Relationship between the antibacterial activity and the acceptor properties of certain penicillins]

Antibacterial activity of 2 natural and 12 semisynthetic penicillins against 5 strains of grampositive bacteria was determined and quantum chemical estimation of their molecules was performed with the Hukkel method. The data were indicative of the fact that antibacterial activity of the penicillins was connected with the acceptor properties of their molecules.     

Advances in enzymatic transformation of penicillins to 6-aminopenicillanic acid (6-APA)

This article elaborates on the important recent developments in the enzymatic transformation of penicillins to 6-aminopenicillanic acid (6-APA), which is the basic raw material for the industrial production of semisynthetic penicillins such as amoxycillin and ampicillin. Particular emphasis is placed on the improvements in purification, stability, and immobilization of the enzymes, (i.e. penicillin acylases) used for these transformations.     

Biotechnological advances on Penicillin G acylase: Pharmaceutical implications,unique expression mechanism and production strategies

In light of unrestricted use of first-generation penicillins, these antibiotics are now superseded by their semisynthetic counterparts for augmented antibiosis. Traditional penicillin chemistry involves the use of hazardous chemicals and harsh reaction conditions for the production of semisynthetic derivatives and, therefore, is being displaced by the biosynthetic platform using enzymatic transformations. Penicillin G acylase (PGA) is one of the most relevant and widely used biocatalysts for the industrial production of β-lactam semisynthetic antibiotics. Accordingly, considerable genetic and biochemical engineering strategies have been devoted towards PGA applications. This article provides a state-of-the-art review in recent biotechnological advances associated with PGA, particularly in the production technologies with an emphasis on using the Escherichia coli expression platform.     

Determination of iodine-containing admixtures in semisynthetic penicillins

Dependence of the values defining the content of iodine sorbing admixtures in semisynthetic penicillins on pH, reaction time and drug aliquots was studied. On the basis of this study a general approach to development of procedures for determining iodine sorbing admixtures in semisynthetic penicillins was suggested. Procedures for determination of iodine sorbing admixtures in carbenicillin, carfecillin, ampicillin, oxacillin, azlocillin and ampiox were developed.     

The role of penicillin amidases in nature and in industry

Penicillin amidase (PA) is the enzyme used commercially for the production of semisynthetic penicillins. During the past decade, a detailed picture of the structure and regulation of the gene encoding this enzyme has emerged, revealing a variety of interesting features that are unique among microorganisms. Clues to the biological role of this enzyme have been provided, as well as new strategies for the commercial production and utilization of PA.     

Drug Resistance as Influenced by Inactivated Sensitivity Discs

Reports of staphylococci resistant to the semisynthetic penicillins stimulated a study of the factors influencing the stability of the drugs in discs. The behavior of penicillin G, methicillin, oxacillin, cloxacillin, and cephalothin discs under different humidity and temperature conditions is described. Humidity was found to be the most significant factor in drug inactivation. Storage of discs in a vacuum desiccator at -20 C provides maximal antibiotic stability.     

β-Lactamase-Free Penicillin Amidase from Alcaligenes sp.: Isolation Strategy, Strain Characteristics, and Enzyme Immobilization

Isolation and characterization of a β-lactamase (EC 3.5.2.6)-free, penicillin amidase (penicillin amidohydrolase, EC 3.5.1.11)-producing organism is reported. The test strain was isolated by an enrichment technique with a substrate other than penicillins. The isolated strain belongs to the genus Alcaligenes. Phenylacetic acid was found to be the inducer of penicillin amidase. The amidase has a broad substrate spectrum. It is very active against penicillin G and semisynthetic cephalosporins, whereas penicillin V and semisynthetic penicillins acted moderately as a substrate. Immobilized cells of Alcaligenes sp. were shown to act as a reversible enzyme. Received: 28 April 1999 / Accepted: 18 May 1999     

Biotechnological applications of penicillin acylases: state-of-the-art

This review describes the most recent developments in the biotechnological applications of penicillin acylases. This group of enzymes is involved mainly in the industrial production of 6-aminopenicillanic acid and the synthesis of semisynthetic beta-lactam antibiotics. In addition, penicillin acylases can also be employed in other useful biotransformations, such as peptide synthesis and the resolution of racemic mixtures of chiral compounds. Particular emphasis is placed on advances in detection of new enzyme specificities towards other natural penicillins, enzyme immobilization, and optimization of enzyme-catalyzed hydrolysis and synthesis in the presence of organic solvents.     

A combined fermentative-chemical approach for the scalable production of pure E. coli monophosphoryl lipid A

Lipid A is the lipophilic region of lipopolysaccharides and lipooligosaccharides, the major components of the outer leaflet of most part of Gram-negative bacteria. Some lipid As are very promising immunoadjuvants. They are obtained by extraction from bacterial cells or through total chemical synthesis. A novel, semisynthetic approach to lipid As is ongoing in our laboratories, relying upon the chemical modification of a natural lipid A scaffold for the fast obtainment of several other lipid As and derivatives thereof. The first requisite for this strategy is to have this scaffold available in large quantities through a scalable process. Here, we present an optimized fed-batch fermentation procedure for the gram-scale production of lipid A from Escherichia coli K4 and a suitable phenol-free protocol for its purification. A study for regioselective de-O-phosphorylation reaction was then performed to afford pure monophosphoryl lipid A with an attenuated endotoxic activity, as evaluated by cytokine production in human monocytic cell line THP-1 in vitro. The reported method for the large-scale obtainment of monophoshoryl lipid A from the fed-batch fermentation broth of a recombinant strain of E. coli may permit the access to novel semisynthetic lipid A immunoadjuvant candidates.     

Engineering novel biocatalytic routes for production of semisynthetic opiate drugs

The morphine alkaloids and their semisynthetic derivatives provide a diverse range of important pharmaceutical drugs. Current production of semisynthetic opiate drugs is by chemical means from naturally occurring morphine, codeine and thebaine. Although various microbial transformations of morphine alkaloids have been identified since the 1960s, more recently there has been considerable effort devoted to engineering biocatalytic routes for producing these important compounds. Such biocatalytic routes are attractive, as they would provide an alternative to the chemical production processes which suffer from limited supply of precursors, often low yields and toxic wastes. The biotransformation of morphine and codeine to the potent analgesic hydromorphone and the mild analgesic/antitussive hydrocodone, respectively, by recombinant Escherichia coli has been demonstrated and the problems encountered when engineering such a system will be discussed.     

β-Lactamase Formation and Resistance of Proteus morganii to Various Penicillins and Cephalosporins

Ten strains of Proteus morganii were selected and the production of β-lactamase was studied. The cellular level of β-lactamase from P. morganii was regulated by the concentration of the inducer in the growth media, and the enzyme activity appeared within 10 min while its highest value was obtained 2 hr after the addition of benzylpenicillin as the inducer. The resistance level to cephaloridine was generally related to the amount of β-lactamase activity among the ten strains of P. morganii, but no relation was found between the ampicillin susceptibilities and the amounts of β-lactamase in their cell-free preparations. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by a crude enzyme was five times higher than that of benzylpenicillin. Semisynthetic penicillins were resistant to hydrolysis and exhibited competitive inhibition. Among the semisynthetic penicillins, dicloxacillin and carbenicillin were powerful competitive inhibitors of the β-lactamase from P. morganii.     

Improved activity and pH stability of E. coli ATCC 11105 penicillin acylase by error-prone PCR

Penicillin G acylase is the key enzyme used in the industrial production of β-lactam antibiotics. This enzyme hydrolyzes penicillin G and related β-lactam antibiotics releasing 6-aminopenicillanic acid, which is an intermediate in the production of semisynthetic penicillins. To improve the enzymatic activity of Escherichia coli penicillin acylase, sequential rounds of error-prone polymerase chain reaction were applied to the E. coli pac gene. After the second round of evolution, the best mutant M2234 with enhanced activity was selected and analyzed. DNA sequence analyses of M2234 revealed that one amino acid residue (K297I), located far from the center of the catalytic pocket, was changed. This mutant (M2234) has a specific activity 4.0 times higher than the parent enzyme and also displayed higher stability at pH 10.     

Efficient use of gentamycin in treating suppurative puerperal mastitis]

A total of 80 patients with postnatal purulent mastitis were treated with gentamicins. The main causative agent was Staph. aureus resistant to the traditionally used antibiotic and sensitive to gentamicin, fusidin, rifampicin and semisynthetic penicillinase-stable penicillins. Gramnegative bacteria contaminated the purulent foci after opening. Gentamicin was highly effective in treatment of postnatal purulent mastitis in cases with mixed Staphylococcus-Proteus infection.     

A common precursor for the two subunits of the penicillin acylase from Escherichia coli ATCC11105

Penicillin acylase (PA) is an industrial enzyme that is used to convert penicillin G into a precursor for semisynthetic penicillins. We have cloned a segment of DNA that codes for the two subunits required for PA activity. We also report the nucleotide sequence of a DNA fragment that codes for (i) the small subunit, (ii) the N-terminal region of the large subunit and (iii) a putative connecting peptide. These results confirm the existence of a common precursor for both peptides.     

Carbenicillin: Activity In Vitro and Absorption and Excretion in Normal Young Men

Carbenicillin is a new semisynthetic penicillin which differs from other penicillins in showing moderate antibacterial activity against Pseudomonas. Its activity in vitro is enhanced at low pH. Serum binding is of low order and does not appreciably alter activity. Strains of Pseudomonas exposed to subinhibitory concentrations of carbenicillin rapidly develop resistance by a mechanism that does not depend upon destruction of the drug. In normal subjects, high levels of anti-Pseudomonas activity are readily obtained in the urine after intramuscular injection. Levels of carbenicillin adequate to inhibit many strains of Pseudomonas can be achieved in serum only with an intravenous administration of a large dose. Carbenicillin appears to exhibit the same low degree of toxicity as do other penicillins.     

Crystal structure of D-hydantoinase from Bacillus stearothermophilus: insight into the stereochemistry of enantioselectivity

Industrial production of antibiotics, such as semisynthetic penicillins and cephalosporins, requires optically pure D-p-hydroxylphenylglycine and its derivatives as important side-chain precursors. To produce optically pure D-amino acids, microbial D-hydantoinase (E.C. 3.5.2.2) is used for stereospecific hydrolysis of chemically synthesized cyclic hydantoins. We report the apo-crystal structure of D-hydantoinase from B. stearothermophilus SD1 at 3.0 A resolution. The structure has a classic TIM barrel fold. Despite an undetectable similarity in sequence, D-hydantoinase shares a striking structural similarity with the recently solved structure of dihydroorotase. A structural comparison of hydantoinase with dihydroorotase revealed that the catalytic chemistry is conserved, while the substrate recognition is not. This structure provides insight into the stereochemistry of enantioselectivity in hydrolysis and illustrates how the enzyme recognizes stereospecific exocyclic substituents and hydrolyzes hydantoins. It should also provide a rationale for further directed evolution of this enzyme for hydrolysis of new hydantoins with novel exocyclic substituents.     

Preparation of optically active D-arylglycines for use as side chains for semisynthetic penicillins and cephalosporins using immobilized subtilisins in two-phase systems

Optically active D-arylglycines, which are of interest for preparation of semisynthetic penicillins and cephalosporins, were isolated from the racemic mixtures of their derivatives using immobilized proteolytic enzyme subtilisin (EC No. 3.4.4. 16). The performance of these reactions in two-phase systems, consisting of water and an immiscible organic solvent, improved the yield, purity, and economics of the process by increasing the substrate solubility and reducing the rate of nonenzymatic hydrolysis. The proportion of the organic phase can be as much as 75% of the overall volume without seriously impairing the enzymatic activity. The optically pure D-and L-arylglycines were liberated from their D- and L-derivatives by acid hydrolysis. The substituent influence of the various arylglycine derivatives on the rate of the enzymatic cleavage reaction was investigated.     

6β-Amidinopenicillanic Acids—a New Group of Antibiotics

SINCE 6-aminopenicillanic acid became available¹, many semisynthetic penicillins carrying an acyl moiety on the 6-amino-group have been prepared. Thereby penicillins with improved oral absorption, resistance to penicillinase and to a lesser degree increased activity towards Gram-negative bacilli have been made available². Many other N-substitutions are possible, however, but these have not so far resulted in useful compounds^{2, 3}. We report here some of our findings on a new type of N-substituted 6-aminopenicillanic acids.     

Prospective Life Cycle Assessment of Epitaxial Graphene Production at Different Manufacturing Scales and Maturity

Epitaxial growth is a potential production process for the new material graphene, where it is grown on silicon carbide (SiC) wafers at high temperatures. We provide first estimates of the life cycle cumulative energy demand, climate change, terrestrial acidification, and eco‐toxicity of this production. For this purpose, we applied prospective life cycle assessment (LCA) for three production scenarios (lab, pilot, and an industrial scenario), which reflect different production scales and technological maturity. The functional unit was one square centimeter of graphene. Results show that the three scenarios have similar impacts, which goes against previous studies that have suggested a decrease with larger production scale and technological maturity. The reason for this result is the dominance of electricity use in the SiC wafer production for all impacts (>99% in the worst case, >76% in the best case). Only when assuming thinner SiC wafers in the industrial scenario is there a reduction in impacts by around a factor of 10. A surface‐area–based comparison to the life cycle energy use of graphene produced by chemical vapor deposition showed that epitaxial graphene was considerably more energy intensive—approximately a factor of 1,000. We recommend producers of epitaxial graphene to investigate the feasibility of thinner SiC wafers and use electricity based on wind, solar, or hydropower. The main methodological recommendation from the study is to achieve a temporal robustness of LCA studies of emerging technologies, which includes the consideration of different background systems and differences in production scale and technological maturity.