PubMed · 2861531
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1985-07-13. Troubles underfoot.. https://pubmed.ncbi.nlm.nih.gov/2861531/
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Genes for rRNA are highly conserved and present in multiple copies in most prokaryotic organisms increasing the number of theoretical sites for homologous recombination. They might be targets for integration events between unrelated microorganisms providing that an efficient genetic transfer is present. We have used a plasmid containing a portion of the 16S rRNA gene from the rrnD operon of Brevibacterium lactofermentum to transform the same strain resulting in non-essential inactivation of various rrn operons. Integration of the transforming DNA occurs in all cases. The system may be used to test possible gene transfer at least among closely related strains and is of great interest for integration of foreign DNA and for mapping.
The kinetic properties of two cholesterol oxidases, one from Brevibacterium sterolicum (BCO) the other from Streptomyces hygroscopicus (SCO) were investigated. BCO works via a ping-pong mechanism, whereas the catalytic pathway of SCO is sequential. The turnover numbers at infinite cholesterol and oxygen concentrations are 202 s-1 and 105 s-1 for SCO and BCO, respectively. The rates of flavin reduction extrapolated to saturating substrate concentration, under anaerobic conditions, are 235 s-1 for BCO and 232 s-1 for SCO (in the presence of 1% Thesit and 10% 2-propanol). With reduced SCO the rate of Delta5-6-->Delta4-5 isomerization of the intermediate 5-cholesten-3-one to final product is slow (0.3 s-1). With oxidized SCO and BCO the rate of isomerization is much faster ( approximately 300 s-1), thus it is not rate-limiting for catalysis. The kinetic behaviour of both reduced COs towards oxygen is unusual in that they exhibit apparent saturation with increasing oxygen concentrations (extrapolated rates approximately 250 s-1 and 1.3 s-1, for BCO and SCO, respectively): too slow to account for catalysis. For BCO the kinetic data are compatible with a step preceding the reaction with oxygen, involving interconversion of reactive and nonreactive forms of the enzyme. We suggest that the presence of micelles in the reaction medium, due to the necessary presence of detergents to solubilize the substrate, influence the availability or reactivity of oxygen towards the enzyme. The rate of re-oxidation of SCO in the presence of product is also too slow to account for catalysis, probably due to the impossibility of producing quantitatively the reduced enzyme-product complexes.
We have studied systematically the effect of the non-ionic surfactants Thesit and Triton X-100, and of propan-2-ol (used as a substrate solubilizer) on the activity of the cholesterol oxidases from Streptomyces hygroscopicus (SCO) and Brevibacterium sterolicum (BCO). Low concentrations of Thesit lead to an activity increase with both enzymes; at higher surfactant concentrations the opposite effect occurs. Triton X-100 inactivates both enzymes at all concentrations. It is deduced that these surfactants exert their effects by interaction with the enzymes and not by affecting micellar phenomena. The effect of propan-2-ol on SCO, in contrast with that on BCO, depends on the buffer concentration (potassium phosphate). Other organic solvents induce results similar to those obtained with SCO and propan-2-ol. A significant difference between the two cholesterol oxidases emerges when stability is tested at 25 degrees C and in the presence of different concentrations of propan-2-ol: BCO activity is rapidly inactivated, whereas SCO still has 70% of the initial activity after 5 h in the presence of 30% propan-2-ol. From our results, SCO seems to be the catalyst of choice in comparison with BCO for the exploitation of cholesterol oxidases in biotechnology and applied biochemistry.