ISSN:0006-3592    

DOI:10.1002/bit.260410102

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

年代:1993

数据来源: WILEY

摘要:

AbstractExpression of recombinant proteins inEscherichia colioften results in the formation of insoluble inclusion bodies, In case of expression of eukaryotic proteins containing cysteine, which may form disulfide bonds in the native active protein, often nonnative inter‐ and intramolecular disulfide bonds exist in the inclusion bodies. Hence, several methods have been developed to isolate recombinant eukaryotic polypeptides from inclusion bodies, and to generate native disulfide bonds, to get active proteins. This article summarizes the different steps and methods of isolation and renaturation of eukaryotic proteins containing disulfide bonds, which have been expressed inE. colias inclusion bodies, and shows which methods originally developed for studying the folding mechanism of naturally occurring proteins have been successfully adapted for reactivation of recombinant eukaryotic proteins. © 1993 John Wiley&Sons, I

ISSN:0006-3592    

DOI:10.1002/bit.260410103

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

年代:1993

数据来源: WILEY

摘要:

AbstractPenicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics.Escherichia coliATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, we constructed various recombinantE. coliHB101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic and (PAA) in the medium. TheE. colistrain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: anE. coliATCC 11105 mutant (E. coliG133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity ofE. coliG271. The host strain,E. coliG133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) intranson the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production byE. coliG133, PGA production by its transformant (E. coliG271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation ofE. coliG133 with a pBR322 derivative containing theHindlllfragment of the PGA gene, showed that the performance ofE. coliG271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficientE. coliHB101 derivatives brought some light on the mechanism of regulation of the PGA gene. © 1993 John Wiley&Sons, Inc

ISSN:0006-3592    

DOI:10.1002/bit.260410104

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

年代:1993

数据来源: WILEY

摘要:

AbstractA packed bed bioreactor was investigated as means for the cultivation of mammalian cells. The packed bed is comprised of porous ceramic particles with pores sufficiently large for cell immobilization as well as for intraparticle convective flow. In this way, the transport of limiting nutrients such as oxygen can be significantly enhanced, allowing maintenance of cell viability and productivity in an environment protective of adverse shear effects. The extent of intraparticle convective medium flow was experimentally quantified relative to the reactor operating conditions, and was found to be the dominant mechanism of nutrient transport to cells immobilized in the particle interior. An approximate linear relationship was obtained between overall reactor productivity and the extent of intraparticle convection. As the latter can be controlled at the single‐particle level through total flow rate control, this relationship is a useful scale‐up tool for the design of bioreactors. The high cell densities and the high volumetric productivities achieved by using small lab‐scale reactors underline the potential of this simple bioreactor configuration for large‐scale cell culture applications. © 1993 John Wiley&S

ISSN:0006-3592    

DOI:10.1002/bit.260410105

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

年代:1993

数据来源: WILEY

摘要:

AbstractThis article deals with the automation of the process of somatic embryogenesis for the propagation of plants. An important problem is the monitoring of the embryo production process in order to decide the time to start harvesting embryos for further processing. The classification algorithm development for somatic embryos of birch (Betula pendulaRoth) showed that automated recognition of embryos at different developmental stages is possible. No globular stage embryos were classified to be heart or torpedo stage and no heart or torpedo stage embryos were classified to be at globular stage. Heart and torpedo stage embryos were classified into three developmental classes by a new index that describes the relation of embryo breadth to the length of the root. The probability of classifying a nonembryo as an embryo was less than 1%, and 14% of the object classified as embryos by a human expert were discarded by the algorithm. A computer vision system suitable for automated monitoring of samples from the bioreactor was constructed. © 1993 John Wiley&Sons, Inc

ISSN:0006-3592    

DOI:10.1002/bit.260410106

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

年代:1993

数据来源: WILEY

摘要:

AbstractInvariance of culture conditions in steady state continuous cultures make these a very valuable tool to study the influence of various culture parameters on cell growth and synthesis of primary and secondary metabolites. The result of a parametric study on production of protease in continuous suspension cultures ofBacillus firmusNRS 783 are reported in this article. This strain is a superior producer of an alkaline protease with major application in the detergent industry. The parameters investigated include dilution rate and concentrations of yeast extract, ammonium, and inorganic phosphate in the bioreactor feed, glucose being the principal carbon source in all experiments. The regulatory effects of the key culture parameters on cell growth, synthesis and secretion of protease, and production of acetic acid are investigated. The relations among the specific cell growth rate, specific utilization rates of the principal carbon, nitrogen, and phosphorous sources, and specific production rates of two nonbiomass products, viz., acetic acid and protease, are examined, and the effects of the manipulated culture parameters on these relations, specific protease activity, and yields of cell mass, protease, and acetic acid on the basis of the principal carbon, nitrogen, and phosphorous sources are studied. An increase in dilution rate led to increases in specific utilization rates of the principal carbon, nitrogen, and phosphorous sources and specific production rates of acetic acid and protease and decreases in bulk activities/concentrations of the three products (acetic acid, cell mass, and protease). As a result, the productivities of the three species were maximized at an intermediate dilution rate. Increased supply of yeast extract (a rich source of amino acids, proteins, and vitamins, besides being an additional source of carbon, nitrogen, and phosphorus) promoted cell mass formation but reduced protease production per unit cell mass. Increased supply of nitrogen and phosphorous sources stimulated protease synthesis up to certain threshold levels and repressed the enzyme synthesis beyond the threshold levels. With increased supply of the nitrogen source, the phosphorous source was more efficiently utilized for cell growth and protease synthesis. Stable maintenance of continuous cultures ofB. firmusover prolonged period is demonstrated in this study. © 1993 John Wiley&Sons, Inc

ISSN:0006-3592    

DOI:10.1002/bit.260410107

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

年代:1993

数据来源: WILEY

摘要:

AbstractA model was formulated to examine the competitive growth of two phenotypes (Leu+and Leu−) and the product formation with recombinantSaccharomyces cerevisiaestrain DBY‐745, which contains the shuttle vector pYGH3‐16‐s with the foreign gene HBsAg (hepatitis B virus surface antigen) as well as experimental fedbatch fermentation data. The important state variables and the process parameters evaluated include (1) the ratio of the plasmid‐free cell concentration to the plasmid‐containing cell concentration (ρ =X−X+), (2) the expression of human hepatitis B surface antigeng(CH), (3) the glucose consumption (S), (4) the ethanol production (/), (5) the change of working volume (V) in the fermentor, (6) the different specific growth rates of two phenotype cells, and (7) the plasmid loss frequency coefficient (α ). These variables and other parameters were carefully defined, their correlations were studied, and a mathematical model using a set of nonlinear ordinary differential equations (ODEs) for fed‐batch fermentation was then obtained based on the theoretical considerations and the experimental results. The extended Kalman filter (EKF) methods was applied for the best estimate of these variables based on the experimentally observable variables: ρV, andg(CH). Each of these variable was affected by random measuring errors under the different operating conditions. Simulation results presented for verification of the model agreed with our observations and provided useful information relevant to the operation and the control of the fedbatch recombinant yeast fermentation. The method of predicting an optimal profile of the cell growth was also demonstrated under the different dissolved oxygen concentrations. © 1993 J

ISSN:0006-3592    

DOI:10.1002/bit.260410108

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

年代:1993

数据来源: WILEY

摘要:

AbstractEvolutionary operation (EVOP) is used as an efficient technique for optimization of three variable experimental parameters using two‐level factorial designs with center points. For metabolic function like enzyme synthesis by microorganisms, small amounts of metal ions, surfactants, and vitamins act as inducers. The desired quantities of these chemicals are, however, species dependent and have to be found out or each microorganism. By employing the EVOP technique, the requirements of these chemicals have been optimized for the production of protease byRhizopus oryzae(RO, IIT KGP). © 1993 John Wiley&Sons, I

ISSN:0006-3592    

DOI:10.1002/bit.260410109

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

年代:1993

数据来源: WILEY

摘要:

AbstractA novel technique which intensifies conventional aqueous two‐phase extraction by conversion of dispersed phase into colloidal gas aphrons (CGAs) has been developed for extraction of an enzyme. In the present work, amyloglucosidase (1,4‐α‐D‐glucan glucohydrolase) was extracted using a polyethylene glycol‐sodium sulfate‐water system. The lighter phase, i.e., polyethylene glycol (PEG) rich phase, was converted into CGAs which were then dispersed into a salt rich phase. The effect of type of surfactant and its concentration, dispersed phase velocity, phase composition, and type of sparger on the dispersed phase mass transfer coefficient was investigated. The results suggests 9–16 times higher values of mass transfer coefficient compared to spray column. The multiorifice sparger at concentrations of 0.33 g/L of cetyl trimethyl ammonium chloride yielded best results. © 1993 John

ISSN:0006-3592    

DOI:10.1002/bit.260410110

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

年代:1993

数据来源: WILEY

摘要:

AbstractBiodegradation of nitroglycerin (NG), an inhibitory, nongrowth substrate present in a multicomponent munition wastewater, was investigated in a pilot‐scale batch reactor operated with both aerobic and anoxic cycles. A mixed culture was initially acclimated by gradual introduction of NG into influent and subsequently exposed to actual NG‐laden production wastewater. System performance revealed that NG was amenable to aerobic biodegradation without adverse impact on removal efficiencies of other pollutants. Temporal NG concentration profiles indicated that an influent concentration of approximately 200 mg/L of NG was reduced to below detection limits in less than 5 h of aeration with no appreciable (<4%) biosorption. Failure of NG‐acclimated cultures to utilize NG as a sole carbon source in bench‐scale reactors suggested that NG behaved as a non‐growth substrate and its degradation possibly occurred by cometabolism. Ethyl acetate present in the waste stream was an adequate growth substrate in terms of both biological and physicochemical properties. High concentrations of NO3‐N, produced as a result of aerobic degradation of NG and other nitrogenous compounds of the waste, were treated in an anoxic phase. Approximately 95 mg/L of NO3‐N was denitrified to below detection limits in 5 h of anoxia without the addition of external carbon sources. Two SRB cycle schemes with different static‐fill times exhibited significant differences in treatment efficiencies. © 1993 John

ISSN:0006-3592    

DOI:10.1002/bit.260410111

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

年代:1993

数据来源: WILEY