Escherichia cell disruption

Tocaj, A., Nandakumar, M. P., Holst, O., and Mattiasson, B. (1999). Flow injection analysis of intracellular /3-galactosidase in Escherichia coli cultivations, using an on-line system including cell disruption, debris separation and immunochemical quantification. Bioseparation 8, 255-267. 

Cereghino GPL, Cereghino JL, Ilgen C et al. (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13 329-332 Chan WK, Belfort M, Belfort G (1991) Protein overproduction in Escherichia coli RNA stabilization, cell disruption and recovery with a cross-flow microfiltration membrane. J Biotechnol... 

A 1000-litre fermenter has been used to produce a continuous feed of Escherichia coli containing a high level of j3-D alactosidase. Investigation of the individual unit operations for the isolation of the enzyme, cell disruption, nucleic acid removal, protein precipitation and solid-liquid separation after each stage, permitted operation of a semi-continuous process which would yield 130 g protein hr comprising 23% j3-D-galactosidase. 

We have speculated on but do not understand the mechanism causing the lytic activity of laetisaric acid. The active twelve carbon metabolite of laetisaric acid may poison a key enzyme in lipid metabolism or disrupt the integrity of the fungal cell membrane by insertion or dissolution as has been shown in Escherichia coli with sodium dodecyl sulfate and Triton X-100 (24 r 25). Why the C-12 molecule is most active remains to be determined. Kinetic studies of lipid metabolism and physicochemical and ultrastructural investigations of membranes treated with the putative active metabolite may answer these questions. 

An important factor complicating the recovery of recombinant proteins from Escherichia coli is their intracellular location. An alternative to the commonly used method of releasing these proteins by mechanical disruption is to chemically permeabilize the cells. The objective of this research was to characterize the protein release kinetics and mechanism of a permeabiliza-tion process using guanidine-HCl and Triton-XIOO. The protein release kinetics were determined as a function of the guanidine, Triton, and cell concentrations. Some of the advantages over mechanical disruption include avoidance of extensive fragmentation of the cells and retention of the nucleic acids inside the cell structure. 

The polypyrrole (Ppy)/dextrin nanocomposite is synthesised via in situ polymerisation and the preparation of this nanocomposite is shown in Figure 5.4. The backbone chain of this nanocomposite polymer contains hydrophobic side chains, which disrupt the microbial cell membrane leading to leakage of the cytoplasm in bacteria including Escherichia coli. Pseudomonas aeruginosa. Staphylococcus aureus and Bacillus subtilis. This material can be implemented in the fields of biomedicine, biosensors and food packaging due to the biodegradable property of dextrin as well as the antibacterial properties of the Ppy [79]. 

As an example, phytoene desaturase was cloned and expressed in recombinant Escherichia coli. To prepare the enzyme, the E. coli cells were disrupted by pressing them through a French Press. After centrifugation, the soluble supernatant fraction was used for enzymatic assays with HPLC recording or recording by optical absorption spectra [26]. 

A batch fermentation of PA-E. coli can produce 5-50 mg tPA/L-broth at harvest. Escherichia. coli may require disruption to release tPA, which is then more difficult to separate. Should a process be synthesized based upon this reaction path, reaction rate data from the laboratory will be needed. Unlike CHO cells, E. coli cells do not add sugar groups (glycosy-lation) to tPA. Like CHO cells, tPA- coli cells are produced and frozen during the research and development phase. 

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