Leakage of recombinant penicillin G acylase from Escherichia coli by adding chloroform under fermentation conditions

A simple method was developed to release periplasmic penicillin G acylase from Escherichia coli BL21(DE3) during the fermentation process. More than 80% of the total penicillin G acylase was released into the broth when 3% (v/v) chloroform was added at 3 h after induction. The activity of extracellular penicillin G acylase reached 20699 U/l. This method was efficient and would facilitate further investigation of penicillin G acylase for industrial applications.     

Role of the intracellular proteolysis in the production of the periplasmic penicillin amidase in Escherichia coli

The yield of periplasmic enzyme, penicillin amidase (PA), from E. coli ATCC 11105 is regulated at the post-translational level by two competitive processes-intracellular proteolysis of newly synthesised pre-pro-PA (ppPA) in the cytoplasm and membrane transport and maturation of ppPA. Intracellular proteolysis results in a significant loss in the yield of active PA. Immunochemical analysis were used to study the influence of the cultivation temperature, phenylacetic acid, and glucose on the proteolysis of the ppPA in the cytoplasm. An increase in glucose concentrations or temperature during the cultivation resulted in a significant loss of PA activity due to the increased rate of intracellular proteolysis of ppPA. Addition of phenylacetic acid reduced the intracellular proteolysis of ppPA and, as a consequence, increased the PA production. Taken together, these data explain a new aspect of regulation of active PA production in E. coli.     

Enhanced production of penicillin G acylase from a recombinant Escherichia coli

Penicillin G acylase (pac) gene was cloned into a stable asd ⁺ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g^–1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg^–1 protein by a novel two-step process.     

Effect of phenylacetic acid on the growth and production of penicillin G acylase of recombinant and host strains derived from Escherichia coli W

The industrial production strain Escherichia coli RE3(pKA18) for penicillin G acylase (PGA) bears simultaneously the pga gene on the chromosome as an inducible gene pgaⁱ, (the inductor is phenylacetic acid, PAA) and on the recombinant plasmid pKA18 as a constitutively expressed gene pga^c. Under non-selective conditions, plasmid-less strains (P⁻) appeared in 17th successive batch culture. However, the population was over taken by P⁻ cells already in fourth culture if the medium was supplemented with PAA. The rate of plasmid loss from the culture depends on the PAA concentration and on the expression of pgaⁱ, not on PGA overproduction from pga^c. PAA at inducing concentration has a negative effect on PGA expression and plasmid stability in the high-expression self-cloning system RE3(pKA18) which results in the reduction of: (1) the specific growth rate of a culture and biomass concentration, (2) the synthesis of PGA (e.g. the specific activity of the strain) and (3) the copy number of the recombinant plasmid and promotion of the plasmid loss from the culture. Segregational stability of pKA18 increases in P⁺ persisting clones and in re-transformed P⁻ clones segregated during the selection in the presence of PAA.     

Bioreactor cultivation of Escherichia coli for production of recombinant penicillin G amidase from Alcaligenes faecalis

The penicillin G amidase (PGA) from Alcaligenes faecalis, which has interesting properties for use in combinatorial biochemistry, was produced by recombinant expression in Escherichia coli. The corresponding gene was cloned into a multicopy vector under the strict regulatory control of the rhamnose inducible promoter. Cells were grown in a synthetic minimal medium in a bioreactor (5 l working vol.), and production of PGA was induced by repeated addition of the inducer rhamnose, that served also as a carbon source. The fermentation yield was about 4500 units PGA activity per liter of culture medium.     

Morphological,growth, and chromosomal changes in bovine pancreatic duct epithelial cells exposed toN-methyl-N′-nitro-N-nitrosoguanidine

Summary Epithelial cells cultured from bovine pancreatic ducts were given a single treatment ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Multinucleated cells and giant cells were observed more frequently in carcinogen-treated cultures than in controls. The MNNG-treated cultures also contained a sizeable population of small, dense cells that were not observed in control cultures. At the concentration of 1.0 μg/ml, MNNG caused an initial depression in the growth rate of the cells followed by growth stimulation for several weeks. The MNNG produced chromosomal damage in the cells as indicated by the observation that a substantial proportion of carcinogen-treated cells were heteroploid and contained a high frequency of metacentric and submetacentric chromosomes and a dicentric marker chromosome. The MNNG treated and control cultures did not acquire the ability to grow in soft agar or to produce tumors after transplantation into athymic, nude mice. This work was supported in part by Public Health Service Contract and Interagency Agreement Y01-CP60204 from the Division of Cancer Cause and Prevention, National Cancer Institute.     

Genetic recombination in Escherichia coli. IV. Isolation and characterization of recombination-deficiency mutants of Escherichia coli K12

19 independent recombination-deficient mutants were isolated. 7 carried mutations that mapped near or in the recB and recC genes between thyA and argA. 10 mutants carried mutations cotransducible with pheA and exhibited no complementation with recA in temporary zygotic diploids.     

Stabilization and functional properties of Escherichia coli penicillin G acylase by covalent conjugation of anionic polysaccharide carboxymethylcellulose

The stabilization of Escherichia coli penicillin G acylase (PGA) conjugated with carboxymethylcellulose (CMC) against temperature and pH was studied. The 2,3-dialdehyde derivative of CMC obtained by periodate oxidation was covalently conjugated to PGA via Schiff's base formation. The inactivation mechanism of both native and CMC-conjugated PGA appeared to obey first order inactivation kinetics during prolonged incubations at 40–60 °C and in the pH range 4–9. Inactivation rate constants of conjugated enzyme were always lower, and half-life times were always higher than that of native PGA. The activation free energy of inactivation (G _i values) of CMC-conjugated enzyme were found to be always higher than that of native PGA at all temperatures and pH values studied as another indicator of enzyme stabilization. Highest stability of CMC-conjugated enzyme was observed as nearly four-fold at 40 °C and pH 8.0. No changes were observed on the temperature and pH profiles of PGA after CMC conjugation. Lower K _m and higher k _cat values of PGA obtained after CMC conjugation indicates the improved effect of conjugation on the substrate affinity and catalytic performance of the enzyme.     

Murein and lipopolysaccharide biosynthesis in synchronized cells of Escherichia coli K 12 and the effect of penicillin G,mecillinam and nalidixic acid

The incorporation of radioactive N-acetylglucosamine into murein and lipopolysaccharide of synchronized cells of Escherichia coli K 12 was followed over 100 min in the presence of antibiotics. At 20 min intervals cell walls were prepared. Lipopolysaccharide and murein sacculi were isolated and the radioactivity was quantified in both polymers. Labelled, newly synthesized murein was characterized according to murein subunits linked to lipoprotein, and the degree of crosslinkage. Furthermore, murein subunits containing anhydromuramic acid were determined, permitting the calculation of the average glycan chain length. The results indicated that penicillin G at 30 g/ml stimulated the incorporation of new murein subunits into sacculi followed by a sudden increase in lipopolysaccharide incorporation into the outer membrane. The degree of crosslinkage in murein synthesized in the presence of 30 g/ml penicillin G was higher than in the control, and almost twice as high as in murein synthesized in the presence of 20 g/ml nalidixic acid. Both antibiotics inhibited cell division at the concentrations indicated. Murein synthesized in the presence of 2 g/ml mecillinam also showed higher crosslinkage. However, about twice as much anhydromuramic acid-containing subunits were observed as in the control. At the same time lipopolysaccharide incorporation into the outer membrane was stimulated two- to three-fold.Abbreviation GlcNAc N-acetylglucosamine     

Effect of organic cosolvent on kinetic resolution of tert-leucine by penicillin G acylase from Kluyvera citrophila

Penicillin G acylase (PGA) from Kluyvera citrophila immobilized on Amberzyml was used for enantioselective hydrolysis of N-phenylacetylated-dl-tert-leucine (N-Phac-dl-Tle) to produce l-tert-leucine (l-Tle). The effects of various organic cosolvents on hydrolysis of N-Phac-dl-Tle have been investigated in aqueous-cosolvent medium. It was founded that the rate of PGA-catalyzed reaction was significantly affected by the presence of 2% (v/v) organic cosolvent concentration. The initial rate fell with increasing logP of the cosolvent, but for logP values less than −0.24 the rate was faster than in purely aqueous medium. Additionally, the relative rate increases with the increase of dielectric constant (ε) of organic cosolvents. The yields of l-Tle in all aqueous-cosolvent systems were above 95% with the enantiomeric excess (ee) of >99%.     

Insight into molecular mechanism underlying the transesterification catalysed by penicillin G amidase (PGA) using a combination protocol of experimental assay and theoretical analysis

Penicillin G amidase (PGA) is one of the most recognised biocatalysts because of its critical application in the antibiotic industry. Herein, the additive effects involved in transesterification catalysed by PGA are explored in detail using a combination of experimental analysis and theoretical modelling. The transesterification ability of PGA is experimentally determined with 17 N-containing compounds as additives, and, on this basis, a series of quantitative structure–activity relationship (QSAR) models are developed from various physicochemical parameters characterising structural variation over the additives. The resulting models exhibit both good stability and predictive power, from which five most important properties that highlight structural basis and reaction mechanism underlying the transesterification are extracted, revealing that the topological property and electrostatic profile of additives exert a significant effect on reaction yield; the charge distribution around additive molecules is the most significant factor controlling reaction yield, and then the topological structure. Furthermore, it is inferred that the additive imidazole might constitute the catalytic triad of Ser, Glu or Asp involved in PGA active site, which appears similar to lipase, rendering PGA with the catalytic ability of transesterfication. The study highlights the potential application of QSAR methodology in the field of enzymatic regulator design.     

Studies on the substrate specificity of the T 4 excision repair endonuclease

Previous studies have shown that the v gene of bacteriophage T4 codes for an endonuclease that specifically attacks pyrimidine dimer sites in UV-irradiated DNA. The present studies have examined the role of this endonuclease in the repair of DNA damaged by nitrogen mustard, N-methyl-N′-nitro-N-nitrosoguanidine (NTG), mitomycin C and 4-nitroquinoline-N-oxide. The observation by Harm that the v gene product of phage T4 facilitates repair of UV damage to the host DNA of excision-repair defective strains enabled us to test whether it does the same with other cellular DNA lesions. It was shown that infection of UV-irradiated E. coliB_s−1 with UV-inactivated phage T4v+ resulted in rescue of a certain fraction of the host cells. However no v gene mediated repair E. coli B_s−1 was observed following treatment with the chemical agents mentioned. Furthermore, though phage T4v₁ is more sensitive to UV-irradiation than phage T4, there was no observed difference in the sensitivity of these phages to nitrogen mustard or NTG. On the basis of these observations it was concluded that the v gene coded endonuclease of T4 is specific for the excision repair of pyrimidine dimers and does not participate in the repair of chemically damaged DNA. In vitro enzymatic degradation of DNA alkylated with nitrogen mustard was observed, but it is probable that this degradation is not part of a repair reaction in vivo.     

Serum trace elements status of rabbits supplemented with Nigella sativa,vitamins C and E,and selenium against damage by N-methyl-N'-nitro-N-nitrosoguanidine

In this study, we investigated the effects of Nigella sativa, vitamins C and E, and selenium on the levels of trace elements in the serum of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-injected rabbits. The rabbits were separated into one control and three experimental groups, each consisting of eight rabbits. MNNG was administered to all rabbits at a dose of 20 mg/kg. Group A received a suspension of N. sativa, group B received a combination of vitamins C and E and selenium, and group C received MNNG without any additional treatment. Group D did not receive any treatment and acted as control. The concentrations of serum zinc, copper, and iron were determined for groups A, B, C, and D. The zinc levels were 155.3±25.8, 304.7±14.22, 117.2±27.9, and 87.0±8 μ/dL for groups A-D, respectively; copper was measured at 234.8±31.9, 214.3±14.2, 196.5±19.3, and 359.2±19.9 μ/dL and iron levels were 276.3±10.71, 260.8±7.15, 211.2±13.47, and 223.4±9.5 μ/dL, in the stated group order. There were statistically significant differences between groups (p<0.05). The results obtained in this work may be of use for monitoring and preventing the nocive effects of N-methyl-N′-nitro-N-nitrosoguanidine and similar carcinogens.     

高产青霉素酰化酶巨大芽胞杆菌的诱变选育及产酶条件优化

【目的】选育高产青霉素G酰化酶(PGA)工业菌株。【方法】采用LiCl-紫外线复合诱变以及常压室温等离子体(ARTP)诱变技术对巨大芽胞杆菌(Bacillus megaterium) ATCC 14945进行处理。处理菌体涂平板后,将长出的菌落接种到液体培养基中,向培养6 h后的二代菌液中添加终浓度为0.1%的苯乙酸,28 °C、250 r/min条件下诱导培养40 h。对离心后获得的上清(粗酶液)采用NIPAB法测定PGA酶活力。以PGA酶活力最高的菌株为材料,对苯乙酸最佳添加量和最佳诱导时间进行优化,采用NIPAB法测定PGA酶活力。采用SDS-PAGE检测诱变前后巨大芽胞杆菌粗酶液中PGA的蛋白特性。【结果】从诱变菌落中筛选到PGA酶活力为39.60 U/mL的菌株12-4,酶活力比出发菌株提高了8.5倍。该菌株在液体培养6 h后添加终浓度为0.2%的苯乙酸,继续培养50 h后,PGA酶活力可达78.45 U/mL,比出发菌株提高了16.8倍。诱变前后菌株培养液中的PGA蛋白均具α、β亚基;诱变后菌株PGA α亚基的量没有明显变化,β亚基的量明显增多;α、β亚基之间的蛋白条带明显增多。【结论】采用诱变技术可提高巨大芽胞杆菌PGA活性,获得的诱变菌株12-4及培养条件对PGA工业化生产具有重要价值。     

Improvement of Kefir yeast by mutation with N-methyl-N- nitrosoguanidine

A commercial culture of Kefir grew well at 30°C and 37°C with NaCl up to 4% (w/v) but at 42°C and 4% (w/v) NaCl it grew very slowly. These conditions were selected for the mutation with N-methyl-N-nitrosoguanidine in order to improve cell yield at high temperatures and salt concentration. The mutated cells had higher biomass and growth rate compared with the original culture. The improvement of Kefir at high temperatures and salt concentrations offers advantages for uses in single cell protein production and alcohol production from whey containing high salt.     

Isolation of unselected mutants of alkaline phosphatase in Escherichia coli through nitrosoguanidine comutation and comparison with natural variants

We have devised a general procedure to isolate enzymatic variants without selecting or screening for related phenotypic peculiarities of the organism. A high mutation rate at phoA, the structural gene for alkaline phosphatase, is found among N-methyl-N'-nitro-N-nitrosoguanidine-induced proC revertants of Escherichia coli. About 1.6% of such revertants lack alkaline phosphatase, and many others exhibit altered enzyme parameters. Three mutants studied in detail had full enzyme activity but differed from the wild type in electrophoretic mobility, thermostability, and, in one case, optimum pH for enzyme activity. Four other phosphatase variants were discovered in a survey of 50 natural E. coli isolates; their electrophoretic mobility and thermostability were different from those of the wild type. Natural and induced enzyme variants are similar enough to suggest the absence of strong selective pressures in natural populations.This work was supported by grants from the Fundación J. March and the Comisión Asesora para la Investigación Científica y Técnica.     

Separation of penicillin G from phenylacetic acid in a supported liquid membrane system

The separation of penicillin G (Pen G) from phenylacetic acid (PAA) by use of a supported liquid membrane (SLM) system with Amberlite LA-2 dissolved in 1-decanol, supported on a microporous polypropylene membrane, was studied. The results show that the individual permeability of each component in mixture was lower than that in a single compartment system and, it suggests a strong transport competition between Pen G and PAA. The SLM system in this study proved to be a promising process for the selective separation of Pen G from PAA. The maximum separation factor was found to be 1.8 under a liquid membrane resistance controlled mechanism. (c) 1994 John Wiley & Sons, Inc.     

Neoplastic transformation and induction of H+,K+-adenosine triphosphatase by N-methyl-N′-nitro-N-nitrosoguanidine in the gastric epithelial RGM-1 cell line

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces gastric cancer in animal models. We established an MNNG-induced mutant of the rat murine RGM-1 gastric epithelial cell line, which we named RGK-1, that could be used as an in vitro model of gastric cancer. This cell line showed signs of neoplasia and transformation, in that it lost contact inhibition and formed tumors in nude mice. The mutant cells also expressed parietal cell-specific H⁺,K⁺-adenosine triphosphatase (H⁺,K⁺-ATPase), which parent RGM-1 did not. The results suggested that parent RGM-1 cells were gastric progenitor cells. This mutant RGK-1 cell line will contribute to future investigation on gastric carcinogenesis and to the development of other pathophysiologic fields.     

Extension of the pre-DNA synthetic phase of the cell cycle as a consequence of DNA alkylation in Chinese hamster cells: a possible mechanism of DNA repair

1.

1.|Alkylation of the DNA of hamster cells with a variety of agents leads to a dose-dependent delay in the onset of DNA synthesis.