ANALYSIS OF PLASMID STABILITY BY THE DIFFERENCE IN COLONY SIZE

Due to the reduced growth rate, plasmid-haboring cells give smaller colonies on agar plates than their plasmid-free counterparts. Based on the difference in colony size, a new method to measure plasmid stability is proposed. The new method is flexible, fast, and agrees well with the replica plating method

EFFECT OF OXYGEN ON ETHANOL FERMENTATION IN PACKED-BED TAPERED-COLUMN REACTOR

In ethanol production with immobilized yeast a major problem is the provision of nutrients to these highly concentrated cells. O2 being one of the nutrients of utmost importance to yeast cells, was fed into a column packed with beads with a cell loading of more than 40 g/l. Since addition of large volume of air or O2 to a cylindrical column reactor would aggravate the problems of pressure build up and channelling caused by the evolving CO2 gas, a tapered-column reactor and pulsed flow of oxygen gas was used. The supplement of O2 gas to the tapered column increased the productivity from 21.1 g ethanol x (l gel x h)-1 to 26.7 g x (l gel x h)-1, when the ethanol concentration at the outlet was about 80 g/l. The yield coefficient of ethanol was also increased from 0.41 g ethanol/g glucose to 0.43 after O2 supplement was started. The effects of frequency and duration of O2 supplement were also determined

PRODUCTION OF L(+)-LACTIC ACID USING IMMOBILIZED RHIZOPUS-ORYZAE - REACTOR PERFORMANCE-BASED ON KINETIC-MODEL AND SIMULATION

The production of L(+)-lactic acid using alginate immobilized Rhizopus oryzae in tapered-column fluidized-bed batch reactor was tested and simulated using the kinetic data taken independently in shake-flask cultures. The data show saturation kinetics with substrate and product inhibitions in linear form. Analysis of the kinetic data gave kinetic constants: V-m, 11.04 g lactic acid/(L-bead. h); K-m, 20.9 g glucose/L; and K-i, 365 g glucose/L for lactic acid production. The product inhibition constant, K-p, was found to be 316 g lactic acid/L. The simulation results showed a good agreement with the experimental results when the initial lag phase was taken into account in the simulation model. Without the adjustment for the initial lag period, the kinetic model showed higher conversion. Starting with a glucose concentration of 150 g/L, it was possible to produce 73 g/L of L(+)-lactic acid in 44.5 h. The lactic acid yield was 64.8% by weight based on the amount of glucose consumed

L-Pyroglutamate spontaneously formed from L-glutamate inhibits growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of Sulfolobus solfataricus in medium containing L-glutamate, we observed formation Of L-pyroglutamic acid (PGA). PGA formed spontaneously from L-glutamate under culture conditions (78 degreesC and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of S. solfataricus growth. The cell growth rate was reduced by, one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition Of L-glutamate or L-aspartate to the medium. PGA was also produced from the L-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78 degreesC and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues Of L-glutamate, such as L-methionine sulfoxide, glutaric acid, succinic acid, and L-glutamic acid gamma -methyl ester, also inhibited the growth of S. solfataricus, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues, N-acetyl-L-glutamate enhanced the growth of S. solfataricus. This compound was stable under cell culture conditions, and replacement of L-glutamate with N-acetyl-L-glutamate in the medium resulted in increased cell density.open1119sciescopu

Penicillin acylase-catalyzed synthesis of cefazolin in water-solvent mixtures: enhancement effect of ethyl acetate and carbon tetrachloride on the synthetic yield

The effects of organic solvents on the penicillin acylase-catalyzed, kinetically controlled synthesis of cefazolin have been examined in various water-solvent mixtures. In the presence of water-miscible solvents, the initial rate and maximum yield of cefazolin (CEZ) synthesis reaction were found to be reduced. The extent of inhibition was increased with increasing hydrophobicity of the solvent in the reaction mixtures. Enzymatic synthesis of cefazolin was also carried out in the water-solvent biphasic systems. Among the water-immiscible solvents tested, ethyl acetate (EtOAc) and carbon tetrachloride (CCl4) were found to markedly improve the yield of cefazolin in the two-phase reaction system. Our study showed that the enhancement effect of EtOAc and CCl4 on the synthetic yield was mainly caused by a reduction of the hydrolysis of acyl donor and product in the two-phase system rather than extraction of the product into the solvent phase. (C) 2000 Elsevier Science B.V. All rights reserved.X1123sciescopu

Inhibitory effect of L-pyroglutamate on extremophiles: correlation with growth temperature and pH

L-Pyroglutamate (PGA) is naturally occurring from L-glutamate solution with accelerated formation rate under high temperature and low pH. Even though PGA has been identified as a neurotoxic agent on brain cells, the effect of PGA on the growth of microorganisms is rarely known. Here various kinds of microorganisms differing in their optimal growth temperature, pH, phylogeny, and isolated biotope were investigated for the effect of PGA. We found that growth of thermoacidophiles, including both archaea and bacteria, was seriously inhibited by the presence of PGA, and the extent of the inhibitory effect was closely related with growth temperature and pH. Interestingly, only microbes that grow at high temperature and low pH are inhibited by PGA, while this compound may stimulate growth rates of organisms that live at neutral pH and low temperature. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.X113sciescopu

DETERMINATION OF KINETIC-PARAMETERS RELATED TO THE PLASMID INSTABILITY - FOR THE RECOMBINANT FERMENTATION UNDER REPRESSED CONDITION

The plasmid stability under the repressed state of cloned gene was studied theoretically as well as experimentally using recombinant E. coli K12-DELTA-H1-DELTA-trp/pPLc23trpA1 as a "host-vector" model system. The important kinetic parameters studied were the plasmid loss rate (Kelvin) describing the rate at which the plasmid-harboring cells lose plasmids and the plasmid-free cells are generated per unit time and the difference in growth rates (DELTA) between the two genotypes. These parameters were carefully defined, studied, and compared with other key kinetic parameters involved in the recombinant fermentation to further our understanding of metabolism of recombinants. The ratio of the concentration of plasmid-free cells to plasmid-harboring cells (OMEGA) was introduced, and the mathematical model was derived and used for the determination of the kinetic parameters associated with plasmid instability. These methods developed based on the theoretical considerations were tested experimentally. The results of these methods were compared, and the best method was selected and recommended. The effect of temperature and dilution rate on kinetic parameters Kelvin and DELTA were also studied in continuous culture, in order to provide some practical information related to the operation and control of recombinant fermentation processes.X1125sciescopu

Disruption of PMR1, encoding a Ca2+-ATPase homolog in Yarrowia lipolytica, affects secretion and processing of homologous and heterologous proteins

The Yarrowia lipolytica PMR1 gene (YlPMR1) is a Saccharomyces cerevisiae PMR1 homolog which encodes a putative secretory pathway Ca2+-ATPase. In this study, we investigated the effects of a YlPMR1 disruption on the processing and secretion of native and foreign proteins in Y. lipolytica and found variable responses by the YlPMR1-disrupted mutant depending on the protein. The secretion of 32-kDa mature alkaline extracellular protease (AEP) was dramatically decreased, and incompletely processed precursors were observed in the YlPMR1-disrupted mutant. A 36- and a 52-kDa premature AEP were secreted, and an intracellular 52-kDa premature AEP was also detected. The acid extracellular protease activity of the YlPMR1-disrupted mutant was increased by 60% compared to that of the wild-type strain. The inhibitory effect of mutations in secretory pathway Ca2+-ATPase genes on the secretion of rice alpha-amylase was also observed in the Y. lipolytica and S. cerevisiae PMR1-disrupted mutants. Unlike rice alpha-amylase, the secretion of Trichoderma reesei endoglucanase I (EGI) was not influenced by the YlPMR1 disruption. However, the secreted EGI from the YlPMR1-disrupted mutant had different characteristics than that of the control. While wild-type cells secreted the hyperglycosylated form of EGI, hyperglycosylation was completely absent in the YlPMR1-disrupted mutant. Our results indicate that the effects of the YlPMR1 disruption as manifested by the phenotypic response depend on the characteristics of the reporter protein in the recombinant yeast strain evaluated.open1117sciescopu