7-aminocephalosporanic Acid Research Articles

Synthesis and preliminary antimicrobial evaluation of new 7-(N-pyrrolyl) derivatives of cephalosporins.

A series of seven new cephalosporins was prepared for preliminary microbiological evaluation by N-acylation of 7-aminocephalosporanic acid with substituted N-pyrrolylcarboxylic acids via mixed anhydrides. The chemical structure of the compounds were confirmed by IR, 1H-NMR and mass spectral data. The 7-(N-pyrrolyl) cephalosporin derivatives were tested in vitro by the disc diffusion method upon 3 strains and subsequent determination of the minimal inhibitory concentration (MIC) of the most active ones upon 29 strains. The products of the series exhibited antibacterial activity. They showed selective potency against gram-positive and were practically inactive against gram-negative microorganisms. The compound 3-[(acetyloxy)methyl]-7-([2-[3-(ethoxycarbonyl)-2-methyl-5-phenyl-1H-1-pyrrolyl]acetyl]amino)-6-oxo-7,7a-dihydro-2H,6H-aceto[2,1-b][1,3]thiazine-4-carboxylic acid (4a) was outlined as more active than the reference cefazolin (CAS 23325-78-2) in regard to S. pyogenes and some strains of S. aureus, the MIC of 4a against S. pyogenes were at least 4-fold lower. The toxicological evaluations of the starting N-pyrrolylcarboxylic acids showed no acute toxicity.

(R)-α-Aminoadipic acid: an interesting chiral pool building block

(R)-alpha-Aminoadipic acid is available on a large scale by enzymatic cleavage from cephalosporin C (CephC) in the production of 7-aminocephalosporanic acid (7-ACA). It can be converted into other interesting enantiomerically pure compounds, e. g. derivatives of (R)-pipecolic acid (R-piperidine-2-carboxylic acid), (R)-6-oxopiperidine-2-carboxylic acid, (R)-1,2,3,4-tetrahydropyridine-2(2H)-carboxylates, and other compounds obtained by further conversions of these products.

Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel

Cephalosporin C (CPC) acylase converts CPC into 7-aminocephalosporanic acid (7-ACA) by single-step enzymatic catalysis. An optimized CPC acylase gene with substituted codons and a reduced GC content was artificially designed, synthesized and overexpressed in recombinant Escherichia coli. The synthetic CPC acylase (sCPCAcy) exhibited 2.3 times more CPC specific deacylation activity with substrate CPC than with substrate glutaryl-7-ACA (GL-7-ACA). Site-directed mutagenesis of the residues around the active center showed that not only the residues that were adjacent to the CPC D-α-aminoadipyl moiety, but also the residues that were in the substrate transport tunnel (Leu666, Ala675, Leu677), played crucial roles in catalysis as the ones locating in active center. Mutant sCPCAcy(Leu666Phe) and sCPCAcy(Leu677Ala) exhibited significantly reduced specific enzymatic activity, while mutant sCPCAcy(Ala675Gly) demonstrated enhanced activity. The specific activity of purified sCPCAcy and sCPCAcy(Ala675Gly) was 10.0 U/mg and 11.3 U/mg, respectively. The optimal CPC acylase productivity of mutant sCPCAcy(Ala675Gly) reached 5349 U/l after 24h in culture, which was a 35% increase over the activity of sCPCAcy.

Determination of cephalosporin-C amidohydrolase activity with fluorescamine

A spectrophotometric procedure for the assay of cephalosporin-C amidohydrolase activity, based on the determination of the 7-aminocephalosporanic acid (7-ACA) produced in the hydrolysis of cephalosporin-C by the enzyme, is described. This procedure can be used to detect 7-ACA over a range of 10 to 200 micrograms mL-1. The same method can be used as a fluorometric procedures with a 100-fold greater sensitivity. At pH 4.5 7-ACA produces a strong fluorophor with fluorescamine, detectable spectrophotometrically at 378 nm and fluorometrically at an excitation of 378 nm and emission of 495 nm. At this pH the fluorophors formed with cephalosporin-C, proteins and aminoadipic acid present minimal absorbance values. The conditions for maximal detection of 7-ACA in the presence of proteins, cephalosporin-C and aminoadipic acid have been determined.

Substitute Technology for Reference Substances in the Analysis of Impurities in Cefonicid for Injection with HPLC Using a Diode Array Detector

With HPLC using a diode array detector (DAD), a method of substitution for reference substances in impurity profiling control was developed that combined peak tracking by the correlation of spectra with application of correction factors for determination of each impurity. For qualitative analysis, two-dimensional (2D) standard spectrochromatographic data produced by HPLC-DAD were compared with sample data to develop 2D chromatographic spectral correlative maps so that the three target impurities were recognized without preparation and injection of reference solutions. For quantitative analysis, correction factors among cefonicid and the three impurities were established. The correction factors were 1.06 for 5-mercapto-1,2,3,4-tetrazole 1-methyl sulfonic acid detected at 255 nm, 0.77 for 7-aminocephalosporanic acid detected at 265 nm, and 0.97 for methoxycefonicid detected at 268 nm. The method could be used in analysis of related substances in cefonicid for injection without recourse to chemical reference standards of the three impurities.

PERMEABILIZATION OF TRIGONOPSIS VARIABILIS FOR ENHANCED D-AMINO ACID OXIDASE ACTIVITY

The permeabilization of T. variabilis for use as a whole-cell biocatalyst has been little studied even though it is one of the most important microbial sources of D-Amino Acid Oxidase (DAO) and is useful for the separation of racemic amino acid mixtures, and the production of α-keto acids and 7-amino cephalosporanic acid. In the present work, for the first time a comparative study has been carried out on the efficiency of the permeabilization of the yeast T. variabilis (CBS 4095) grown in a culture medium with D-methionine as an inducer of the synthesis of DAO using different detergents, organic solvents, and freeze-thaw cycles. The best results were obtained with treatment with cetyltrimethylammonium bromide (CTAB), 2% of wet weight of cells, at 45°C for 30 min. The efficiency of the permeabilization procedure was seen to depend on the detergent/cell ratio, being independent of the concentration of detergent in the reaction medium over a broad range: 0.016–0.8% (w/v). The main advantages of the CTAB procedure are the high activity and reproducibility obtained, together with the fact that it can be used with either fresh or frozen cells with almost identical results, unlike the other detergents studied (CHAPS, Triton X-100, Sarcosyl), and especially treatment with toluene-ethanol at 0°C, where a previous sample-freezing step is determinant in the cell permeabilization process. The permeabilization procedure reported here can be used for both accurate in vivo quantification of DAO activity in T. variabilis and to prepare a whole-cell biocatalyst with a high level of activity and stability.

Characteristic of immobilized cephalosporin C acylase and its application in one-step enzymatic conversion of cephalosporin C to 7-aminocephalosporanic acid

Cephalosporin C (CPC) acylase is an enzyme which hydrolyzes CPC to 7-aminocephalosporanic acid (7-ACA) directly, and therefore has great potential in industrial application. In this study, the CPC acylase from a recombinant Escherichia coli was purified to high purity by immobilized metal affinity chromatography, and the CPC acylase was covalently attached to three kinds of epoxy supports, BB-2, ES-V-1 and LX-1000EP. The immobilized CPC acylase with LX-1000EP as the support shows the highest activity (81 U g−1) suggesting its potential in industrial 7-ACA production. The activity of immobilized enzyme was found to be optimal at pH between 8.5 and 9.5 and to increase with temperature elevation until 55 °C. Immobilized CPC acylase showed good stability at pH between 8.0 and 9.5 and at temperature up to 40 °C. To avoid product degradation, the production of 7-ACA utilizing immobilized enzyme was carried out at 25 °C, pH 8.5 in a designed reactor. Under optimal reaction conditions, a very high 7-ACA yield of 96.7% was obtained within 60 min. In the results of repeated batch production of 7-ACA, 50% activity of the initial cycle was maintained after being recycled 24 times and the average conversion rate of CPC reached 98%.

Biosynthesis of Cephalosporin-C Acylase Enzyme: Optimal Media Design, Purification, and Characterization

7-aminocephalosporanic acid (7-ACA) is the key intermediate for the synthesis of semisynthetic cephalosporin antibiotics and enzyme cephalosporin-C acylase (CPC acylase) plays an important role in the conversion of cephalosporin-C to 7-ACA. With an aim to increase the yield of 7-ACA production by Micrococcus luteus, a stepwise strategy, statistical medium was applied for optimizing the medium composition for the production of CPC acylase. Purified enzyme was found to be of 80 kDa. The optimum pH and temperature for the production of 7-ACA were 7.6 and 340C, respectively. The Km and Vmax were estimated to be 9.43 mg/mL and 7.65 U/mL, respectively.

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 gamma-glutamyltranspeptidase from Bacillus subtilis for 7-ACA production, since the wild-type gamma-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 gamma-glutamyltranspeptidase of B. subtilis.

Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin.

α-Amino ester hydrolases (AEH) are a small class of proteins, which are highly specific for hydrolysis or synthesis of α-amino containing amides and esters including β-lactam antibiotics such as ampicillin, amoxicillin, and cephalexin. A BLAST search revealed the sequence of a putative glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase 93% identical to a known AEH from Xanthomonas citri. The gene, termed gaa, was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913 and the corresponding protein was expressed into Escherichia coli. The purified protein was able to perform both hydrolysis and synthesis of a variety of α-amino β-lactam antibiotics including (R)-ampicillin and cephalexin, with optimal ampicillin hydrolytic activity at 25 °C and pH 6.8, with kinetic parameters of k(cat) of 72.5 s(-1) and K(M) of 1.1 mM. The synthesis parameters α, β(o), and γ for ampicillin, determined here first for this class of proteins, are α = 0.25, β(o) = 42.8 M(-1), and γ = 0.23, and demonstrate the excellent synthetic potential of these enzymes. An extensive study of site-directed mutations around the binding pocket of X. campestris pv. campestris AEH strongly suggests that mutation of almost any first-shell amino acid residues around the active site leads to inactive enzyme, including Y82, Y175, D207, D208, W209, Y222, and E309, in addition to those residues forming the catalytic triad, S174, H340, and D307.

Environmentally Safe Production of 7-ACA by Recombinant Acremonium chrysogenum

7-Amino cephalosporanic acid (7-ACA), which is currently obtained by chemical deacylation from cephalosporin C (CPC), is a major intermediate for industrial production of β-lactam antibiotics. 7-ACA can also be produced from CPC by enzymatic route including two-step and one-step procedures. In our research, an ecs gene coding for CPC acylase was synthesized and cloned into pET-28a(+) to construct an E. coli expression plasmid pYG232. E. coli BL21(DE3) bearing pYG232 was induced by IPTG and successfully expressed the recombinant ECS (88.9 kDa). Under the optimal conditions: 0.5 mg/ml purified ECS protein, 5 mg/ml CPC, 100 mM Tris-Cl (pH 9.6), supplement with 7 mM Zn(2+), slightly shaking for 6 h at 25°C, the transformation productivity was 54.4%. Then, ecs was cloned downstream of an A. chrysogenum endogenous promotor, PpcbC, to construct pYG233 for expression in A. chrysogenum. pYG233 was introduced into a CPC high-producer via integrative transformation of protoplasts. Two independent bleomycin-resistant transformants were investigated by PCR, Southern blotting, quantitative RT-PCR, western blotting, and fermentation. Although these two transformants both have one copy of integrated ecs, they showed different expression level of ECS protein and 7-ACA production. When concentration of CaCO(3) was reduced to 50 mM, ZnSO(4) was increased to 7 mM, CuSO(4) was eliminated from the fermentation media, and the pH was adjusted to 8.0 at day 4 during fermentation, 7-ACA production of one of the transformants could reach 1701 μg/ml, indicated that more than 30% of CPC produced by this high-producer have been transformed into 7-ACA directly in vivo. This is the highest 7-ACA production by direct fermentation ever reported.

Determination of cephalosporin acylase activity by biological and colorimetric method in bacteria

The effective production of 7-aminocephalosporanic acid (7-ACA) is a matter of concern in the pharmaceutical industry because it is a starting material for the synthesis of semi synthetic cephalosporin. Therefore screening for new source of cephalosporin acylase positive bacteria is very important. The cephalosporin acylase can be found in several Pseudomonas sp. and other bacteria. To facilitate the attempts of obtaining the microorganisms with higher cephalosporin acylase activity from natural environments, development of new and specific methods for screening environmental microorganisms with cephalosporin acylase activity is very important. In this study, a biological and colorimetric method was evaluated for determination of cephalosporin acylase product in bacteria. Samples were cultured in general and selective media, and the routine biochemical laboratory tests were used for diagnosis of Pseudomonas sp. All of the isolated strains were tested for cephalosporin acylase by a biological and colorimetric method. A total of 180 Pseudomonas sp. out of 350 samples were isolated. Two strains capable of producing cephalosporin acylase were identified from 180 candidates. The Pseudomonas bacteria isolated in this study is a source for cloning and cephalosporin acylase enzyme production.

A single Phe54Tyr substitution improves the catalytic activity and thermostability of Trigonopsis variabilisd-amino acid oxidase

The industrial importance of Trigonopsis variabilisd-amino acid oxidase (TvDAAO) is represented by its biocatalytic oxidative deamination of cephalosporin C (CPC) to yield glutaryl-7-aminocephalosporanic acid (GL-7-ACA). The process has been incorporated into a two-step bioconversion to produce 7-aminocephalosporanic acid, the crucial synthetic nucleus for several semi-synthetic cephalosporin antibiotics. A homology model of TvDAAO indicated that residue F54 is in a close proximity to the in silico docked CPC. Substitution of this F54 to Tyr (F54Y) resulted in 6-fold improvement in k(cat,app) and approximately 2.5-fold increase in K(i) of GL-7-ACA. Heat treatment (55 degrees C, 60 min) did not decrease the activity of F54Y. It is suggested that the Tyr substitution might initiate hydrogen bond formation with the amino group of CPC and facilitate deamination. Faster substrate turnover, reduced GL-7-ACA inhibition and improved thermostability of the F54Y substitution render it a useful candidate in industrial production of semi-synthetic cephems.

Improved cross-linked enzyme aggregates for the production of desacetyl β-lactam antibiotics intermediates

Cross-linked enzyme aggregates (CLEAs) are reported for the first time for a recombinant acetyl xylan esterase (AXE) from Bacillus pumilus. With this enzyme, CLEAs production was most effective using 3.2 M (80%-saturation) ammonium sulfate, followed by cross-linking for 3 h with 1% (v/v) glutaraldehyde. Particle size was a key determinant of the CLEAs activity. The usual method for generating particles, by short-time vortexing was highly inefficient in terms of enzyme activity yields. In contrast, the use of long-time vortexing increased activity recovery, and a novel approach consisting in the utilization of a commercial mechanical cell disruptor which is based on a reciprocating movement recovered all the enzyme activity in few seconds. In the CLEAs thus produced, the enzyme was much more resistant to shear, heat and extreme pH values than the soluble enzyme. The CLEAs were highly effective in transforming fully 7-amino cephalosporanic acid and cephalosporin C into their corresponding desacetyl derivatives, which are important advanced intermediates in the production of semisynthetic β-lactam antibiotics. The operational stability of such CLEAs was remarkable, with a half life of 45 cycles. Therefore, the new methodology used here should decrease the industrial cost of the CLEAs, both in terms of biocatalyst production and reusability.

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.

An Improved Process for the Preparation of Diphenylmethyl 7β-Phenylacetamido-3-hydroxymethyl-3-cephem-4-carboxylate

An efficient and improved process for the preparation of diphenylmethyl 7β-phenylacetamido-3-hydroxymethyl-3-cephem-4-carboxylate was developed. With the commercially available 7-aminocephalosporanic acid (7-ACA) as starting material, up to 73.5% overall isolated yield of the titled compound was synthesized in two steps via direct phenylacetylation with phenylacetyl chloride, followed by basic hydrolysis and esterification with diphenyldiazomethane. The newly developed process obviated the use of protecting groups, reduced the environmental footprint, and could be easily controlled and conveniently scaled up for this pivotal intermediate in cephalosporin chemistry.

Determination of cephalosporin acylase activity by biological and colorimetric method in bacteria

The effective production of 7-aminocephalosporanic acid (7-ACA) is a matter of concern in the pharmaceutical industry because it is a starting material for the synthesis of semi synthetic cephalosporin. Therefore screening for new source of cephalosporin acylase positive bacteria is very important. The cephalosporin acylase can be found in several Pseudomonas sp. and other bacteria. To facilitate the attempts of obtaining the microorganisms with higher cephalosporin acylase activity from natural environments, development of new and specific methods for screening environmental microorganisms with cephalosporin acylase activity is very important. In this study, a biological and colorimetric method was evaluated for determination of cephalosporin acylase product in bacteria. Samples were cultured in general and selective media, and the routine biochemical laboratory tests were used for diagnosis of Pseudomonas sp. All of the isolated strains were tested for cephalosporin acylase by a biological and colorimetric method. A total of 180 Pseudomonas sp. out of 350 samples were isolated. Two strains capable of producing cephalosporin acylase were identified from 180 candidates. The Pseudomonas bacteria isolated in this study is a source for cloning and cephalosporin acylase enzyme production.

Continuous production of 7-Aminocephalosporanic acid by immobilized cells of Pseudomonas diminuta

The continuous production of 7-ACA with immobilized whole cells of P. diminuta was carried out in a tubular glass reactor at optimal conditions. The biocatalyst was prepared by gel entrapment using chitosan, geletin and agar as immobilizing agents. The micro-organism was characterized in terms of cell-to-carrier ratios, diffusional properties, as well as storage and operational stability, etc., towards the production of 7-ACA. It was found that a cell to carrier ratio of 0.45 and a flow rate of 120 mL h−1, respectively, were most suitable for the conversion of CPC to 7-ACA. Operational stability was highest with chitosan, with a half life of 2106 h followed by gelatin, then agar as immobilizing supports.

Properties and applications of microbial D-amino acid oxidases: current state and perspectives

D: -Amino acid oxidase (DAAO) is a biotechnologically relevant enzyme that is used in a variety of applications. DAAO is a flavine adenine dinucleotide-containing flavoenzyme that catalyzes the oxidative deamination of D-isomer of uncharged aliphatic, aromatic, and polar amino acids yielding the corresponding imino acid (which hydrolyzes spontaneously to the alpha-keto acid and ammonia) and hydrogen peroxide. This enzymatic activity is produced by few bacteria and by most eukaryotic organisms. In the past few years, DAAO from mammals has been the subject of a large number of investigations, becoming a model for the dehydrogenase-oxidase class of flavoproteins. However, DAAO from microorganisms show properties that render them more suitable for the biotechnological applications, such as a high level of protein expression (as native and recombinant protein), a high turnover number, and a tight binding of the coenzyme. Some important DAAO-producing microorganisms include Trigonopsis variabilis, Rhodotorula gracilis, and Fusarium solani. The aim of this paper is to provide an overview of the main biotechnological applications of DAAO (ranging from biocatalysis to convert cephalosporin C into 7-amino cephalosporanic acid to gene therapy for tumor treatment) and to illustrate the advantages of using the microbial DAAOs, employing both the native and the improved DAAO variants obtained by enzyme engineering.

Development of the semi-synthetic penicillins and cephalosporins

Semi-synthetic penicillins and cephalosporins both derive from their respective chemical nuclei, 6-aminopenicillanic acid (6-APA) and 7-aminocephalosporanic acid (7-ACA). Work leading to their isolation was being carried out in parallel, but following very different pathways, during the last half of the 1950s. The development of 6-APA was reviewed recently in this journal, and in the present article I take a closer look at early work on ‘penicillin amidase’ and revisit the steps that led to 7-ACA.