摘要:

AbstractThe potential of bacteriophage λ as an expression vector for a large scale production of cloned‐gene proteins was evaluated in batch and continuous bioreactors using a temperature‐sensitive mutant in theclgene, which allows a simple manipulation of temperature as a means to control the phage in the lysogenic or lytic state. A temperature switch from 32°C (or below) to 38°C (or above) forces the phage to go from the lysogenic state to the lytic state. Temperature cycling and a two‐reactor system were used for continuous cultures. For the latter the first reactor is maintained in the lysogenic state at a lower temperature to stably maintain the foreign DNA in the host cell, while the second reactor is maintained in the lytic state to force replication of the cloned‐gene and overproduction of its products. The results are promising but suggest a greater potential for a mutant which lacks theQgene which is responsible for host cell lysis and packaging of phage

ISSN:0006-3592    

DOI:10.1002/bit.260370402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe oxidation of cinnamyl alcohol to cinnamaldehyde by horse liver alcohol dehydrogenase (LADH) was carried out in nearly anhydrous organic solvents and in solvents containing from 0.1 to 10% added water. In nearly anhydrous solvents containing less than 0.02% water, the oxidation rate increased as the water solubility in the solvent decreased, but the reaction did not require active LADH. Moreover, the highest activity in nearly anhydrous heptane was obtained by lyophilizing the enzyme from a solution of pH 2.0, even though LADH exhibits virtually no enzymatic activity in water at this pH. The catalytic activity of LADH was restored and increased dramatically as small amounts of water were added to each solvent. In conjunction with the activity measurements, electron paramagnetic resonance (EPR) spectroscopy and two active‐site directed spin labels were used to examine solvent‐dependent structural features of LADH. The EPR spectra indicated that LADH became more rigid as the dielectric constant of the solvent decreased. The degree of rigidity also depended on the pH from which the enzyme was lyophilized, indicating that the ionization state of the enzyme can have an important influence on its dynamics in organic solvents. Finally, adding 1% water to organic solvents had no apparent effect on the enzyme's conformation or flexibility near the spin label, even though enzyme activity was an order of magnitude higher when 1% water was pres

ISSN:0006-3592    

DOI:10.1002/bit.260370403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe effects of growth rate on cloned gene product synthesis in recombinantSaccharomyces cerevisiaehave been studied in continuous culture. The plasmid employed contains a yeastGAL10‐CYC1hybrid promoter directing expression of theE. coli lacZgene. β‐Galactosidase production was therefore controlled by the yeast galactose regulatory circuit, and the induction process and its effects were studied at the various dilution rates. At all dilution rates plasmid stability decreased with induction oflacZgene expression. In some instances, two induced “steady states” were observed, the first 10–15 residence times after induction and the second after 40–50 residence times. The second induced steady state was characterized by greater biomass concentration and lower β‐galactosidase specific activity relative to the first induced “steady‐state.” β‐Galactosidase specific activity and biomass concentration increased as dilution rate was reduced, and despite lower flow rate and plasmid stability, overall productivity (activity/L/hr) was substantially higher at low dilution rate. Important factors influencing all of the trends were the glucose and galactose (inducer) concentrations in the vesse

ISSN:0006-3592    

DOI:10.1002/bit.260370404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe effects of plasmid promoter strength and origin of replication on cloned gene expression in recombinantSaccharomyces cerevisiaehave been studied in batch and continuous culture. The plasmids employed contain theEscherichia coli lacZgene under the control of yeast promoters regulated by the galactose regulatory circuit. The synthesis of β‐galactosidase was therefore induced by the addition of galactose. The initial induction transients in batch culture were compared for strains containing plasmids with 2μ andARS1origins. As expected, cloned gene product synthesis was much lower with theARS1plasmid: average β‐galactosidase specific activity was an order of magnitude below that with the 2μ‐based plasmid. This was primarily due to the low plasmid stability of 7.5% when the plasmid origin of replication was theARS1element. The influence of plasmid promoter strength was studied using the yeastGAL1, GAL10, and hybridGAL10‐CYC1promoters. The rate of increase in β‐galactosidase specific activity after induction in batch culture was 3–5 times higher with theGAL1promoter. Growth rate under induced conditions, however, was 15% lower than in the absence oflacZexpression for this promoter system. The influence of plasmid promoter strength on induction behavior and cloned gene expression was also studied in continuous fermentations. Higher β‐galactosidase production and lower biomass concentration and plasmid stability were observed for the strain bearing the plasmid with the strongerGAL1promoter. Despite the decrease in biomass concentration and plasmid stability, overall productivity in continuous culture using the GAL1 promoter was three times that obtained with th

ISSN:0006-3592    

DOI:10.1002/bit.260370405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractA new plasmid construct has been used in conjunction with selective recycle to successfully maintain otherwise unstable plasmid‐bearingE. colicells in a continuous bioreactor and to produce significant amounts of the plasmid‐encoded protein β‐lactamase. The plasmid is constructed so that pilin expression, which leads to bacterial flocculation, is under control of thetacoperon. The plasmid‐bearing cells are induced to flocculate in the separator, whereas cell growth and product synthesis occur in the main fermentation vessel without the inhibiting effects of pilin production. Selective recycle allows for the maintenance of the plasmid‐bearing cells by separating flocculent, plasmid‐bearing cells from nonflocculent, segregant cells in an inclined settler, and recycling only the plasmid‐bearing cells to the reactor. As a result, product expression levels are maintained that are more than ten times the level achieved without selective recycle. All experimental data agree well with theoretic

ISSN:0006-3592    

DOI:10.1002/bit.260370406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractUsing data from the literature, a method is adopted for determining the empirical composition and the unit carbon formula for driedEscherichia coliK‐12 cells by summing the quantities of C, H, O, N, P, and S in each of the major classes of macromolecular substances comprising the cellular biomass. With these data and the molar growth yield of cells on succinic acid, equations are written representing the anabolism and catabolism ofE. coliK‐12 on this quantity of substrate. The enthalpy change accompanying catabolism can be calculated directly using standard enthalpies of formation because there is no term representing cellular substance. The enthalpy change accompanying anabolism is calculated to be very small or zero using microcalorimetric and other data from which the enthalpy of formation of a unit quantity of living cellular substance can be obtained. This indicates that the net enthalpy change accompanying the growth process (anabolism plus catabolism) is the same as that calculated for catabolism alone, in agreement with the same conclusion by several investigators using direct microcalorimetry. The method described here of determining the unit carbon formula and the quantity of ash remaining after cellular combustion is compared to that conventionally used in which cellular P and S is considered either to be negligible or to be a part of the ash. It is concluded that equations representing anabolism and the growth process can be written more accurately using the presently described method, leading to more accurate thermodynamic calculati

ISSN:0006-3592    

DOI:10.1002/bit.260370407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe anaerobic baffled reactor (ABR) contains a granulated, mixed anaerobic culture segregated into compartments. Operation of four reactors under a range of hydraulic retention times showed that this novel reactor design offers highly efficient performance in the conversion of carbon in the feed stream to methane and carbon dioxide. The design parameter varied was the number of compartments. COD removal at 20 h retention time was routinely over 95% in all reactors, with low washout of biomass. Very high specific reaction rates were achievable (although with a loss of efficiency) at low biomass concentrations and high loading rates. In order to optimize volumetric reaction rates, a tradeoff has to be made between high biomass concentration, granule size, and the resulting mass transfer limitations. Formate is shown to be an important intermediate in the process under conditions of high loading.

ISSN:0006-3592    

DOI:10.1002/bit.260370408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractSoybeans (Glycine max) contain an α‐galactosidase that makes up a small fraction of the total protein of the seed. The properties of this enzyme are of interest because of its potential to convert the galactooligosaccharides, stachyose and raffinose, in soybean meal to sugars digestible in the human gastro intestinal tract and thereby increase potential uses of this vegetable protein source in human and animal foods. Study of this enzyme required the isolation of milligram quantities of electrophoretically pure protein from ground soybeans and therefore, scaleup of laboratory procedures by a factor of 300 times. Large scale acid precipitation, ammonium sulfate precipitation, and centrifugal recovery of the precipitated protein allowed α‐galactosidase to be isolated from 45.5 kg soybean meal containing 17.1 kg protein, to obtain an enzyme extract with a specific activity of 90 to 100. A novel combination of strong anion exchange and cation exchange chromatography followed by Concanavalin‐A affinity chromatography with a methyl α‐D mannoside gradient gave α‐galactosidase with an average specific activity of 56,000. Ion exchange chromatography preceding Concanavalin‐A affinity chromatography allowed elimination of a relatively costly melibiose affinity chromatography step (which followed the Concanavalin‐A column In the laboratory procedure) thereby making

ISSN:0006-3592    

DOI:10.1002/bit.260370409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe effect of initial KLa on the batch culture of Catharanthus roseus ID1 in a 12.5‐L bioreactor has been examined. Optimum KLa values were found for both growth and alkaloid accumulation. Different sparger designs were used to alter the KLa values. High KLa values caused increased aggregation of the cultures, depressed biomass yields, and altered patterns of alkaloid accumulatio

ISSN:0006-3592    

DOI:10.1002/bit.260370410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

ISSN:0006-3592    

DOI:10.1002/bit.260370411

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY