Acylase activity

Preparation of o-Amino Acids Using the W-Acylase Activity of Penicillin-G Acylase 85... 

Fig. 8.1. Screening for penicillin G acylase activity. A) Screening in agar plate formats using 6-nitro-3-(phenylacetamido)-benzoic acid (NIPAB) [106], Colonies secreting Penicillin G acylase activity stain a NIPAB-filter yellow. B) Screening in solution using phenylacetyl-MCA and periplasmic extracts without (open symbols) or with ) penicillin G acylases from Kluyvera citrophila, Proteus rettgeri and Escherichia coli respectively [65],...

Organisms were obtained by screening for growth on ACA as C and N source (which requires acylase activity) and testing for L-phenylalanine accumulation. The enzymatic activi for forming L-phenylalanine from ACA was coirsidered to be indudble since diis activity was not detected when ACA was omitted. 

In spite of usefulness of the simplification obtained by decreasing the experimental substrate concentration, many studies are aimed at the investigation of kinetic properties of immobilized biocatalysts within broader concentration ranges. In a previous paper [29], cells of Escherichia coli with penicillin acylase activity were immobilized by entrapment in calcium pectate gel and tested on the transformation of penicillin G to 6-amino penicillanic acid. Figure 9 shows experimental data from a microcalorimetric investigation of the penicillin G transformation in steady state. As appreciable particle-mass transfer was expected, the mathematical model that includes particle-mass balance was used. 

In an attempt to find a suitable source of amino acid acylase, Sanzyme, essentially an alpha-amylase complex extracted from a strain of Aspergillus oryzae was explored for the presence of this enzyme. Aspergillus oryzae is known to contain amylase, however, there is no report available for the presence of this enzyme in Sanzyme strain. It was found out that the crude Sanzyme samples contain 7.8 units of amino acylase activity [2], where one unit is defined as 1 micro mole of acetyl methionine hydrolysed/hr/mg of enzyme. It was also found out that the purified form of amino acylase from Sanzyme contain 28.4 units of activity [3] which is high when compared to any other amino acylase reported in literature. 

Acetyl DL-methionine which is used as a substrate for amino acylase activity determination was prepared by acetylation of DL-methionine with acetic anhydride in acetic acid [5]. The rate of enzymatic hydrolysis was determined by measuring the liberated amino acid by ninhydrin method [6] where ascorbic acid was used as oxidizing agent instead of sodium cyanide. The activity curve of pure amino acylase enzyme is shown in Fig. 1 as a continuous line. For determining the effect of metal ions on the activity of amino acylase the following procedure was adopted. 

Youshko Ml, Bukhanov AL, Svedas VK (2003) Study of the nucleophile binding in the penicillin acylase active center. Kinetic analysis. Biochemistry (Moscow) 68(3) 334-338... 

The reaction scheme for enzymatic synthesis of 7-ACA from c halosportn C is shown in Figure 1. The pathway involves the oxidative deamination of cephalosporin C into 7 P (5cephalosporanic acid (keto-AD-7-ACA) by DAO. A portion of the product further reacts nonenzymatically with hydrogen peroxide, which is formed as a by-product in the above reaction, to give GL-7-ACA. GL-7-ACA is hydrolyzed by GL-7-ACA acylase to 7-ACA. C7 side chains of cephalosporin C, keto-AD-7-ACA, and GL-7-ACA ate hydrolyzed by GC acylase to 7-ACA. However, the CC acylase activity for cephalo xirin C is low compared with that for GL-7-ACA. 

Matsuda et al. (25,26) cloned a CC acylase gene, acyll, from Pseitdomoms sp. SE83. The DMA sequence contained a single open reading frame (2322 bp) for two nonidentical subunits (25.4 and 58.2 kDa), predicting a common precursor (83.6 kDa). The CC acylase hydrolyzed cephalosporin C directly to 7-ACA, and the CC acylase activity for cephalosporin C was low (5%) compared with that for GL-7-ACA (I(X)%). 

Substrate lecificities of N176 and V22 CC acylases are shown in Table 2. The and values of N176 CC acylase for cephalosporin C at pH 8.7 were 21.5 mM and 5.93 sec-i, respectively. N176 CC acylase activity for keto-AD-T-ACA was relatively high compared with that for GL-7-ACA. It appears that ketO AD-7-ACA may be hydrolyzed by VZ2 and SE83 CC acylases at the same approximate rates. 

We reported that Hm transformants of A. chrysogenum expressing only the DAO gene, as well as some transformants of pHDVll having DAO and weak acylase activities, prO duced GL 7 ACA and GL 7-ADACA (6,7). However, those products were detected as products of different retention times compared with those of standard samples under a newly developed analysis condition of HPLC. Those products were isolated and ana-lyzed by fast atom bombardment mass spectroscopy (FAB-MS). Additionally, the a-hydroxyadipyl side chains of those products were analyzed by an isomer... 

In atopic dermatitis, there is evidence that dry skin and eczema are related sphingomyelin is not used for synthesizing the ceramides due to an abnormal sphingomyelin acylase activity. Instead, it is metabolized into the pro-inflammatory sphingosyl-phosphorylcho-line that enhances arachidonic acid or eicosanoid release and increases membrane ICAM-i expression in human keratinocyte cultures (Murata et al. 1996). 

Hydrogen bonding catalysts comprise oligopeptides and molecular receptors. Among the first group, the tetrapeptides used in Reference (28) are good examples. These have acylase activity and allow kinetic resolution of the substrate, in this case racemic rans-2-A -acetylamidocyclohexanol (28). 

Ninkovic, M. Riester, D. Wirsching, E Dietrich, R. Schwienhorst, A. Fluorogenic assay for penicillin G acylase activity. Anal. Biochem. 2001, 292, 228-233. 

Scheper, T. Weiss, M. Schuegerl, K. Two new fluorogenic substrates for the detection of penicillin-G-acylase activity. Anal. Chim. Acta 1986,182, 203-206. 

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