Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: CAS:957-68-6 (7-ACA)
 46 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using directed evolution, we have selected an adipyl acylase enzyme that can be used for a one-step bioconversion of adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) to 7-ADCA, an important compound for the synthesis of semisynthetic cephalosporins. The starting point for the directed evolution was the glutaryl acylase from Pseudomonas SY-77. The gene fragment encoding the beta-subunit was divided into five overlapping parts that were mutagenized separately using error-prone PCR. Mutants were selected in a leucine-deficient host using adipyl-leucine as the sole leucine source. In total, 24 out of 41 plate-selected mutants were found to have a significantly improved ratio of adipyl-7-ADCA versus glutaryl-7-ACA hydrolysis. Several mutations around the substrate-binding site were isolated, especially in two hot spot positions: residues Phe-375 and Asn-266. Five mutants were further characterized by determination of their Michaelis-Menten parameters. Strikingly, mutant SY-77(N266H) shows a nearly 10-fold improved catalytic efficiency (k(cat)/K(m)) on adipyl-7-ADCA, resulting from a 50% increase in k(cat) and a 6-fold decrease in K(m), without decreasing the catalytic efficiency on glutaryl-7-ACA. In contrast, the improved adipyl/glutaryl activity ratio of mutant SY-77(F375L) mainly is a consequence of a decreased catalytic efficiency toward glutaryl-7-ACA. These results are discussed in the light of a structural model of SY-77 glutaryl acylase.
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PMID:Altering the substrate specificity of cephalosporin acylase by directed evolution of the Beta -subunit. 1219 40

beta-Lactam acylases are crucial for the synthesis of semisynthetic cephalosporins and penicillins. Unfortunately, there are no cephalosporin acylases known that can efficiently hydrolyse the amino-adipic side chain of Cephalosporin C. In a previous directed evolution experiment, residue Asn266 of the glutaryl acylase from Pseudomonas SY-77 was identified as being important for substrate specificity. In order to explore the function of this residue in substrate specificity, we performed a complete mutational analysis of position 266. Codons for all amino acids were introduced in the gene, 16 proteins that could be functionally expressed in Escherichia coli were purified to homogeneity and their catalytic parameters were determined. The mutant enzymes displayed a broad spectrum of affinities and activities, pointing to the flexibility of the enzyme at this position. Mutants in which Asn266 was changed into Phe, Gln, Trp and Tyr displayed up to twofold better catalytic efficiency (k(cat)/K(m))than the wild-type enzyme when adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) was used as substrate, due to a decreased K(m). Only mutants SY-77(N266H) and SY-77(N266M) showed an improvement of both catalytic parameters, resulting in 10- and 15-times higher catalytic efficiency with adipyl-7-ADCA, respectively. Remarkably, the catalytic activity (k(cat)) of SY-77(N266M) when using adipyl-7-ADCA as substrate was as high as when glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) was used, and approaches commercially interesting activity. SY-77(N266Q), SY-77(N266H) and SY-77(N266M) mutants showed a modest improvement in hydrolysing Cephalosporin C. Since these mutants also have a good catalytic efficiency when adipyl-7-ADCA is used and are still active towards glutaryl-7-ACA, they can be regarded as broad substrate acylases. These results demonstrate that the combination of directed evolution for the identification of important positions, together with saturation mutagenesis for finding the optimal amino acid, is a very effective method for finding improved biocatalysts.
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PMID:Mutational analysis of a key residue in the substrate specificity of a cephalosporin acylase. 1517 65

To compare the DAAO expression level in different Pichia pastoris host strains, the gene encoding DAAO from Trigonopsis variabilis was cloned into plasmid pPIC3.5k and then transformed into P. pastoris GS115 and KM71 respectively. The positive transformants PDK13 (MutS) and PD27 (Mut+) were obtained by PCR analysis. Their optimal and different expression conditions were investigated. To compare with PD27, PDK13 was determined to poss a slower consumption of methanol, a longer induction time, a lower oxygen request and apparently higher expression of DAAO. The highest expression levels were reached up to 2700, 2500 IU/L in shaking flask and 10140, 8463.5 IU/L in fermentor respectively. The over-expression of DAAO can meet its large demand for production of 7-ACA, alpha-keto acid and L-amino acid. In addition, the phenylpyruvate and L-phenylalanine were obtained by crude DAAO reacting with DL-phenylalanine at 37 degrees C for 3h.
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PMID:[Compared D-amino acid oxidase expression in different Pichia pastoris host strains]. 1596 91

We have compared the interplay of several antimicrobial agents and aminoacids on the neutrophil respiratory burst in response to formylmethionyl-leucyl-phenylalanine (fMLP), a chemoattractant. Mainly, an inhibitory effect has been observed in the penicillin family of agents and an enhancing effect in the cephalosporin family of agents. The molecules in which the sulfur numbered 1 in the 6-APA or 7-ACA nucleus was replaced by a carbon or an oxygen, had a different effect as compared with the other members of the family. The modulatory effects of ampicillin and cephalothine were not significant at a concentration lower than 10 mg/l and the effect of cephalothine looked maximum at 20-40 mg/l. If studies in cell-free systems demonstrated that the inhibitory effect of some antimicrobials could be due to a direct oxidant-scavenger activity mainly of HOCl, only hypotheses are proposed to explain the enhancing activity of the others. It could be in relation with (i) a synergistic effect upon fMLP receptor leading to an increase in H(2)O(2)/HOCl production or (ii) the generation of new oxydant products originating in cephalosporin lysis under HOCl attack, which would be able to react with luminol. The interplay of antimicrobial agents with the respiratory burst measured outside the cells probably has no therapeutic consequences because the bactericidal activity of neutrophils is achieved inside phagosomes where few agents are known to come into and where chemical conditions are different. On the opposite, in clinical use, this interplay could be interesting to study for a prevention of side effects.
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PMID:[Modulatory effect of antimicrobial agents or aminoacids on the oxidative burst of polymorphonuclear neutrophils triggered by formylmethionyl-leucyl-phenylalanine (fMLP)]. 1637 77