Cloning and sequencing of a novel glutaryl acylase β-subunit gene ofPseudomonas cepacia BY21 from bioinformatics

Pseudomonas cepacia BY21 was found to produce glutaryl acylase that is capable of deacylating glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) to 7-aminocephalosporanic acid (7-ACA), which is a starting material for semi-synthetic cephalosporin antibiotics. Amino acids of the reported glutaryl acylases from variousPseudomonas sp. strains show a high similarity (>93% identity). Thus, with the known nucleotide sequences ofPseudomonas glutaryl acylases in GenBank, PCR primers were designed to clone a glutaryl acylase gene fromP. cepacia BY21. The unknown β-subunit gene of glutaryl acylase from chromosomal DNA ofP. cepacia BY21 was cloned successfully by PCR. The β-subunit amino acids ofP. cepacia BY21 acylase (GenBank accession number AY948547) were similar to those ofPseudomonas diminuta KAC-1 acylase except that Asn408 ofP. diminuta KAC-1 acylase was changed to Leu408.     

Isolation and characterization of soil strains producing glutaryl-7-aminocephalosporanic acid acylase

A search was undertaken to screen microorganisms that produce an enzyme capable of deacylating glutaryl-7-aminocephalosporanic acid to 7-aminocephalosporanic acid in soil samples. The screening was carried out by preparing enrichment cultures containing glutaryl-7ACA and cephalosporin C as selective carbon sources. A non-β-lactam model compound, glutaryl-p-nitroanilide, was synthesized as a substrate suitable for the rapid screening of microorganisms isolated from the enrichment cultures. Two isolates exhibiting acylase activity, designated BY7.4 and BY8.1, were identified as strains ofPseudomonas species.Pseudomonas BY8.1 showed higher acylase activity toward Gl-7ACA thanPseudomonas BY7.4. Environmental conditions for the optimal acylase activity ofPseudomonas BY8.1 were shown to be pH 9 and 30°C.     

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;     

Cephalosporin C acylase: dream and(/or) reality

Cephalosporins currently constitute the most widely prescribed class of antibiotics and are used to treat diseases caused by both Gram-positive and Gram-negative bacteria. Cephalosporins contain a 7-aminocephalosporanic acid (7-ACA) nucleus which is derived from cephalosporin C (CephC). The 7-ACA nucleus is not sufficiently potent for clinical use; however, a series of highly effective antibiotic agents could be produced by modifying the side chains linked to the 7-ACA nucleus. The industrial production of higher-generation semi-synthetic cephalosporins starts from 7-ACA, which is obtained by deacylation of the naturally occurring antibiotic CephC. CephC can be converted to 7-ACA either chemically or enzymatically using d-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase. Both these methods show limitation, including the production of toxic waste products (chemical process) and the expense (the enzymatic one). In order to circumvent these problems, attempts have been undertaken to design a single-step means of enzymatically converting CephC to 7-ACA in the course of the past 10 years. The most suitable approach is represented by engineering the activity of a known glutaryl-7-aminocephalosporanic acid acylase such that it will bind and deacylate CephC more preferentially over glutaryl-7-aminocephalosporanic acid. Here, we describe the state of the art in the production of an effective and specific CephC acylase.     

Enhancement of glutaryl-7-aminocephalosporanic acid acylase activity of γ-glutamyltranspeptidase of Bacillus subtilis

Semisynthetic cephalosporins, the best-selling antibiotics worldwide, are derived from 7-aminocephalosporanic acid (7-ACA). Currently, in the pharmaceutical industrie, 7-ACA is mainly produced from cephalosporin C by sequential application of D -amino acid oxidase and cephalosporin acylase. Here we study the potential of industrially amenable enzyme γ-glutamyltranspeptidase from Bacillus subtilis for 7-ACA production, since the wild-type γ-glutamyltranspeptidase of B. subtilis has inherent glutaryl-7-aminocephalosporanic acid acylase activity with a k_cat value of 0.0485 s^-1. Its activity has been enhanced by site directed and random mutagenesis. The k_cat/K_m value was increased to 3.41 s^-1 mM^-1 for a E423Y/E442Q/D445N mutant enzyme and the kcat value was increased to 0.508 s^-1 for a D445G mutant enzyme. Consequently, the catalytic efficiency and the turnover rate were improved up to about 1000-fold and 10-fold, compared with the wildtype γ-glutamyltranspeptidase of B. subtilis.     

Bioconversion of cephalosporin C with D-amino acid oxidase from the yeastRhodosporidium toruloides

Summary A D-amino acid oxidase-producing yeast,Rhodosporidium toruloides CCRC 20306, was used to convert cephalosporin C (Ceph C) into -ketoadipyl cephalosporanic acid. A part of Ceph C could be directly converted into glutaryl-7-aminocephalosporanic acid (GL-7-ACA) by permeated cells of CCRC 20306. There were unknown side products formed during the conversion. The side products could be substantially reduced in amount by heating the cell extract containing D-alanine at 55°C for 5 min for five cycles prior to the conversion. Esterase activity present in the cell extract, which might be one of the causes of the side reaction, was greatly diminished by heat treatment.     

Construction and application of fusion proteins of d-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase for direct bioconversion of cephalosporin C to 7-aminocephalosporanic acid

Two fusion proteins of D-amino acid oxidase (DAAO) and glutaryl-7-aminocephalosporanic acid acylase (GLA) were designed to simplify the bioconversion process of cephalosporin C to 7-aminocephalosporanic acid (7-ACA), which is conventionally produced in a two-step enzymatic process. Two recombinant plasmids, pET-DLA and pET-ALD, were constructed to express fusion proteins of DAAO-linker-GLA (DLA) and GLA-linker-DAAO (ALD), respectively. When the recombinant plasmids were expressed in E. coli, the fusion protein DLA was not correctly folded and only DAAO activity could be detected. ALD, however, possessed activities of both DAAO and GLA, which directly catalyze the conversion of cephalosporin C into 7-ACA.     

Cloning and co-expression of D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase genes in Escherichia coli

To convert cephalosporin C to 7-aminocephalosporin (7-ACA), a D-amino acid oxidase (DAAO) gene from Trigonopsis variabilis and a glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) gene from Pseudomonas were cloned and expressed in recombinant Escherichia coli. For DAAO recombinant strain BL21(DE3)/pET-DAAO, a high DAAO activity of 250 U ml⁻¹ was obtained by a fed-batch culture. A GL-7-ACA acylase gene, in which the signal peptide sequence was deleted, was also successfully expressed in a recombinant E. coli BL21(DE3)/pET-ACY with a high expression level of 3000 U l⁻¹. A novel recombinant strain, BL21(DE3)/pET-DA, harboring both genes of DAAO and GL-7-ACA acylase, was further constructed, and a rather high DAAO activity of 140 U ml⁻¹ and GL-7-ACA acylase activity of 950 U l⁻¹ were simultaneously obtained. This recombinant strain, in which two genes are co-expressed, made it possible to catalyze cephalosporin C into 7-ACA directly.     

Morphological,physiological and enzymatic characteristics of cephalosporin acylase-producing Arthrobacter strain 45-8A

A bacterial strain producing cephalosporin acylases was isolated from soil. The morphological and physiological properties of this strain suggest that it belongs to the genus Arthrobacter, and the isolate was therefore designated Arthrobacter strain 45-8A. Substrate specificity of the enzyme was examined. The enzyme can convert both cephalosporin C and 7-(4-carboxylbutan-amino)cephalosporanic acid to 7-aminocephalosporanic acid. An interesting feature of the acylases is their temperature-dependent regulation. Activity of acylases was detected in strain 45-8A grown at temperature below 30 °C, but was not observed at higher temperature. Arthrobacter strain 45-8A did not exhibit -lactamase activity, even though its resistance to cephalosporin C was very strong (>2000 g/ml). This is quite beneficial for its application in the manufacture of 7-aminocephalosporanic acd.Abbreviations used NBHAB 2-Nitro-5-(6-bromohexanoylamino)-benzoic acid - NIPAB 2-Nitro-5-phenylacetaminobenzoic acid - CPC cephalosporin C - GL-7ACA 7-(4-carboxybutanamino)cephalosporanic acid - 6-APA aminopenicillanic acid - 7-ACA 7-aminocephalosporanic acid - PDAB p-Dimethylaminobenzaldehyde     

Two-step immobilized enzyme conversion of cephalosporin C to 7-aminocephalosporanic acid

The first large-scale production of 7-aminocephalosporanic acid (7ACA) from cephalosporin C (CPC) using a wholly enzymatic synthesis method is reported here. We produced 7ACA from CPC in as high a molar yield as 85% using the immobilized enzymes D-amino acid oxidase (D-AOD) and glutaryl-7-ACA acylase (GL-acylase). In the first reactor, CPC is converted to keto-adipyl-7-aminocephalosporanic acid (keto-7ACA) using an immobilized D-AOD isolated from a yeast, Trigonopsis variabilis. The keto-7ACA is then spontaneously converted to glutaryl-7-aminocephalosporanic acid (GL-7ACA) via a chemical reaction with hydrogen peroxide. The hydrogen peroxide is also a product of the D-AOD reaction. Near quantitative conversion of the keto-7ACA to GL-7ACA was observed. The second reactor converts GL-7ACA to 7ACA using an immobilized GL-acylase, which was isolated from a reconbinant Escherichia coli. The final 7ACA crystalline product is a high quality product. The reactions are conducted under very mild aqueous conditions: pH 8.0 and 20 degrees to 25 degrees C. The production of desacetyl side products is minimal. This process is currently being implemented on an industrial scale to produce 7ACA. (c) 1995 John Wiley & Sons, Inc.     

Cloning and high expression of glutaryl 7-aminocephalosporanic acid acylase gene from Pseudomonas diminuta

The gene coding for the glutaryl 7-aminocephalosporanic acid (GL 7-ACA) acylase from Pseudomonas diminuta KAC-1 was cloned and expressed in Escherichia coli. The acylase gene was composed of 2160 base pairs and encoded a polypeptide of 720 amino acid residues. The E. coli BL21 carrying pET2, the plasmid construct for high expression of GL 7-ACA acylase gene, produced this enzyme at approx. 30% of the total proteins with 3.2 units activity mg protein^–1. Growth at temperature below 31 °C and deletion of signal peptide increased the processing of precursor acylase to active enzyme in the recombinant E. coli cells.     

Enzymatic Modifications of Cephalosporins by Cephalosporin Acylase and Other Enzymes

ABSTRACT

Semisynthetic cephalosporins are important antibacterials in clinical practice. Semisynthetic cephalosporins are manufactured by derivatizing 7-aminocephalosporanic acid (7-ACA) and its desacetylated form. Microbial enzymes such as D-amino acid oxidase, glutaryl-7-ACA acylase and cephalosporin esterase are being used as biocatalysts for the conversion of cephalosporin C (CEPH-C) to 7-ACA and its desacetylated derivatives. Recent developments in the field of enzymatic modifications of cephalosporin with special emphasis on group of enzymes called as cephalosporin acylase is discussed in this review. Aspects related to screening methods, isolation and purification, immobilization, molecular cloning, gene structure and expression and protein engineering of cephalosporin acylases have been covered. Topics pertaining to enzymatic modifications of cephalosporin by D-amino acid oxidase, cephalosporin methoxylase and β -lactamase are also covered.     

Screening and characterization of microorganisms with glutaryl-7 ADCA acylase activity

A screening of microorganisms producing glutaryl-7 ADCA acylase, an enzyme able to hydrolyse glutaric acid selectively from glutaryl-3-deacetoxy-7-aminocephalosporanic acid (glutaryl-7 ADCA), has been carried out in soil samples. Five microorganisms expressing acylase activity were isolated and classified as Bacillus cereus, Achromobacter xylosooxidans, Bacillus sp., Pseudomonas sp. and Pseudomonas paucimobilis. The screening was carried out by preparing enrichment cultures containing glutaryl-7-ADCA or cephalosporin C as the selective carbon source. Four model compounds (adipoyl-, glutamyl- and glutaryl-p-nitroanilide and glutarylcoumarin), mimicking the glutaryl-7 ADCA -lactam moiety, were synthesized as substrates suitable for the rapid screening of the microorganisms (2500) isolated from the enrichment cultures. A total of 300 strains were active on the model substrates and only 5 displayed acylase activity on glutaryl-7 ADCA. The fermentation parameters, such as pH and inducer concentration, for the optimal acylase expression and acylase specificity towards the model substrates were different for each strain.     

产GL-7-ACA酰化酶重组菌的构建及高表达研究

戊二酰基-7-氨基头孢烷酸(GL-7-ACA)酰化酶是7-氨基头孢烷酸(7-ACA)两步酶法生产中的关键酶。成功构建组成型表达的产GL-7-ACA酰化酶重组大肠杆菌JM105/pMKC-ACY,并对其高表达条件进行了研究,得到了组成简单、廉价的国产培养基配方及操作简便、易于实现工业化的发酵工艺。在优化条件下,上罐补料高密度发酵的酶活高达6668.9U/L,是优化前的12.4倍,产率最高可达275.5U/(L.h),达到了工业生产的要求。     

Deacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues

Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), mainly by environmentally toxic chemical deacylation of cephalosporin C (CPC). Thus, the enzymatic conversion of CPC to 7-ACA by cephalosporin acylase (CA) would be very interesting. However, CAs use glutaryl-7-ACA (GL-7-ACA) as a primary substrate and the enzymes have low turnover rates for CPC. The active-site residues of a CA were mutagenized to various residues to increase the deacylation activity of CPC, based on the active-site conformation of the CA structure. The aim was to generate sterically favored conformation of the active-site to accommodate the D-alpha-aminoadipyl moiety of CPC, the side-chain moiety that corresponds to the glutaryl moiety of GL-7-ACA. A triple mutant of the CA, Q50betaM/Y149alphaK/F177betaG, showed the greatest improvement of deacylation activity to CPC up to 790% of the wild-type. Our current study is an efficient method for improving the deacylation activity to CPC by employing the structure-based repetitive saturation mutagenesis.     

Thermal and Operational Characteristics of Glutaryl-7-aminocephalosporanic acid Acylase Immobilized on Silica Gel Modified by Epoxide Silanization

Summary In this study, an investigation was performed into the thermal and operational characteristics of glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase (EC 3.5.1.-) immobilized on silica gel that had been modified by epoxide silanization. The pH values for the optimum activity of free and immobilized GL-7-ACA acylase were almost the same. However, the pH-dependent activity profile for the immobilized GL-7-ACA acylase is considerably expanded. Both free and immobilized enzymes generally had the highest activity at 50 °C. In thermodynamic studies, it was found that immobilization using epoxide silanization made GL-7-ACA acylase thermodynamically stable. In the results of repeated batch production of 7-ACA, 89.0 and 83.5% of the 7-ACA produced at the initial cycle were maintained after 20 times of recycle at 25 °C and 30 °C, respectively. Hence it was suggested that mass production of 7-ACA at 25 °C using immobilized GL-7-ACA acylase by epoxide silanization would be possible on a large scale.     

Characterization of an industrial biocatalyst: immobilized glutaryl-7-ACA acylase

A batch of the immobilized industrial biocatalyst glutaryl-7-ACA acylase (GA), one of the two enzymes involved in the biotransformation of cephalosporin C (CefC) into 7-aminocephalosporanic acid (7-ACA), was characterized. K(m) value for glutaryl-7-ACA was 5 mM. Enzyme activity was found to be optimal at pH between 7 and 9.5 and to increase with temperature and in buffered solutions. To avoid product degradation, optimal reaction conditions were obtained working at 25 degrees C using a 50-mM phosphate buffer, pH 8.0. Immobilized GA showed good stability at pH value below 9 and at temperature up to 30 degrees C. The inactivation of immobilized GA in the presence of different amounts of H(2)O(2), a side product that might be present in the plant-scale industrial solutions of glutaryl-7-ACA, was also investigated, but the deactivation rates were negligible at H(2)O(2) concentration that might be reached under operative conditions. Finally, biocatalyst performance in the complete two-step enzymatic conversion process from CefC to 7-ACA was determined on a laboratory scale. Following the complete conversion of a 75 mM solution of CefC into glutaryl-7-ACA catalyzed by an immobilized D-amino acid oxidase (DAAO), immobilized GA was used for the transformation of this intermediate into the final product 7-ACA. This reaction was repeated for 42 cycles. An estimation of the residual activity of the biocatalyst showed that 50% inactivation of immobilized GA was reached after approximately 300 cycles, corresponding to an enzyme consumption of 0.4 kU per kg of isolated 7-ACA.     

Immobilization of a cephalosporin acetylesterase by containment within an ultrafiltration device

A cephalosporin acetylesterase produced by Bacillus subtilis catalyzes the deacetylation of 7-aminocephalosporanic acid (7-ACA). Previous reports from our laboratory described the kinetic constants that characterize the reaction: K_m = 2.8 × 10^?3M, K_ia acetate = 5 × 10^?2M, and K_id deacetyl-7-ACA = 3.6 × 10^?2M. These constants were used to predict the time course of the reaction using the following equation for dual competitive product inhibition. where S_t = mg/ml 7-ACA, A_t = mg/ml acetate, D_t = mg/ml deacetyl-7-ACA. The predicted time course closely matched the time course measured experimentally. The equation also was solved without the inhibition terms and the solution indicated that product inhibition caused about a 30% increase in the time required for complete (>97%) hydrolysis of a 24 mg/ml 7-ACA solution. The esterase was immobilized by containment within an ultrafiltration device. With this technique the enzyme was reused 20 times over an 11 day span to deacetylate 7-ACA solutions containing 4 to 24 mg/ml 7-ACA. The specific activity after the 20th use was the same as the activity prior to the first use, indicating little enzyme inactivation occurred.     

Single-pot conversion of cephalosporin C to 7-aminocephalosporanic acid in the absence of hydrogen peroxide

In this study, d-amino acid oxidase (DAAO) and catalase (CAT) in the permeabilized recombinant Pichia pastori cells were well investigated. It appeared that their thermal stability was negatively correlated with the apparent enzymatic activities. The frozen-melted cells presented the best stability and the lowest apparent activities of DAAO and CAT, whereas the cetyltrimethylammonium bromide (CTAB) permeabilized cells displayed the weakest stability and the highest apparent activities of the two enzymes. Simultaneous action of DAAO and CAT in the CTAB-permeabilized cells and glutaryl-7-aminocephalosporanic acid acylase (GA) immobilized on carrier contributed to the conversion of cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA) with a yield of 76.2%. During such a reaction cycle, no visible activity loss occurred at the immobilized GA, whereas the loss rates of DAAO and CAT activities were about 0.029 and 1.13 U min⁻¹, respectively. Nevertheless, this problem could be easily solved by continuous feeding of the new permeabilized cell suspension at the rate of 6 ml h⁻¹ to the reactor. Following such a fed-batch strategy, these permeabilized cells and the immobilized GA could be efficiently reused for 6 and 15 reaction cycles, respectively, yielding around 76% 7-ACA at each reaction cycle.     

Active site residues of cephalosporin acylase are critical not only for enzymatic catalysis but also for post-translational modification

Cephalosporin acylase (CA) is a recently identified N-terminal hydrolase. It is also a commercially important enzyme in producing 7-aminocephalosporanic acid (7-ACA), a backbone chemical in synthesizing semi-synthetic cephalosporin antibiotics. CA is translated as an inactive single chain precursor, being post-translationally modified into an active enzyme. The post-translational modification takes place in two steps. The first intramolecular autocatalytic proteolysis takes place at one end of the spacer peptide by a nucleophilic Ser or Thr, which in turn becomes a new N-terminal Ser or Thr. The second intermolecular modification cleaves off the other end of the spacer peptide by another CA. Two binary structures in complex with glutaryl-7-ACA (the most favored substrate of CAs) and glutarate (side chain of glutaryl-7-ACA) were determined, and they revealed the detailed interactions of glutaryl-7-ACA with the active site residues (Y. Kim and W. G. J. Hol (2001) Chem. Biol., in press). In this report: 1) we have mutated key active site residues into nonfunctional amino acids, and their roles in catalysis were further analyzed; 2) we performed mutagenesis studies indicating that secondary intermolecular modification is carried out in the same active site where deacylation reaction of CA occurs; and 3) the cleavage site of secondary intermolecular modification by another CA was identified in the spacer peptide using mutational analysis. Finally, a schematic model for intermolecular cleavage of CA is proposed.