摘要:

AbstractThe latex particles composed of poly(styrene/N‐isopropylacrylamide/glycidyl methacrylate) [P(St/NIPAM/GMA)] and poly(styrene/N‐isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] were prepared by emulsifier‐free emulsion polymerization. These latex particles with submicrometer size showed the thermosensitivity originated from the thermo‐sensitive nature of NIPAM. That is, the minimum NaCI concentration for flocculation of these latex particles [critical flocculation concentration (CFC)]decreased significantly with increasing temperature and reached constant values at above the critical temperature [critical flocculation temperature (CFT)]. At a certain NaCl concentration, the thermo‐sensitive latex particles were flocculated by raising temperature, and conversely, the flocculated thermo‐sensitive latex particles were completely dispersed by lowering temperature. Bovine serum albumin (BSA) was covalently immobilized onto the P(St/NIPAM/GMA) and P(St/NIPAM/MMA) latex particles with high efficiency. The BSA‐immobilized P(St/NIPAM/GMA) and P(St/NIPAM/MAA) latex particles (immunomicrospheres) showed the similar dependencies of CFC on temperature to the bare latex particles. These thermo‐sensitive immunomicrospheres were successfully used for the immunoaffinity purification of anti‐BSA antibodies from antiserum. © 1994 Jo

ISSN:0006-3592    

DOI:10.1002/bit.260440102

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

年代:1994

数据来源: WILEY

摘要:

AbstractThis research investigates the effect of solids retention time (SRT) on the acid‐phase anaerobic digestion of primary sludge. A series of experiments were conducted using two continuous‐flow 3‐L units with the following configuration: a completely mixed reactor (CMR) with clarifier and solids recycle and an upflow anaerobic sludge blanket (UASB) reactor. Results show that C2to C5volatile fatty acids (VFA) were the predominant compounds formed. At a constant hydraulic retention time (HRT) of 12 h, variation in SRT from 10 to 20 days resulted in a slight increase in VFA production in both systems, but at a shorter SRT (5 days) a drastic drop in acid production was observed. In addition, the percent distribution of VFA was to some extent affected by the change in SRT. On the other hand, organic matter degradation [measured by the chemical oxygen demand (COD) specific solubilization rate or the percent volatile suspended solids (VSS) reduction] appeared to be independent of SRT, at least in the range investigated. The percent soluble COD in the form of VFA, however, increased steadily with increasing SRT, approaching the 90% level at 20 days. The remaining soluble COD in the effluent from these systems may be mainly attributed to metabolic intermediates and unused soluble substrate. © 1994 John Wiley&Son

ISSN:0006-3592    

DOI:10.1002/bit.260440103

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

年代:1994

数据来源: WILEY

摘要:

AbstractApproaches to increasing the productivity of benzophenanthridine alkaloids in suspension cultures inEscherichia californicawere made in an airlift fermentor under different culture conditions. Elicitation with yeast extract elicitor reduced the time required to obtain a certain amount of alkaloid production. In a two‐phase airlift fermentor with compounded silicone fluid, total alkaloid concentration in silicone fluid was 153.1 mg/L and that in the aqueous cellular phase was 8.2 mg/L at day 21 from inoculation. The large accumulation capacity of silicone fluid made it possible to store correspondingly large amounts of total alkaloid and increased the alkaloid production. Act day 21 from inoculation, the volumetric alkaloid productivity and the netproduction in a two‐phase airlift fermentor were 1.4 and 1.5 times higher than those of normal airlift fermentor operation. This performance was furthermore enhanced by elicitation. Elicitation in two‐phase airlift fermentor operation increased the volumetric productivity and the new production 3.3‐ and 3.5‐fold compared to those of normal airlift fermentor operation. © 1994 John Wiley

ISSN:0006-3592    

DOI:10.1002/bit.260440104

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

年代:1994

数据来源: WILEY

摘要:

AbstractContinuous fermentation with cell recycle proved very effective in increasing the ethanol volumetric productivity of the thermophilic facultative anaerobe,Bacillus stearothermophilusstrain LLD‐15, on sucrose at 70°C. When complete cell recycle was used, cell viability decreased after a few residence times and sucrose consumption was reduced. Operation using a constant bleed rate resulted in greater stability and higher ethanol volumetric productivities. A mathematical model based on maintenance energy requirements provided an adequate description of the system. © 1994 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260440105

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe protein components of three industrial recombinant expression systems:Escherichia coli, Saccharomyces cerevisiae, and a mammalian cell culture supernatant of CHO cells were characterized in terms of their molecular weight, isoelectric point, and relative surface hydrophobicity. Identification of individual proteins was done by reference to their position in protein band profiles by polyacrylamide gel electrophoresis (PAGE) of the crude material. This permitted a rapid and facile assignment of quantitative values for these three parameters to all the major protein components in these materials. Because it is the indigenous proteins in expression systems that will form the bulk of any impurities in the product, once the values of these parameters are known for any target recombinant protein, the data obtained will enable appropriate expression systems to be chosen for minimizing amounts of potential contaminants and reducing downstream processing requirements and costs. The data will also indicate which fractionation steps (i.e., charge, size or hydrophobicity‐based) are likely to be best for distinguishing between target and contaminant proteins, thus aiding and early removal of the maximum quantities of undesired protein to bring subsequent bioseparation steps down in scale and cost and up in terms of efficiency. © 1994 John Wiley&Sons, I

ISSN:0006-3592    

DOI:10.1002/bit.260440106

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

年代:1994

数据来源: WILEY

摘要:

AbstractA simple method to prepare size‐regulated spheroids has been successfully developed by combining a temperature responsive polymer, poly‐N‐isopropyl‐acrylamide (PNIPAAm), conjugated with collagen and ultraviolet (UV) irradiation with photomasks. The coating layer composed of PNIPAAm conjugated with collagen functions as a cell substratum at 37°C, then when lowering the temperature of culture medium the cells attached to it detach as a self‐supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature LCST; about 30°C, but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. On the other hand, UV effectively immobilized collagen in the coating layer because UV generates crosslinkages in collagen molecules. Crosslinkages were quantitatively introduced by controlling the energy of UV‐irradiation thus the ability of human dermal fibroblasts to attach to and detach from the surface was tightly controlled. When the collagen content in the coating layer was 9 μg/cm2(collagen ratio, 4.5%), UV‐irradiation energy of 2000 J/m2was suitable to obtain 100% of the attachability and detachability. However, the cells did not attach to the nonirradiated surface at this collagen content because insufficient collagen was immobilized. Using photomakes to apply UV‐irradiation, it was possible to obtain cell‐adhesive areas(irradiated areas) and nonadhesive areas (nonirradiated areas) on the same surface. Consequently, spheroids of any size and in any number from one dish were prepared. The viability of cells in spheroids 350 μm in diameter was maintained at a high level for 28 days; however, viability of spheroids 800 μm in diameter rapidly decreased for 2 days. The size was very important to maintain the viability. This novel method is useful to develop size‐regulated spheroids for different applications; for example, in toxicology tests. ©

ISSN:0006-3592    

DOI:10.1002/bit.260440107

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

年代:1994

数据来源: WILEY

摘要:

AbstractRecombinant proteins were harvested from Chinese hamster ovary (CHO) cells by a controlled release process, which increased the purity and concentration of the harvested protein. Recombinant human melano‐transferrin (p97) was expressed linked to the outer surface of CHO cells by a glycosyl‐phosphatidylinositol (GPI) membrane anchor. Cells were grown to confluence in T‐flask culture, and the p97 harvested by replacing the growth medium for 30 min with phosphate‐buffered saline (PBS) containing 10 mU/mL phosphatidylinositol‐phospholipase C (PI‐PLC). The GPI anchor was selectively cleaved by PI‐PLC. In fresh medium, the CHO cells regained over 95% of their p97 expression within 40 h. The process was repeated for eight harvests. Harvested protein concentrations varied from 1.5 to 3.8 μg/mL due to difficulties in maintaining stable confluent T‐flask cultures. Harvesting from cells growing on porous microcarriers was investigated to increase p97 product concentrations and to overcome culture stability problems. Semicontinuous cultures were maintained in spinners for up to 76 days with average bioreactor cell densities of over 107cell/mL. The p97 was harvested at up to 100 μg/mL and 30% purity with protein production remaining stable for 4 harvest cycles. Production of high levels of p97 from CHO cells was maintained at 0.5% serum. © 1994 Jo

ISSN:0006-3592    

DOI:10.1002/bit.260440108

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

年代:1994

数据来源: WILEY

摘要:

AbstractIn this article, we describe the use of 1‐ to 2‐μm sized affinity microparticles for the isolation and purification of IgG from artificial IgG‐human serum albumin mixtures and clarified hybridoma cell culture supernatants by affinity cross‐flow filtration. Affinity microparticles were prepared from cell wall fragments ofClostridium thermohydrosulfuricum L111‐69, in which the peptidoglycan‐containing layer was completely covered with a hexagonally ordered S‐layer lattice. After crosslinking the S‐layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein. A. Quantitative determination confirmed that Protein A molecules formed a monomolecular layer on the outermost surface of the S‐layer lattice. Affinity microparticles were found to withstand high centrifugal and shear forces and revealed no Protein A leakage or S‐layer protein release under cross‐flow conditions between pH 2 to 12. The IgG‐binding capacity of affinity microparticles was investigated under crossflow conditions and compared with that obtained in batch adsorption processes. ©

ISSN:0006-3592    

DOI:10.1002/bit.260440109

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe formation of native disulfide bonds during in vitro protein folding can be limiting in obtaining biologically active proteins. Thus, optimization of redox conditions can be critical in maximizing the yield of renatured, recombinant proteins. We have employed a folding model, that of the β subunit of human chorionic gonadotropin (hCG‐ β), to investigate in vitro oxidation conditions that facilitate the folding of this protein, and have compared the in vitro rates obtained with the rate of folding that has been observed in intact cells. Two steps in the folding pathway of hCG‐β were investigated: the rate‐limiting events in the folding of this protein, and the assembly of hCG‐β with, hCG‐α. The rates of these folding events were determined with and without protein disulfide isomerase (PDI) using two different types of redox reagents: cysteamine and its oxidized equivalent, cystamine, and reduced and oxidized glutathione. Rates of the rate‐limiting folding events were twofold faster in cysteamine/cystamine redox buffers than in glutathione buffers in the absence of PDI. Optimal conditions for hCG‐β folding were attained in a 2 mMglutathione buffer, pH 7.4, that contained 1 mg/mL PDI and in 10μMcysteamine/cystamine, pH 8.7, without PDI. Under these conditions, the half‐time of the ratelimiting folding event was 16 to 20 min and approached the rate observed in intact cells (4 to 5 min). Moreover, folding of the β subunit under these conditions yields a functional protein, based on its ability to assemble with the α subunit. The rates of assembly of hCG‐β with hCG‐α in the cysteamine/cystamine or glutathione/PDI redox buffers were comparable (t1/2/sb>= 9 to 12 min). These studies show that rates of folding and assembly events that involve disulfide bond formation can be optimized by a simple buffer system composed of cysteamine and cystamin

ISSN:0006-3592    

DOI:10.1002/bit.260440110

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

年代:1994

数据来源: WILEY

摘要:

AbstractScanning calorimetry measurements of different amounts of chymotrypsin in water alone gave a temperature of denaturation (Td) value of 54°C. However, when high‐molecular‐weight poly(ethylene glycol) was added to aqueous solutions of chymotrypsin, the thermostability of the enzyme was enhanced. For example, the addition of 20% (w/w) of poly(ethylene glycol) of molecular weight of 100,000 increased theTd/value to 66°C. In toluene containing various amounts of added water, ethyl cellulose was used to improve the thermostability of chymotrypsin. For this system, aTdvalue of 82°C was obtained with a 20% (w/w/) concentration of ethyl cellulose and 2% (v/v) of added water. Polymers in these solvents interact with water, which could otherwise denature the enzyme; polymers also from complexes with enzyme molecules to produce a more stable structure. © 1994 John Wiley&So

ISSN:0006-3592    

DOI:10.1002/bit.260440111

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

年代:1994

数据来源: WILEY