Biotechnology Letters 24: 1045–1048, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Chromogenic analogues of penicillin dihydroF and penicillin K for thecontinuous spectrophotometric determination of aliphatic penicillinacylase activity

Miguel Arroyo∗, Raquel Torres-Guzman, Jesus Torres-Bacete, Isabel de la Mata, Marıa PilarCastillon & Carmen AcebalDepartamento de Bioquımica y Biologıa Molecular I, Facultad de Ciencias Biologicas,Universidad Complutensede Madrid, Spain∗Author for correspondence (Fax: +34-91-394 46 72; E-mail: arroyo@bbm1.ucm.es)

Received: 20 March 2002; Revisions requested 5 April 2002; Revisions received 17 April 2002; Accepted 18 April 2002

Key words: chromogenic substrates, penicillin acylase, Streptomyces lavendulae

Abstract

The synthesis of 2-nitro-5-[(hexanoyl)-amino]-benzoicacid and 2-nitro-5-[(octanoyl)-amino]-benzoicacid as chro-mogenic substrates for the determination of aliphatic penicillin acylase activity is described. During enzymatichydrolysis, the released chromophore, 2-nitro-5-amino-benzoic acid, was detected at 405 nm. Penicillin acylasefrom Streptomyces lavendulae had an appreciable activity towards these substrates, which can then be used todetect penicillin acylases able to cleave hexanoyl and octanoyl residues off synthetic amides as well as penicillindihydroF and penicillin K, their natural analogues.

Abbreviations: DMSO – dimethyl sulfoxide; NABA – 2-nitro-5-amino-benzoic acid; NIHAB – 2-nitro-5-[(hexanoyl)-amino]-benzoic acid; NIOAB – 2-nitro-5-[(octanoyl)-amino]-benzoic acid; NIPAB – 2-nitro-5-[(phenylacetyl)-amino] benzoic acid; NIPOAB – 2-nitro-5-(2-phenoxy-acetylamino) benzoic acid; 6-APA –6-aminopenicillanic acid.

Introduction

Penicillin acylases (EC 3.5.1.11) can be classified ac-cording to their selectivity for cleaving the amidebond of penicillins. Their activity is usually mea-sured by the detection of 6-aminopenicillanic acid(6-APA) released during the hydrolysis. Commonmethods for 6-APA detection include reaction withp-dimethylaminobenzaldehyde (Shewale et al. 1987),ninhydrin (Baker 1979) or fluorescamine (Baker1983). Such methods, which require the collection ofsamples of the reaction medium and subsequent quan-titative determination of the product, are laborious,time-consuming and sometimes less sensitive thanmethods of continuous assay. The concept of replac-ing natural substrates with chromogenic analogues is

widely used in enzymology. It has been successfullyapplied for the assay of penicillin G acylase (Alkemaet al. 1999) where penicillin G was replaced bythe commercially available 2-nitro-5-[(phenylacetyl)-amino] benzoic acid (NIPAB), and also for penicillinV acylase (Kerr 1993) where penicillin V was re-placed by 2-nitro-5-(2-phenoxy-acetylamino) benzoicacid (NIPOAB). Such chromogenic substrates are spe-cific for quantitative determination of penicillin acy-lase activity by detecting, at 405 nm, the 2-nitro-5-amino benzoic acid (NABA) chromophore, which isreleased during the hydrolysis. The synthesis of otheramides of 2-amino-5-nitrobenzoic acid would allowthe use of new chromogenic substrates for the detec-tion of penicillin acylases with different specificitiesthan the ones described above. In the present work, we

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Fig. 1. Aliphatic natural penicillins and their analogue chromogenic substrates.

describe the synthesis of chromogenic analogs of peni-cillin dihydroF and penicillin K (NIHAB and NIOAB,respectively, Figure 1) for the rapid and continuousspectrophotometric determination of novel penicillinactivities. Such substrates can be hydrolyzed by peni-cillin acylase from Streptomyces lavendulae, an extra-cellular enzyme that can also cleave the amide bondsof penicillin V, F, dihydroF and K (Torres et al. 1999).

Materials and methods

Materials

Penicillin acylase from Streptomyces lavendulaeATCC 13664 was produced by fermentation and pu-rified as reported elsewhere (Torres et al. 1998). 2-Nitro-5-amino benzoic acid was from TCI (Tokyo,Japan), phenoxyacetyl chloride and pyridine were sup-plied by Fluka (Switzerland), hexanoyl and octanoylchlorides were from Aldrich (Milwaukee, USA).

Chemistry

Melting points were determined with a Büchi 530hot stage apparatus and are uncorrected. Proton NMRspectra were recorded on a Bruker AC 300 spec-trometer at 303 ◦K. Chemical shifts were recordedin parts per million (δ) downfield from tetramethyl-silane (TMS). IR spectra were recorded in KBr witha Perkin–Elmer 780 spectrophotometer. Silica gelused for flash column chromatrography was Kiesel-gel 60 (230–400 mesh) (Merck, Germany). Evapora-tions were performed at reduced pressure in a Büchiconcentrator-evaporator.

General procedure for the synthesis of chromogenicamides

2-Nitro-5-amino benzoic acid (5.49 mmol) dissolvedin 2 ml pyridine and hexanoyl or octanoyl chloride(5.86 mmol) was added dropwise. The reaction mix-ture was heated at 100 ◦C for 4 h and then allowedto reach room temperature. Water (5 ml) was addedand the aqueous layer was extracted twice with ethylacetate, and the combined organic layers were washedwith 3 M HCl and then water. The organic layerwas dried over anhydrous MgSO4, filtered and evap-orated to yield a crude product, which was purifiedas follows. When using hexanoyl chloride, the crudeproduct was purified by flash column chromatogra-phy, eluting with chloroform/ethanol 80:20 (v/v) toyield 2-nitro-5-[(hexanoyl)-amino]-benzoic acid (NI-HAB) as an off-white solid (400 mg, 1.43 mmol, 26%yield). Spectroscopic data for NIHAB are: IR (KBr)3325, 1710, 1680 cm−1; 1H NMR (MeOD) δ 7.98–7.87 (m, 3H, Ar), 2.41 (t, 2H, CH2), 1.7 (m, 4H,CH2), 1.37 (m, 4H, CH2), 0.92 (t, 3H, CH3). Meltingpoint for NIHAB was 120–122 ◦C. On the other hand,when using octanoyl chloride, the crude product wasredissolved in ethyl acetate, and 2-nitro-5-[(octanoyl)-amino]-benzoic acid (NIOAB) precipitated with theaddition of cyclohexane. The obtained off-white solid(350 mg, 1.14 mmol) was filtered, and the final yieldwas 21%. Spectroscopic data for NIOAB are: IR(KBr) 3325, 1710, 1680 cm−1; 1H NMR (MeOD) δ

7.99–7.83 (m, 3H, Ar), 2.4 (t, 2H, CH2), 1.69 (m, 2H,CH2), 1.34 (m, 6H, CH2), 0.9 (t, 3H, CH3). Meltingpoint for NIOAB was 126–128 ◦C.

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Table 1. Increment of absorbance at 405 nm during hydrolysis of chromogenicsubstrates catalyzed by penicillin acylase from Streptomyces lavendulae.

Rate of hydrolysis (� Abs405 nm min−1)∗Substrate concentration NIPOAB NIHAB NIOAB

1 mM 0.012 0.003 0.009

6 mM 0.026 0.004 0.012

25 mM 0.049 0.013 0.019

∗The hydrolysis rate was linear for at least 15 min of reaction.

Synthesis of 2-nitro-5-(phenoxy-acetylamino)-benzoicacid, NIPOAB

NIPOAB was synthesized according to the methoddescribed by Kerr (1993).

Enzyme assayAll spectrophotometric assays were performed at40 ◦C at 405 nm. At this wavelength, the extinction co-efficient of 2-nitro-5-amino-benzoic acid is 8980 M−1

cm−1 and for NIHAB and NIOAB are 79 M−1 cm−1

and 57 M−1 cm−1, respectively, whereas for NIPOABis 75 M−1 cm−1 (Kerr 1993). The synthetic amideswere at 1 M in DMSO. In a quartz cuvette (total vol-ume of 1 ml) were mixed the chromogenic amide in a1–25 mM final concentration and 1.6 µg of pure peni-cillin acylase in 100 mM potassium phosphate bufferpH 8 at 40 ◦C. The change in absorbance at 405 nmwas linear for at least 15 min of reaction.

Results and discussion

Streptomyces lavendulae produces an extracellularpenicillin acylase which has a high hydrolytic activityagainst penicillin V and other natural aliphatic peni-cillins, such as penicillin K, penicillin F and penicillindihydroF (Torres et al. 1999). As shown in Table 1,the enzyme can also hydrolyze NIPOAB, NIHAB andNIOAB at different concentrations. At standard assayconditions, the rate of hydrolysis was linear for at least15 min and the amount of released 2-nitro-5-aminobenzoic acid was higher when substrate concentra-tion was increased in the assay medium. All theseamides are poorly soluble in water, the protocol foractivity determination includes their solubilization inDMSO prior to the assay. The aqueous solubility ofthese chromogenic amides is related to the hydropho-bicity of their acyl chain and may be improved byadding a higher amount of DMSO in the reaction

medium, taking into the account that the final con-centration of the organic solvent must be maintainedbelow the threshold concentration that could lead toenzyme deactivation (Arroyo et al. 2000).

The applicability of NIHAB and NIOAB couldbe used for the determination of new penicillin acy-lase activities in crude extracts after fermentation.Such determination of enzyme activity with the chro-mogenic analogues here described, would avoid thefalse estimation of other substances with amino groupsin the medium, that could be determined by othertraditional methods for 6-APA detection such as p-dimethylaminobenzaldehyde and fluorescamine. Re-sults obtained with penicillin acylase from Strepto-myces lavendulae seem to support the usefulness ofthese new substrates for detecting those enzymes withacylase activity towards other natural penicillins suchas penicillin dihydroF and penicillin K. In addition,the availability of these new compounds would allowthe development of rapid assays for screening novelacylase-producing bacteria with the help of NIHAB orNIOAB test papers. These test papers have been suc-cessfully used for the detection of microorganisms ca-pable of producing penicillin G acylases (Zhang et al.1991) or cephalosporin acylases (Zhang et al. 1986):the paper is briefly applied to bacterial colonies on theagar surface, which are subsequently scored individu-ally on the paper by the yellowness that corresponds tothe released 2-nitro-5-amino benzoic acid. The appli-cation of these chromogenic substrates would be alsoimportant for kinetic studies of invisible substrate en-zymatic conversion (Alkema et al. 1999) as well asfor stopped-flow spectrophotometric studies. Simul-taneous provision of both chromogenic and invisiblereactions with penicillin dihydroF or K catalyzed bythe same penicillin acylase (e.g. from Streptomyceslavendulae) would provide useful information aboutsubstrate specificity and catalytic mechanism of theenzyme.

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Acknowledgements

We want to thank Dr Ana Gradillas from the Univer-sidad San Pablo CEU, for helping us in the synthe-sis of NIHAB and NIOAB. Financial support fromEuropean FEDER project 2FD97-1842 is gratefullyacknowledged.

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