摘要:

AbstractPhotosynthetic reaction centers, isolated and purified from the facultative phototrophic bacteriumChloroflexus aurantiacus, were immobilized in optically transparent lipidic cubic phases composed of 42% (w/w) 1‐palmitoyl‐2‐hydroxy‐sn‐glycero‐3‐phosphocholine and 58% (w/w) water. The immobilized photosynthetic protein retains its native properties, as indicated by visible and circular dichroic spectra. The ground state visible spectrum of the immobilized reaction centers is very similar to the corresponding spectrum in aqueous solution, indicating that the protein pigments are not extracted into the lipidic regions of the cubic phase. The secondary structure of the protein is maintained in the immobilized state, as determined by far‐UV circular dichroism spectroscopy in the 200‐ to 250‐nm range. Moreover, immobilized reaction centers retain their photochemical activity: a reversible photo‐oxidation of the primary electron donor (P) is seen upon continuous illumination. Furthermore, the entrappment of reaction centers does not affect the kinetics of charge recombination between the photo‐oxidized primary donor (P+) and the photoreduced primary quinone acceptor, generated by a short flash of light. Reaction centers devoided of the secondary quinone acceptor can be easily reconstituted in cubic phases by means of their coimmobilization with 1,4‐naphtoquinone. Indeed, the kinetics for charge recombination in reconstituted reaction centers is dramatically slower than the corresponding kinetics in the unreconstituted protein. Interestingly, immobilized reaction centers are significantly stabilized as compared with reaction centers in aqueous solution: the integrity of the protein in the cubic phase is maintained for at least 5 months, whereas in water solution 50% of the activity is lost within 2 months. ©

ISSN:0006-3592    

DOI:10.1002/bit.260460202

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

年代:1995

数据来源: WILEY

摘要:

AbstractIn this article, the extraction of cytochrome c utilizing various nonionic surfactant microemulsions has been tested to determine the effect of surfactant structure on protein partitioning. Surfactants tested include a linear alcohol ethoxylate (Neodol 91–2.5), two alkyl phenol ethoxylates (lgepal CO‐520, Trycol 6985), and a series of alkyl sorbitan esters that are either ethoxylated (Tweens) or un‐ethoxylated (Spans). Initial attempts to extract hemoglobin into Neodol 91–2.5 Winsor II microemulsions (oil‐continuous) appeared successful based on heme estimation. Careful analysis showed that the hemoglobin had dissociated prior to extraction and that only the heme was extracted with false positive results. In fact, Neodol 91–2.5 microemulsions were unable to extract a variety of proteins with differing biophysical properties. Among all the other nonionic surfactant microemulsions tested only those made using sorbitan esters extracted significant amounts of cytochrome c. The partition coefficients achieved in this study are more than an order of magnitude higher than that seen previously in the literature for comparable sorbitan systems. However, this partition coefficient is extremely sensitive to ionic strength. At an ionic strength as low as 0.001M, the partition coefficient is reduced to that seen in previous studies. We have found that protein partitioning in sorbitan ester microemulsions is not a function of water content. In addition, extraction is not a function of either alkyl chain length, or polyethylene oxide molecular weight. Hence, the sorbitan group appears to have an important role in extraction, possibly through a weak electrostatic protein–surfactant interaction. © 1995 John W

ISSN:0006-3592    

DOI:10.1002/bit.260460203

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

年代:1995

数据来源: WILEY

摘要:

AbstractAlkyl β‐D‐glucosides were synthesized fromD‐glucose and alcohols by reverse hydrolysis using the commercially available almond β‐D‐glucosidase in 9:1 (v/v) acetonitrile–water medium. The main characteristics of this enzyme‐catalyzed glucosylation were established by using 2‐hydroxybenzyl alcohol. The reaction is entirely regio‐ and stereoselective. The solvent plays a fundamental role because, by decreasing the water concentration in the medium, the shift of the reaction equilibrium toward synthesis is realized without using an excessive amount of alcohol. Nevertheless, a minimum amount of water is necessary to maintain the enzyme activity. In contrast to the use of the enzyme in aqueous medium, the pH of the added water in acetonitrile did not influence the synthesis. Using this procedure, we have conducted systematic glucosylation of numerous alcohols and we have investigated enzyme specificity and alcohol reactivity. The enzyme has a pronounced affinity for the alcohols containing a phenyl group, and enantioselectivity for the aglycon is obtained with 1‐phenylethyl alcohol. Moreover, by using almond β‐D‐glucosidase it was also possible to synthesize alkyl β‐D‐galactosid

ISSN:0006-3592    

DOI:10.1002/bit.260460204

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

年代:1995

数据来源: WILEY

摘要:

AbstractBased on a review of thePenicillium chrysogenumbiochemistry a stoichiometric model has been set up. The model considers 61 internal fluxes and there are 49 intracellular metabolites which are assumed to be in pseudo–steady state. In addition to the intracellular fluxes the model considers the uptake of 21 amino acids. From the stoichiometric model the maximum theoretical yield of penicillin V is calculated to 0.43 mol/mol glucose. If biosynthesis of cysteine is by direct sulfhydrylation rather than by transsulfuration, the maximum theoretical yield is about 20% higher, i.e., 0.50 mol/mol glucose. The theoretical yield decreases substantially if α‐aminoadipate is converted to 6‐oxo‐piperidine‐2‐carboxylic acid (OPC). If only 40% of the α‐aminoadipate is recycled, the maximum theoretical yield is 0.31 mol/mol glucose. The uptake rates of glucose, lactate, γ‐aminobutyrate, and 21 amino acids were measured during fed‐batch cultivations. The rates of formation of penicillin V, δ‐(L‐α)‐aminoadipyl‐L‐cysteinyl‐D‐valine (ACV), OPC, and the pool of isopenicillin N, 6‐APA, and 8‐HPA were also measured. Finally the synthesis rates of the biomass constituents RNA/DNA, protein, lipid, carbohydrate, and amino carbohydrate were measured. From these measured rates and the stoichiometric model the metabolic fluxes through the different intracellular pathways are calculated. The calculations show that penicillin formation is accompanied by a large flux through the pentose phosphate (PP) pathway due to a large requirement for nicotinamide‐adenine dinucleotide phosphate (NADPH) used in the biosynthesis of cysteine. If cysteine is added to the medium, the flux through the PP pathway decreases. From the stoichiometric modelYxATPis calculated to 87 mmol adenosine triphosphate (ATP)/g dry weight (DW), and from the flux calculationsmATPis found to 3 mm

ISSN:0006-3592    

DOI:10.1002/bit.260460205

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

年代:1995

数据来源: WILEY

摘要:

AbstractA prototype disc stack centrifuge was tested for the separation of mammalian cell cultures from 80‐ and 2000‐L fermentations. The clarification capacity for mammalian cells was excellent, but some smaller particles remained in the supernatant and reduced its usefulness for downstream processing. In order to identify the source of such particle formation, several parameters were assessed and minimum particle size for separation was calculated. An analysis of particle distribution was performed. Temperature and pressure effects inside the centrifuge bowl were measured. Some modifications of mechanical engineering can be suggested for the improvement of the use of standard disc stack centrifuges for mammalian cells. © 1995 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260460206

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

年代:1995

数据来源: WILEY

摘要:

AbstractA fully predictive mathematical description of a three‐phase, tapered, fluidized‐bed bioreactor is developed. This mathematical model includes the effects of the tapered bed, variable dispersion coefficient, and variable solid holdup upon the concentration profiles developed in the bed. In addition, the effect of the concentration profile which is developed inside the biocatalyst bead is included by means of an effectiveness factor calculation. Using accepted correlations for the dispersion coefficient and for the liquid, gas, and solid holdup in the bed, the model is fully predictive. The model was found to adequately predict experimental obtained concentration profiles. Then, the model was used to examine the various phase holdups through the bed and the degree to which the dispersion coefficient varied through the bed. The effect of changes in these calculated variables upon the reaction rate is discussed. © 1995 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260460207

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon‐γ (IFN‐γ), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN‐γ. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN‐γ within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN‐γ are heterogeneous in their environment, with variable access to O2and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell–cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell–cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. © 1995 J

ISSN:0006-3592    

DOI:10.1002/bit.260460208

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

年代:1995

数据来源: WILEY

摘要:

AbstractBiological reduction of nitrate and nitrite was studied with a continuously operated cyclic reactor. The medium was fed to the reactor during the first phase of the cycle, and the effluent was drawn from the reactor during the third phase of the cycle; reaction occurred throughout the cycle. The process was described mathematically based on kinetic expressions revealed in an independent study. The model equations were subjected to detailed analysis with numerical codes based on the bifurcation theory for forced systems. The analysis has shown that in the operating parameter space there are extensive regions where the system can reach up to three different periodic states. The results of this analysis are shown in the form of two‐dimensional operating diagrams. Numerical results have also shown that under certain operating conditions nitrate can be completely eliminated, while nitrite remains practically untreated. An experimental unit was designed, constructed, and used in experiments with a strain ofPseudomonas denitrificans[American Type Culture Collection (ATCC) 13867] under different operating conditions. The experimental results confirmed the theoretical predictions both qualitatively and quantitatively. Conditions under which complete reduction of both nitrate and nitrite is achieved, were found and experimentally verified. The results of this study suggest a methodology for analysis and design of cyclically operated bioreactors employed in denitrification of wastewaters. © 1995 John Wiley&Sons, I

ISSN:0006-3592    

DOI:10.1002/bit.260460209

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

年代:1995

数据来源: WILEY

摘要:

AbstractBy using trypsin as the model protein and AOT as the model surfactant, the effect of a variety of solvents on protein transfer and activity recovery during the liquid–liquid reversed micellar extraction was investigated. It was found that several solvents, including isooctane, octane, heptane, and kerosene, had a similar effect on the recovery of trypsin activity after a full cycle of forward and backward extraction, and could all be used as the solvents for AOT‐reversed micelles in trypsin extraction. Two other solvents (hexane and cyclohexane), however, were not so efficient. © 1995 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260460210

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe catalytic properties of bovine liver catalase have been investigated in organic solvents. In tetrahydrofuran, dioxane, and acetone (all containing 1% to 3% of water), the enzyme breaks downtert‐butyl hydroperoxide several fold faster than in pure water. Furthermore, the rate of catalase‐catalyzed production oftert‐butanol fromtert‐butyl hydroperoxide increases more than 400‐fold upon transition from aqueous buffer to ethanol as the reaction medium. The mechanistic rationale for this striking effect is that in aqueous buffer the rate‐limiting step of the enzymatic process involves the reduction of catalase's compound I bytert‐butyl hydroperoxide. In ethanol, and additional step in the reaction scheme becomes available in which ethanol, greatly outcompeting the hydroperoxide, is oxidized by compound I regenerating the free enzyme. In solvents, such as acetonitrile or tetrahydrofuran, which themselves are not oxidizable by compound I, catalase catalyzes the oxidation of numerous primary and secondary alcohols withtert‐butyl hydroperoxide to the corresponding aldehydes or ketones. The enzymatic oxidation of some chiral alcohols (2,3‐butanediol, citronellol, and menthol) under these conditions occurs enantioselectively. Examination of the enantioselectivity for the oxidation of 2,3‐butanediol in a series of organic solvents reveals a considerable solvent dependence. © 1995

ISSN:0006-3592    

DOI:10.1002/bit.260460211

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

年代:1995

数据来源: WILEY