Purification and characterization of cloned isopenicillin N synthetase

Isopenicillin N synthetase (IPS) cloned from Cephalosporium acremonium has been isolated from transformed Escherichia coli and purified to homogeneity. The resulting, abundant, recombinant protein, whilst undergoing slightly different N-terminal processing to that observed for the fungally-derived protein, has identical kinetics for the conversion of LLD-aminoadipoyl-cysteinyl-valine to isopenicillin N. Recombinant IPS converts analogue substrates into unusual beta-lactam antibiotics in exactly the same way as the fungal protein.     

Substrate specificity of isopenicillin N synthase.

Highly purified isopenicillin N synthase (IPNS) from two sources (naturally occurring in Penicillium chrysogenum and that expressed in Escherichia coli via a cloned gene derived from Cephalosporium acremonium) have been isolated and utilized in vitro to test synthetic modifications of the natural substrate, (L-alpha-amino-delta-adipyl)-L-cysteinyl-D-valine (ACV). A very sensitive procedure utilizing the ability of beta-lactams to induce the synthesis of beta-lactamase was employed to determine whether an ACV analogue could serve as a substrate for IPNS. A wide variety of amino and carboxyl terminal tripeptide substitutions were examined and found to elicit positive beta-lactamase induction profiles. However, none of these modifications were found to function as efficiently as a substrate as ACV. One of the beta-lactam products which was formed from the reaction of IPNS and the tripeptide analogue was independently synthesized and evaluated for antibacterial activity. Modification of the L-cysteine residue in the second position of ACV resulted in tripeptides that were unable to serve as substrates. Conversion of the D-valine residue in the third position of ACV to an aromatic amino acid or to a highly electronegative residue such as trifluorovaline resulted in elimination of substrate activity and creation of an inhibitor of the enzyme.     

Studies on the biosynthesis of isopenicillin N with a cell-free preparation of Penicillium chrysogenum

When delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine was added to a cell-free system prepared by lysis of Penicillium chrysogenum protoplasts, "compounds X and Y" were detected after analysis on a cation-exchange column. The chromatographic position as well as results of experiments with double labelled tripeptides showed "compound X" to be the penicilloic acid of isopenicillin N. LLD-Tripeptide labelled with tritium at carbon-2 of th valine part was incorporated into isopenicillin N with retention of label. "Compound Y" retained all hydrogens on the valine part of the peptide, but lost half of the tritium on carbon-3 of the cysteine part. The results are consistent with the hypothesis that the LLD-tripeptide is converted into isopenicillin N via a monocyclic beta-lactam and without a dehydrovalinyl intermediate. Extensive transacylase activity was observed between isopenicillin N and 6-aminopenicillanic acid.     

Further studies on microbiological ring-expansion of penicillin N.

The rate of microbiological ring-expansion of penicillin N to deacetoxycephalosporin C using protoplast lysates of the antibiotic-negative mutant Cephalosporium acremonium M-0198 has been increased some 70-fold over that of our earlier system. We confirmed the stimulatory effects of FeSO4 and ascorbate described by Hook et al. (Biochem. Biophys. Res. Commun. 87: 258, 1979); the optimum concentrations found were 0.04 mM FeSO4 and 0.67 mM ascorbate. Adenosine triphosphate concentration was lowered to 0.83 mM; phosphoenolpyruvate and pyruvate kinase were eliminated. The optimum pH and temperature for the reaction were 7.2 and 25 degrees C, respectively. Alpha-ketoglutarate and MnCl2 showed no marked effect on the reactions, MgSO4 and KCl were mildly stimulatory, and CuSO4 and ZnSO4 were very inhibitory. Penicillin N was optimal at a concentration of 0.07 mM. Specific ring-expansion activity reached its peak 13 hours after growth ceased and then disappeared rapidly.     

Carbon catabolite regulation of the conversion of penicillin N into cephalosporin C

Cephalosporin C biosynthesis by Cephalosporium acremonium was delayed until most glucose in the medium was used. Addition of increasing concentrations of glucose up to 55 g/liter decreased cephalosporin C biosynthesis but stimulated growth. Sequential formation of penicillin N (an intermediate in the cephalosporin C biosynthetic pathway) and cephalosporin C was found when the culture was developed synchronously. Little cephalosporin C formation was observed until most penicillin N had already been formed. The sequential formation of penicillin N and cephalosporin C was due to the sequential formation of the "penicillin N synthetase system" and the "cephalosporin C synthetase system". Cells grown in the presence of glucose showed an increased accumulation of penicillin N and clear reduction of the conversion of penicillin N to cephalosporin C. Resting cell studies indicated that the glucose effect was due to the repression of one or more of the enzymes converting penicillin N into cephalosporin C. Little inhibition by glucose of the activity of these enzymes, once formed, was observed. Glucose did not effect significantly the pool sizes of either precursor amino acids of cephalosporin (alpha-aminoadipic acid and valine) or methionine (an inducer of penicillin N and cephalosporin C biosynthesis). On the basis of these data it is suggested that glucose catabolism specifically represses the enzyme system converting penicillin N into cephalosporin C.     

Effect of methionine on cephalosporin C and penicillin N production by a mutant of Cephalosporium acremonium.

A mutant with enhanced potential to utilize sulfate for cephalosporin C production was isolated from a strain of Cephalosporium acremonium. The mutant displayed potency levels more than twofold that of the parent in the presence of sulfate but its productivity was severely inhibited by more than 0.5% of methionine which gave high cephalosporin C production with the parent. In a complex medium norleucine stimulated cephalosporin C production by the mutant in the presence of sulfate, whereas it showed no effect on the parent. In an incubation system with sulfur-starved cells of the mutant, L-methionine, but not the D-isomer, gave lower cephalosporin C production and a delayed production of penicillin N. However, it exhibited a stimulatory effect in the presence of valine or alpha-aminoadipic acid, the constituent amino acids of the antibiotic. Norleucine showed a similar effect to that of L-methionine in the presence of sulfate. On the basis of these results, characteristics of the mutant are discussed in connection with the effect of methionine.     

A preparative method for the purification of isopenicillin N from genetically blocked Acremonium chrysogenum strain TD189: studies on the degradation kinetics and storage conditions

A protocol for preparative isopenicillin N (IPN) purification, a highly interesting and hitherto unavailable intermediate of the penicillin and cephalosporin biosynthetic pathway due to its high unstability, is described. Culture broths of Acremonium chrysogenum TD189, a strain blocked in cephalosporin biosynthesis that accumulates this metabolite, were treated with acetone and filtered though charcoal and a hydrophobic resin in a single step as tandem columns. The cleared broth was then lyophilized and passed though a Sephadex G-25 column. The last step was the purification to homogeneity of IPN in a semipreparative HPLC equipment and, optionally, a desalting step by Sephadex G-10 column. Once purified, a complete analysis of the stability of the compound and the conditions for its long-term storage was carried out. Our results suggest a first-order model for IPN decomposition for all the pH and temperature analyzed. IPN is more stable at neutral pH, and once lyophilized, can be stored under vacuum and -75?° C with a half-life of 770 days.     

Preparation and evaluation of N(3)-O-toluyl-fluorouracil-loaded liposomes

This study was aimed at developing a liposome delivery system for a new and potential antitumor lipophilic prodrug of 5-fluorouracil (5-Fu)-N(3)-O-toluyl-fluorouracil (TFu), intended to improve the bioavailability and therapeutic efficacy of 5-Fu by oral and intravenous administration. TFu-loaded liposomes were prepared by a modified film dispersion-homogenization technique, the formulation and manufacture parameters were optimized concerning the drug encapsulation efficiency. TFu-loaded liposomes were characterized according to particle size, size distribution, zeta potential, drug entrapment efficiency, drug loading and physical stability, respectively. In vitro release characteristics, in vivo pharmacokinetic properties and bioavailabilities were also investigated. The formulated liposomes were found to be relatively uniform in size (400.5 +/- 9.6 nm) with a negative zeta potential (-6.4 +/- 0.8 mV). The drug entrapment efficiency and loading were (88.87 +/- 3.25%) and (8.89 +/- 0.19%), respectively. The physical stability experiments results indicated that lyophilized TFu-loaded liposomes were stable for at least 9 months at 4 degrees C. In vitro drug release profile of TFu-loaded liposomes followed the bi-exponential equation. The results of the pharmacokinetic studies in mice indicated that the bioavailability of TFu-loaded liposomes was higher than the suspension after oral administration, and was bioequivalent comparing with TFu 50% alcohol solution after intravenous (i.v.) administration. These results indicated that TFu-loaded liposomes were valued to develop as a practical preparation for oral or i.v. administration.     

青霉素V钾片与青霉素G注射剂联合治疗小儿呼吸道感染

目的 为了减轻每天数次静脉注射青霉素给患儿带来的痛苦与不便,本研究比较了青霉素G静脉注射与青霉素V钾片联合治疗小儿急性肺炎、急性扁桃体炎的疗效。方法 完全随机将200例患儿分为青霉素V钾片治疗组(治疗组,n=97)和青霉素G注射剂常规治疗组(对照组,n=112)。治疗组青霉素1次静脉滴注(简称静滴)后加青霉素V钾片进行治疗;对照组青霉素每天2次静滴治疗。治疗组急性扁桃体炎患儿64例,肺炎患儿33例,用青霉素G每次10万～20万U·kg-1加入50～100mL生理盐水中静脉滴注,每天上午1次,然后口服青霉素V钾片每次15 mg·kg-1,每天3次;对照组急性扁桃体炎患儿68例,肺炎患儿44例,用青霉素G静脉滴注,每天2次(每次10万～20万U·kg-1),观察临床疗效。结果 治疗组与对照组对于小儿肺炎、急性扁桃体炎在退热和缓解症状、体征及炎性细胞恢复等方面的临床疗效相似。结论 青霉素G静脉滴注1次加青霉素V钾片治疗可以替代单用青霉素多次静注治疗小儿肺炎和扁桃体炎。且具有应用方便、减轻患儿疼痛等优点。