Assay of penicillin acylase in organic media

Summary The synthetic substrate 6-nitro-3-(phenylacetamido) benzoic acid (NIPAB) is an appropriate substrate for assaying penicillin acylase activity in reversed micellar systems of Aerosol - OT in isooctane. Accumulation of 6-nitro-3-aminobenzoic acid (NABA) produced by the enzymatic hydrolysis of NIPAB, followed by the increase in absorbance at 405 nm, was linear at 4 to 20 mM for up to 30 minutes and 15 °C to 40 °C.Abbreviations PA penicillin acylase (penicillin amidohydrolase EC 3.5.1.11) - AOT Aerosol OT (sodium bis- (2-ethylhexyl) sulfosuccinate) - NIPAB 6-nitro-3-(phenylacetamido)-benzoic acid - NABA 6-nitro-3-aminobenzoic acid - BSA bovine serum albumin     

Substrate specificity of penicillin acylase from Streptomyces lavendulae

The kinetic parameters of several substrates of penicillin acylase from Streptomyces lavendulae have been determined. The enzyme hydrolyses phenoxymethyl penicillin (penicillin V) and other penicillins with aliphatic acyl-chains such as penicillin F, dihydroF, and K. The best substrate was penicillin K (octanoyl penicillin) with a k(cat)/K(m) of 165.3 mM(-1) s(-1). The enzyme hydrolyses also chromogenic substrates as NIPOAB (2-nitro-5-phenoxyacetamido benzoic acid), NIHAB (2-nitro-5-hexanoylamido benzoic acid) or NIOAB (2-nitro-5-octanoylamido benzoic acid), however failed to hydrolyse phenylacetil penicillin (penicillin G) or NIPAB (2-nitro-5-phenylacetamido benzoic acid) and penicillins with polar substituents in the acyl moiety. These results suggest that the structure of the acyl moiety of the substrate is more determinant than the amino moiety for enzyme specificity. The enzyme was inhibited by several organic acids and the extent of inhibition changed with the hydrophobicity of the acid. The best inhibitor was octanoic acid with a K(i) of 0.8 mM. All the results, taking together, point to an active site highly hydrophobic for this penicillin acylase from Streptomyces lavendulae.     

2-Nitro-5-(6-bromohexanoylamino)benzoic acid test paper method for detecting microorganisms capable of producing cephalosporin acylases.

A novel method for detecting microorganisms capable of producing cephalosporin C (CPC) acylase and/or 7-(4-carboxybutanamido)cephalosporanic acid (GL-7-ACA) acylase has been developed. The method is based on the degradation of 2-nitro-5-(6-bromohexanoylamino)benzoic acid (NBHAB), a chromogenic substrate, into yellow 2-nitro-5-aminobenzoic acid by the action of the CPC acylase or the GL-7-ACA acylase. This method is very sensitive and quite specific, and has been successfully applied to screen the acylases from a variety of bacteria. A large number of colonies isolated on a plate surface from more than 67 samples and several known bacteria were tested by the NBHAB paper. Five NBHAB-positive strains and isolates were obtained. They were further examined by the reaction of their bacterial cells upon CPC and GL-7-ACA, respectively, and by thin-layer chromatography in order to distinguish the CPC acylase from the GL-7-ACA acylase.     

Separation and purification of penicillin acylase from Escherichia coli using AOT reverse micelles

Summary The extraction of penicillin acylase by reverse micellar solutions of a surfactant was studied. A 50 mM solution of dioctyl sodium sulphosuccinate in isooctane extracted 46% of the enzyme activity in a crude periplasmic extract of induced cells of E. coli ATCC 9637. The increase in the specific activity of the final enzyme preparation, after stripping of the organic phase at pH 7.5, in the presence of 1 M KCl, was 8 - fold.Abbreviations PA penicillin acylase (penicillin amidohydrolase EC 3.5.1.11) - AOT Aerosol OT (dioctyl sodium sulphosuccinate) - NIPAB 6-nitro-3-(phenylacetamido)-benzoic acid - NABA 6-nitro-3-aminobenzoic acid - BSA bovine serum albumin - SDS sodium dodecylsulphate     

Kinetics of enzyme acylation and deacylation in the penicillin acylase-catalyzed synthesis of beta-lactam antibiotics.

Penicillin acylase catalyses the hydrolysis and synthesis of semisynthetic beta-lactam antibiotics via formation of a covalent acyl-enzyme intermediate. The kinetic and mechanistic aspects of these reactions were studied. Stopped-flow experiments with the penicillin and ampicillin analogues 2-nitro-5-phenylacetoxy-benzoic acid (NIPAOB) and d-2-nitro-5-[(phenylglycyl)amino]-benzoic acid (NIPGB) showed that the rate-limiting step in the conversion of penicillin G and ampicillin is the formation of the acyl-enzyme. The phenylacetyl- and phenylglycyl-enzymes are hydrolysed with rate constants of at least 1000 s-1 and 75 s-1, respectively. A normal solvent deuterium kinetic isotope effect (KIE) of 2 on the hydrolysis of 2-nitro-5-[(phenylacetyl)amino]-benzoic acid (NIPAB), NIPGB and NIPAOB indicated that the formation of the acyl-enzyme proceeds via a general acid-base mechanism. In agreement with such a mechanism, the proton inventory of the kcat for NIPAB showed that one proton, with a fractionation factor of 0.5, is transferred in the transition state of the rate-limiting step. The overall KIE of 2 for the kcat of NIPAOB resulted from an inverse isotope effect at low concentrations of D2O, which is overridden by a large normal isotope effect at large molar fractions of D2O. Rate measurements in the presence of glycerol indicated that the inverse isotope effect originated from the higher viscosity of D2O compared to H2O. Deacylation of the acyl-enzyme was studied by nucleophile competition and inhibition experiments. The beta-lactam compound 7-aminodesacetoxycephalosporanic acid (7-ADCA) was a better nucleophile than 6-aminopenicillanic acid, caused by a higher affinity of the enzyme for 7-ADCA and complete suppression of hydrolysis of the acyl-enzyme upon binding of 7-ADCA. By combining the results of the steady-state, presteady state and nucleophile binding experiments, values for the relevant kinetic constants for the synthesis and hydrolysis of beta-lactam antibiotics were obtained.     

The use of chromogenic reference substrates for the kinetic analysis of penicillin acylases.

Determination of kinetic parameters of penicillin acylases for phenylacetylated compounds is complicated due to the low K(m) values for these substrates, the lack of a spectroscopic signal, and the strong product inhibition by phenylacetic acid. To overcome these difficulties, a spectrophotometric method was developed, with which kinetic parameters could be determined by measuring the effects on the hydrolysis of the chromogenic reference substrate 2-nitro-5-[(phenylacetyl)amino]benzoic acid (NIPAB). To that end, spectrophotometric progress curves with NIPAB in the absence and presence of the phenylacetylated substrates and their products were measured and analyzed by numerical fitting to the appropriate equations for competing substrates with product inhibition. This analysis yielded kinetic constants for phenylacetylated substrates such as penicillin G, which are in close agreement with those obtained in independent initial velocity experiments. Using NIPAB analogs with lower k(cat)/K(m) values, kinetic parameters for the hydrolysis of cephalexin and penicillin V were determined. This method was suitable for determining the kinetic constants of penicillin acylases in periplasmic extracts from Escherichia coli, Alcaligenes faecalis, and Kluyvera citrophila. The use of chromogenic reference substrates thus appears to be a rapid and reliable method for determining kinetic constants with various substrates and enzymes.     

Chromogenic analogues of penicillin dihydroF and penicillin K for the continuous spectrophotometric determination of aliphatic penicillin acylase activity

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

一株产多种β-内酰胺类抗生素酰化酶菌株的筛选

为了从大量的候选菌株中快速筛选头孢菌素酰化酶产生菌,设计并合成了一系列头孢菌素酰化酶的底物类似物。这些酰胺类的底物类似物由二部分组成,一部分为与头孢菌素相同或相似的侧链,另外一部分为发色基团或便于检测的基团。它们被酰化酶水解酰胺键以后可以方便快速的检测,因此用于对大量菌株进行快速筛选。采用这些化合物筛选到6株酰化酶阳性菌株。其中菌株ZH0650能够同时水解GL-7ACA和多个底物类似物。进一步研究表明,该菌至少产生3种酰化酶,AD-NABA酰化酶,青霉素G酰化酶和头孢菌素C酰化酶。我们初步纯化了AD-NABA酰化酶和青霉素G酰化酶,并对头孢菌素C酰化酶的活力进行了鉴定。这是首次报道的可以产生青霉素G酰化酶和头孢菌素酰化酶等多种酰化酶的菌株,具有良好的应用前景。     

青霉素G酰化酶α亚基Ser177的突变对酶活性的影响

The technique of cassette and site-specific mutagenesis were used to study the role of residue No. 177 in penicillin G acylase (PGA, EC 3.5.1.11). Ser is conserved at residue No. 177 in all penicillin binding proteins. We got a series of mutants in which the amino acid at residue No. 177 was replaced by other amino acids through the site-specific and cassette mutagenesis, and we characterized the mutants by colony hybridization, NIPAB paper test and DNA sequence analysis. These mutants all show no activity of enzyme, even if the Ser residue was replaced by Thr, Gly and Ala respectively. The results show that Ser residue may be essential for substrate-binding or catalysis of PGA.     

青霉素G酰化酶α亚基Ser177的突变对酶活性的影响

用盒式突变和定点突变对大肠杆菌青霉素G酰化酶α亚基177位ser进行了突变研究,结果发现所挑选的突变体均无酶的活力,这一结果可能可以用来解释Ser 177附近肽段和一些青霉素结合蛋白青霉素结合区在一级结构上保持同源性的原因。     

Fluorogenic assay for penicillin G acylase activity

A simple, highly sensitive, and rapid assay for high-throughput screening of penicillin G acylase-producing bacteria is presented. The method is based on the specific release of fluorescent 7-amino-4-methyl-coumarin through cleavage of phenylacetyl-4-methyl-coumaryl-7-amide by penicillin G acylase. The present method is suitable for screening pure enzymes as well as various penicillin G acylases like those from Escherichia coli, Proteus rettgeri, and Kluyvera citrophila in cell extracts. In addition, the new substrate was used for rapid assay of amidase activity in nondenaturing polyacrylamide gels.     

Simple screening method for isolation of penicillin acylase-producing bacteria

A new screening method for bacteria capable of producing penicillin acylase is described. The method is based on the use of Serratia marcescens sensitive to 6-aminopenicillanic acid but comparatively resistant to benzylpenicillin. It is simple, quite specific, and requires no special equipment. It can also be used to screen for phenoxymethylpenicillin acylase activity. We also suggest an acidimetric method for rapid detection of cloned genes in genetic engineering studies of penicillin acylase.     

Simple screening method for isolation of penicillin acylase-producing bacteria.

A new screening method for bacteria capable of producing penicillin acylase is described. The method is based on the use of Serratia marcescens sensitive to 6-aminopenicillanic acid but comparatively resistant to benzylpenicillin. It is simple, quite specific, and requires no special equipment. It can also be used to screen for phenoxymethylpenicillin acylase activity. We also suggest an acidimetric method for rapid detection of cloned genes in genetic engineering studies of penicillin acylase.     

Cloning and characterization of penicillin V acylase from Streptomyces mobaraensis

We report on the molecular cloning and characterization of penicillin V acylase (PVA) from an actinomycete, Streptomyces mobaraensis (Sm-PVA), which was originally isolated as an acylase that efficiently hydrolyzes the amide bond of various N-fatty-acyl-l-amino acids and N-fatty-acyl-peptides as well as capsaicin (8-methyl-N-vanillyl-6-nonenamide). In addition, the purified Sm-PVA hydrolyzed penicillin V with the highest activity (k(cat)) among the PVAs so far reported, penicillin G, and 2-nitro-5-phenoxyacetamide benzoic acid. The BLAST search revealed that the Sm-PVA precursor is composed of a polypeptide that is characteristic of enzymes belonging to the beta-lactam acylase family with four distinct segments; a signal sequence (43 amino acids), an alpha subunit (173 amino acids), a linker peptide (28 amino acids), and a beta subunit (570 amino acids). The mature, active Sm-PVA is a heterodimeric protein with alpha and beta subunits, in contrast to PVAs isolated from Bacillus sphaericus and B. subtilis, which have a homotetrameric structure. The amino acid sequence of Sm-PVA showed identities to PVA from S. lavendulae, N-acylhomoserine lactone-degrading acylase from Streptomyces sp., cyclic lipopeptide acylase from Streptomyces sp., and aculeacin A acylase from Actinoplanes utahensis with 68, 67, 67, and 41% identities, respectively.     

Penicillin acylase from the hybrid strains Escherichia coli 5K(pHM12): enzyme formation and hydrolysis of beta-lactam antibiotics with whole cells.

Penicillin acylase formation by the hybrid strain Escherichia coli 5K(pHM12) was studied under different culture conditions and reached 200 to 250 mumol of 6-aminopenicillanic acid per min per g of bacteria (wet weight) for penicillin G. The Km of whole-cell acylase was determined with 9 to 11 mM for penicillin G at a pH optimum of 7.8 at 45 degrees C. A competitive product inhibition for phenylacetic acid of Ki = 130 mM was found. 6-Aminopenicillanic acid acts as a noncompetitive inhibitor, with a Ki of 131. The temperature optimum of the reaction lies at 54 degrees C. Penicillin G inhibits the reaction at Ki(S) = 1,565 to 1,570 mM. Whole-cell acylase reacts on a wide spectrum of penicillins and cephalosporins, but those substrates with a delta-aminoadipyl rest are not hydrolized. beta-Lactamase activity of less than 1% relative to the acylase activity was found at reaction temperatures between 28 and 45 degrees C. After a comparison of different methods for the estimation of beta-lactamase activity, we found that high-pressure liquid chromatography is to be preferred. During batch fermentation of E. coli 5K(pHM12), problems of plasmid stability in the host strain arose which were overcome by the addition of 4 mg of tetracycline per liter to the medium as a selective marker.     

青霉素G酰化酶基因Ser177的定点突变

本研究用缺口以链法对肠杆菌青霉素Ｇ酰化酶（ＰＧＡ）基因Ｓｅｒ１７７进行寡核苷酸定点突变。通过ＮＩＰＡＢ（２－硝基－５－苯乙酰胺苯甲酸）试纸法筛选和测序鉴定,获得突变体Ｃｙｓ１７７,Ｇｌｙ１７７,Ａｒｇ１７７和Ａｓｎ１７７。它们的ＰＧＡ活性均已丧失。酶蛋白电泳分析表明突变体蛋白在体内正常表达。推测ＰＧＡ Ｓｅｒ１７７秀可能位于酶底物结合中心,是酶活性所必需,不能被置换。     

Penicillin acylase from the hybrid strains Escherichia coli 5K(pHM12): enzyme formation and hydrolysis of beta-lactam antibiotics with whole cells

Penicillin acylase formation by the hybrid strain Escherichia coli 5K(pHM12) was studied under different culture conditions and reached 200 to 250 mumol of 6-aminopenicillanic acid per min per g of bacteria (wet weight) for penicillin G. The Km of whole-cell acylase was determined with 9 to 11 mM for penicillin G at a pH optimum of 7.8 at 45 degrees C. A competitive product inhibition for phenylacetic acid of Ki = 130 mM was found. 6-Aminopenicillanic acid acts as a noncompetitive inhibitor, with a Ki of 131. The temperature optimum of the reaction lies at 54 degrees C. Penicillin G inhibits the reaction at Ki(S) = 1,565 to 1,570 mM. Whole-cell acylase reacts on a wide spectrum of penicillins and cephalosporins, but those substrates with a delta-aminoadipyl rest are not hydrolized. beta-Lactamase activity of less than 1% relative to the acylase activity was found at reaction temperatures between 28 and 45 degrees C. After a comparison of different methods for the estimation of beta-lactamase activity, we found that high-pressure liquid chromatography is to be preferred. During batch fermentation of E. coli 5K(pHM12), problems of plasmid stability in the host strain arose which were overcome by the addition of 4 mg of tetracycline per liter to the medium as a selective marker.     

Molecular biology of β-lactam acylases

-Lactam acylases such as penicillin G acylases, penicillin V acylases and glutaryl 7-aminocephalosporanic acid acylases are used in the manufacture of 6-aminopenicillanic acid, 7-aminodesacetoxycephalosporanic acid and 7-aminocephalosporanic acid (7-ACA). Genetically-engineered strains producing 1050 U/g, 3200 U/g and 7000 to 10,000 U/I of penicillin G acylase, penicillin V acylase and glutaryl-7-ACA acylase, respectively, have been developed. The penicillin G acylase studied to date and the glutaryl-7-ACA acylase from Pseudomonas sp. share some common features: the active enzyme molecules are composed of two dissimilar subunits that are generated from respective precursor polypeptide; the proteolytic processing is a post-translational modification which is regulated by temperature; and the Ser residue at the N-terminus of the -sub-unit (Ser²⁹⁰; penicillin G acylase numbering) is implicated as the active site residue. Protein engineering, to generate penicillin G acylase molecules and their precursors with altered sequences, and the structure-function correlation of the engineered molecules are discussed.The authors are with Research and Development, Hindustan Antibiotics Ltd, Pimpri, Pune 411 018, India;     

胞外青霉素酰化酶产生菌的选育

利用纸片显色方法,从土壤甲诀速筛选出98株产胞外青霉素酰化酶的菌种,经复筛其中10株酶活力较高,经鉴定均属于巨大芽孢杆菌。经单株分离得46号菌,用这株菌进行了产酶条件的研究,在最适产酶条件下,酶话力比开始提高了3．6倍。在此基础上又进行了物理化学因素处理,得突变株UL-81,酶活力达720u／1 Ooml发酵液。对原株和突变株进行比较,发现UL-81菌落、细胞形态、诱导剂苯乙酸用量及添加时间等明显不同于原株。在500L罐发酵酶活达8 20u／1OOml发酵液,为开始酶活的16倍。