No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Genetic Transformation of Immobilized Competent Cells
Abstract
This study describes the investigation of the possibility of genetic transformation of already immobilized competent cells
by plasmids. The preliminary prepared competent cells were entrapped into granules of an insoluble carrier, a cryogel of poly(vinyl
alcohol). The specific activity of organophosphate hydrolase and ampicillin resistance conferred by pOPf1 plasmid were used
as markers of successful transformation of the immobilized competent cells. The effect of main experimental conditions of
transformation usually used for free cells, i.e., time of incubation of cells with DNA solution, temperature, and time of
heat shock, on the transformation efficiency of the immobilized competent cells has been studied. A num ber of important factors
of preparation of immobilized transformed cells, i.e., the concentration of im mobilized competent cells inside the granules,
the concentration of DNA solution used for transformation, have been shown to affect the OPH-activity of the final immobilized
transformants. The possibility of transformation of the immobilized competent cells by both single- and double-stranded plasmid
DNA molecules has been demonstrated.
The main dynamic characteristics of biochemical methanol formation by the oxidation of methane using a biocatalyst were studied.
The biocatalyst is based on cells of bacteria Methylosinus sporium B-2121, both suspended in a medium and immobilized in the poly(vinyl alcohol) cryogel. The change in the methane concentration and
the biocatalyst amount affects the productivity of the system, the maximal concentration of methanol in the cultural liquid,
and the rate of methanol accumulation. The most part of the dynamic characteristics are described by extremal curves. The
experimental conditions were optimized prior to experiments. The use of the immobilized biocatalyst makes it possible to enhance
the productivity of the process more than fivefold compared to that of the free cells and to achieve the highest methanol
concentration in the medium: 62±2 mg L−1.
Free and immobilized cells ofC. glutamicum were analyzed for production of L-lysine. Non-growing free cells as well as immobilized ones produced higher levels of lysine than growing free cells, when cultured for 24 h in a medium containing 80 g/L glucose as a carbon source. The effect of initial biomass and yeast extract concentrations was investigated in order to improve lysine production.
A biosensor to quantify L-proline within 10(-5)-10(-3) mole/L concentration is described. Immobilized Pseudomonas sp. cells grown in a medium containing L-proline as the only source of carbon and nitrogen were used to create the biosensor. The cells oxidized L-proline specifically consuming O2 and did not react with other amino acids and sugars. The change in oxygen concentration was detected with a Clark oxygen membrane electrode. The cells were immobilized by entrapment in polyvinyl alcohol (PVA) cryogel. The resultant biocatalyst had a high mechanical strength and retained its L-proline-oxidizing ability for at least two months.
A rapid and simple technique was developed for conjugation between group N and group D streptococci by using cells entrapped within calcium alginate gel beads. With this method, the frequencies of transfer of lactose metabolism from Streptococcus lactis ME2 to S. lactis LM2302 were comparable to those achieved with agar surface matings. Conjugal transfer of the chloramphenicol and erythromycin resistance plasmid pVA797::Tn917 from S. faecalis V1229 to S. faecalis V1102 in alginate beads occurred at frequencies comparable to those achieved with filter matings. The results demonstrated efficient conjugal transfer of plasmid DNA among alginate-immobilized streptococcal cells and suggested that this method could be used as an alternative to conventional solid-surface and filter matings with these organisms.
There has been an increasing interest in the use of immobilized cells for the production of pharmaceuticals as well as for products such as high fructose syrup or ethanol. Some of these compounds are now produced on an industrial scale whereby the cells are used in a resting or growing state or in a nonviable form as natural carriers of the enzyme(s) involved in the synthesis. The advantages of immobilized cell technology should also apply to microorganisms modified by recombinant DNA techniques to produce a variety of eukaryotic proteins such as hormones. We describe here the properties of immobilized Bacillus subtilis cells carrying plasmids encoding rat proinsulin. Cell proliferation normally coupled to DNA replication is undesirable in immobilized cell systems as "clogging' of the system occurs due to cells growing outside the beads. Therefore, different ways were investigated to inhibit cell division while allowing continued protein synthesis. We found that the addition of certain antibiotics in the growth medium, such as novobiocin which inhibits DNA replication, fulfills these requirements, allowing proinsulin synthesis and excretion to take place over a period of several days.
A new biosensor for the direct detection of organophosphorus (OP) neurotoxins has been developed utilizing cryoimmobilized, recombinant E. coli cells capable of hydrolyzing a wide spectrum of OP pesticides and chemical warfare agents. The biological transducer was provided by the enzymatic hydrolysis of OP neurotoxins by organophosphate hydrolase which generates two protons through a reaction in which P-O, P-F, P-S or P-CN bonds are cleaved, and the proton release corresponded with the quantity of organophosphate hydrolyzed. This stoichiometric relationship permitted the creation of a potentiometric biosensor for detection of OP neurotoxins and a pH-based assay was developed as a direct function of the concentration of OP neurotoxins and the immobilized biomass. In these studies utilizing paraoxon as the substrate, neurotoxin concentration was determined with two different types of measuring units containing immobilized cells: (1) a stirred batch reactor; and (2) a flow-through column minireactor. A pH glass electrode was used as the physical transducer. The linear detection range for paraoxon spanned a concentration range of 0.25-250 ppm (0.001-1.0 mM). The response times were 10 min for the batch reactors and 20 min for the flow-through systems. It was possible to use the same biocatalyst repetitively for 25 analyses with a 10 min intermediate washing of the biocatalyst required for reestablishing the starting conditions. The cryoimmobilized E. coli cells exhibited stable hydrolytic activity for over 2 months under storage in 50 mM potassiumphosphate buffer at +4 degrees C and provide the potential for the development of a stable biotransducer for detecting various OP neurotoxins.
Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a homodimeric enzyme that catalyzes the hydrolysis of organophosphorus pesticides and nerve agents. We have analyzed the urea- and guanidinium chloride-induced equilibrium unfolding of OPH as monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence. These spectral methods, which monitor primarily the disruption of protein secondary structure and tertiary structure, respectively, reveal biphasic unfolding transitions with evidence for an intermediate form of OPH. By investigating the protein concentration dependence of the unfolding curves, it is clear that the second transition involves dissociation of the monomeric polypeptide chains and that the intermediate is clearly dimeric. The dimeric intermediate form of OPH is devoid of enzymatic activity, yet clearly behaves as a partially folded, dimeric protein by gel filtration. Therefore, we propose an unfolding mechanism in which the native dimer converts to an inactive, well-populated dimeric intermediate which finally dissociates and completely unfolds to individual monomeric polypeptides. The denaturant-induced unfolding data are described well by a three-state mechanism with delta G for the interconversion between the native homodimer (N2) and the inactive dimeric intermediate (I2) of 4.3 kcal/mol while the overall standard state stability of the native homodimer relative to the unfolded monomers (2U) is more than 40 kcal/mol. Thus, OPH is a remarkably stable protein that folds through an inactive, dimeric intermediate and will serve as a good model system for investigating the energetics of protein association and folding in a system where we can clearly resolve these two steps.