can be removed efficiently by a lipase from Mucor miehei and an acetyl esterase from the flavedo of oranges under exceptionally mild conditions (pH 5-6).110,11 121 Acetyl esterase discriminated between acetyl and longer acyl side chains (Scheme 8). 

C-Esterases are found in the IUBMB classification under the entry acetyl-esterase (acetic ester acetylhydrolase, EC 3.1.1.6). Doubts have been expressed about the existence of C-esterases [56], Indeed, their activation by sodium 4-(hydroxymercurio)benzoate is not reproducible, and paraoxon has at least slight inhibitory effects on these esterases. 

Tsujiyama, S. and Nakano, N. (1996). Distribution of acetyl esterase in wood-rotting fungi. Mycoscience, 37(3), 289-294. 

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