Identification of a penicillin V acylase processing fungus

In our studies with the penicillin V acylase of Bovista plumbea strains NRRL 3501 and NRRL 3824, we wanted to receive spores of these fungi. Surprisingly the fruiting bodies obtained in our work were not identical with those characteristic for Bovista plumbea. We identified them as Pleurotus ostreatus. For this reason we have to correct the name of the fungi known as Bovista plumbea NRRL 3501 and NRRL 3824.     

Production of penicillin by immobilized films ofPenicillium chrysogenum

Summary Various materials were tested and polycarbonate was chosen as the support material for attachment and film growth ofP. chrysogenum in rotating disc fermenters. During batch runs using a penicillin production medium attached biomass increased at a linear rate and penicillin G was produced by the immobilized mycelium over a six day period.     

Beijerinckia indica var.penicillanicum penicillin V acylase: enhanced enzyme production by catabolite repression-resistant mutant and effect of solvents on enzyme activity

Summary Beijerinckia indica var.penicillanicum mutant UREMS-5, producing 168% more penicillin V acylase, was obtained by successive treatment with UV, -irradiation and ethylmethane sulfonate. Penicillin V acylase production by the mutant strain was resistant to catabolite repression by glucose. Incorporation of glucose, sodium glutamate and vegetable oils in the medium enhanced enzyme production. The maximum specific production of penicillin V acylase was 244 IU/g dry weight of cells. Effect of solvents on hydrolysis of penicillin V by soluble penicillin V acylase and whole cells was studied. Methylene chloride, chloroform and carbon tetrachloride significantly stimulated the rate of penicillin V hydrolysis by whole cells.     

Utilization of side-chain precursors for penicillin biosynthesis in a high-producing strain of Penicillium chrysogenum

Utilization of the side-chain precursors phenoxyacetic acid (POA) and phenylacetic acid (PA) for penicillin biosynthesis by Penicillium chrysogenum was studied in shake flasks. Precursor uptake and penicillin production were followed by HPLC analysis of precursors and products in the medium and in the cells. P. chrysogenum used both POA and PA as precursors, producing phenoxymethylpenicillin (penicillin V) and benzylpenicillin (penicillin G), respectively. If both precursors were present simultaneously, the formation of penicillin V was blocked and only penicillin G was produced. When PA was added at different times to cells that were induced initially for POA utilization and were producing penicillin V, the POA utilization and penicillin V formation were blocked, whereas the cells started utilizing PA and produced penicillin G. The blocking of the POA turnover lasted for as long as PA was present in the medium. If POA was added to cultures induced initially for PA utilization and producing penicillin G, this continued irrespective of the presence of POA. Utilization of POA increased concomitant with depletion of PA from the medium. Analysis of cellular pools from a growing cell system with POA as precursor to which PA was added after 48 h showed that the cellular concentration of POA was kept high without production of penicillin V and at a concentration comparable to the concentration in the medium. The cellular concentration of POA was higher than the concentration of PA that was utilized for penicillin G production. Correspondence to: S. Havn Eriksen     

Penicillin production by glucose-derepressed mutants ofPenicillium chrysogenum

Summary Wild-type strains ofPenicillium chrysogenum produce lower penicillin V titers in media containing excess glucose. Two mutant strains were isolated and shown to produce normal penicillin V titers in the presence of excess glucose. These strains, designated as glucose-repression insensitive (GRI) mutants, produced higher penicillin V titers than the wild-type strain in media containing lactose as the main carbohydrate source. In lactose-based media, the production of penicillin V was depressed to a much lesser extent by in-cycle additions of glucose with the GRI mutants when compared to the wild-type strain. In short-term biosynthesis experiments using washed cells in a medium containing glucose as the sole carbon source, the GRI mutants produced penicillin V at a faster rate than the wild-type strain. In fed-batch fermentations in 14-liter fermentors, where glucose was fed continuously and pH controlled, both GRI mutants produced more than 10% higher penicillin V titers than the wild-type strain. These results suggest that isolation of GRI mutants is an effective way to select for higher producing strains and that the synthesis of penicillin synthesizing enzymes in GRI mutants may be less repressed by glucose than in wild-type strains.     

Biosynthesis of penicillin V acylase by Fusarium sp.: effect of culture conditions

Penicillin V acylase was produced, both intracellularly and extracellularly, by Fusarium sp. SKF 235 grown in submerged fermentation. When neopeptone was added to the medium, >95% of the penicillin V acylase was extracellular. In the absence of a complex organic nitrogen source, the fungus produced low levels of totally intracellular penicillin V acylase. MgSO₄ was essential for synthesis of the enzyme, which was induced by phenoxyacetic acid and penicillin V. The maximum yield of penicillin V acylase was 430 IU/g dry cell wt. The optimum pH value and temperature for the penicillin V acylase were 6.5 and 55°C, respectively.     

Morphology of Penicillium chrysogenum strains immobilized in calcium alginate beads and used in penicillin fermentation

Summary The morphology of two strains of Penicillium chrysogenum immobilized in calcium alginate and used in penicillin fermentation was examined. The degree and distribution of mycelial growth inside and on the surface of the beads depended on the strain, the cultivation media and the fermentation time. P. chrysogenum ATCC 12690 developed as a mycelial network inside the beads. The growth tendency of P. chrysogenum S₁ in micropellets was directed to the outer surface of the beads. At the end of the production phase only a trace of mycelia and no micropellets in the center of alginate beads were observed, while the outer surface and the subsurface were completely covered with mycelia.     

Decreased production ofpara-hydroxypenicillin V in penicillin V fermentations

Summary Penicillin V (phenoxymethyl penicillin) is produced by industrial strains ofPenicillium chrysogenum in the presence of phenoxyacetic acid (POAc), a side-chain precursor for the penicillin V molecule. The wild-type strain ofP. chrysogenum produces an undesirable penicillin byproduct,para-hydroxypenicillin V (p-OH penicillin V), in addition to penicillin V, viapara-hydroxylation of POAc and subsequent incorporation of thep-OH phenoxyacetic acid into the penicillin molecule. Most of thep-OH penicillin V is produced late in cycle when the POAc concentration in the medium is nearly depleted. The level ofp-OH penicillin V produced by the control strain ranges up to 10–15% of the total penicillins produced. 3-Phenoxypropionic acid andp-bromophenylacetic acid partially inhibit the formation ofp-OH penicillin V with a minimal effect on penicillin V productivity. Mutants deficient in their ability to hydroxylate POAc were found to produce lower levels ofp-OH penicillin V. Multi-step mutation and screening, starting with the wild-type strain, have culminated in isolation of mutants which producep-OH penicillin V as 1% of the total penicillins with no adverse effect on penicillin V productivity.     

Role of antimicrobial agents in simultaneous saccharification and fermentation of paddy malt mash to ethanol by mixed cultures of Saccharomyces cerevisiae PH03 and Zymomonas mobilis ZM4

Summary Among various antimicrobial plant extracts, chemicals and antibiotics used for simultaneous saccharification and fermentation, penicillin G prevented contamination and did not inhibit amylase activity and growth of the synergistic co-cultures Saccharomyces cerevisiae PH03 and Zymomonas mobilis ZM4 during a 7-day fermentation of paddy malt (25.0%) mash (18.0% dextrose equivalent) to ethanol at 30°C and pH 5.5. The treatment yielded 10.1% (v/v) ethanol from the mash which was significantly more than that of the boiled and fermented mash (9.3% v/v) and equal to that of the mash boiled and fermented (10 2% v/v) after added amylases treatment. Most of the other compounds (kanamycin, streptomycin, polymyxin, tetracycline) had growth inhibitory effect especially on Z.mobilis.     

Stimulation of cell-free ring expansion of penicillin N by sonication and Triton X-100

Summary Cell-free protoplast lysates of Cephalosporium acremonium convert penicillin N to deacetoxycephalosporin C by ring expansion. Filtration eliminates 90% of their activity, indicating that the enzyme activity is particulate. The surfactant Triton X-100 or sonication of the lysate stimulates protoplast lysate activity. Active extracts could be produced by merely sonicating intact mycelia.     

Expression ofBacillus subtilis endoglucanase gene inLactobacillus acidophilus

Summary ABacillus subtilis gene which codes for endoglucanase was transferred intoLactobacillus acidophilus by electroporation with plasmids containing the gene. The endoglucanase gene expressed well in the transformed cells and most of the gene product was found in the culture medium. The efficiency of the endoglucanase production by the transformed.L. acidophilus cells depended greatly on the choice of the vector plasmids on which the gene to be inserted.     

Elicitation of anthocyanin production in cell cultures of carrot (Daucus carota L) by using elicitors and abiotic stress

Summary A cell line of carrot (Daucus carota L) which produces anthocyanin was subjected to various elicitors and abiotic stresses: The elicitors tested were culture filtrates (CF) and cell extracts (CE) of certain bacteria and yeasts. The abiotic stresses were salts of certain metal ions. The production increase obtained with cell extracts of Bacillus cereus. Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus were 49, 72, 45 and 41% respectively over the control. Maximum elicitation was obtained with elicitor derived from cell extract of the yeast Rhodotorula rubra where it enhanced anthocyanin production by two fold. The abiotic stress agents Ca, Mn, Zn, Co, Fe & V enhanced anthocyanin production. Of all the metal ions tested Ca was the most effective. The elicitation process was governed by the type and level of elicitor.     

Endocytic uptake of Escherichia coli spheroplasts by Neurospora crassa slime cells

Summary Interaction of Escherichia coli spheroplasts with Neurospora crassa slime cells was examined by transmission electron microscopy after treatment with polyvinyl alcohol followed by dilution with the high pH-high Ca buffer. Bacterial spheroplasts were found either adhering to the flat surface, associating with the invaginating surface, or residing within the intracellular vesicle of fungal protoplasts. In addition, bacterial spheroplasts free of the surrounding vesicles and those in the course of breakdown were observed in the fungal cytoplasm. It was concluded that Escherichia coli spheroplasts are taken up by Neurospora crassa protoplasts almost exclusively via endocytosis. This is the first cytological evidence for the endocytic activity of fungal cells.     

Immobilization of whole Escherichia coli containing penicillin amidase using cross-linking agents and fillers

To obtain a catalyst with good mechanical stress stability and other operational characteristics, cross-linked aggregates of whole Escherichia coli containing penicillin amidase were reinforced with surface modified precipitated silica and chitosan. The immobilized cells plus mixed fillers possess better performance characteristics i.e. higher stability at 4°C and 30°C, least protein leaching capacity and good settling characteristics. Most suitable conditions to prepare catalyst with mixed fillers were: chitosan, 0.3: silica, 0.2 g/g d.wt cell; and minimum moisture content in catalyst, 30% (w/w).     

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     

Conversion of cephalosporin-c into 7-phenoxy-acetamido-cephalosporanic acid by acyltransferase of mutants of Penicillium chrysogenum

Summary We have tested the acyl-transferase of penicillin producing strains and penicillin-less mutants of Penicillium chrysogenum for their ability to exchange the d--aminoadipic acid side chain of cephalosporin-C with phenoxyacetic acid. We found the reaction in both types of strains.     

Expression of interferon genes in murine macrophages: Possible role of endogenous interferon in the modulation of cell differentiation

The expression of interferon (IFN)- gene was studied in mouse peritoneal macrophages (PM) harvested from normal mice (lps ⁿ ) or LPS-hyporesponsive mice (lps ^d ). A strong direct correlation between the LPS response of PM and their capacity to expressing basal levels of IFN was found. The results suggest that the constitutive expression of IFN- gene can play an important role not only in the resistance to viral infection but also in the modulation of cell differentiation.     

Specific differences in tolerance to exogenous berberine among plant cell cultures

The tolerance of plant cells to exogenously administered berberine, an antimicrobial isoquinoline alkaloid, was studied using berberine-producing and nonproducing cell suspension cultures. Both Coptis japonica and Thalictrum flavum cells, which have an intrinsic ability to synthesize berberine, took up exogenous berberine from the culture medium by an energy-requiring active transport to accumulate it exclusively in vacuoles. By contrast, T. minus cells, which excrete indigenous berberine mostly into the medium, did not take up exogenously supplied berberine, indicating that the alkaloid transport in this species is unidirectional. No inhibition of cell growth by exogenous berberine was observed in the three berberine-producing cell cultures. On the other hand, a small amount of exogenous berberine strongly inhibited cell growth in the berberine-free cultures of Datura innoxia, Catharanthus roseus, and Paeonia albiflora. The berberine taken up actively by Datura cells could not be transported into vacuoles but was dispersed in the cytoplasm, causing a severe inhibition of cell growth.     

Point mutations change specificity and kinetics of metal uptake by ZupT from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The ZIP (ZRT-, IRT-like Protein) protein ZupT from Escherichia coli is a transporter with a broad substrate range. Phenotypic and transport analysis showed that ZupT, in addition to Zn(II), Fe(II) and Co(II) uptake, is also involved in transport of Mn(II) and Cd(II). Competition experiments with other substrate cations suggested that ZupT has a slight preference for Zn(II) and kinetic parameters for Zn(II) in comparison to Co(II) and Mn(II) transport support this observation. Metal uptake into cells by ZupT was optimum at near neutral pH and inhibited by ionophores. Bicarbonate or other ions did not influence metal-uptake via ZupT. Amino acid residues of ZupT contributing to substrate specificity were identified by site directed mutagenesis. ZupT with a H89A exchange lost Co(II) and Fe(II) transport activity, while the S117V mutant no longer transported Mn(II). ZupT with E152D was impaired in overall metal uptake but completely lost its ability to transport the substrates Zn(II) and Mn(II). These experimental findings expand our knowledge on the substrate specificity of ZupT and provide further insight into the function of ZupT as a bacterial member of the vastly distributed and important ZIP family.     

Protoplast formation and cell wall regeneration inClostridium thermocellum

Summary A protocol was developed for the production ofClostridium thermocellum protoplasts and the regeneration of protoplasts into vegetative cells. Protoplasts were prepared by exposure of whole cells to lysozyme (125 ug/ml for 10 min. at 45 C). Protoplasts plated on regeneration agar reverted to walled cells with a frequency of up to approximately 0.3 % of input cells.