الفهرس يوجد فقط 14 صفحة متاحة للعرض العام
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المستخلص
Two hundred and thirty microbial isolates were recovered from 210
pathological specimens from a variety of origins and were screened for
phospholipase production by the egg-yolk plate method. Only 37 isolates
have shown high phospholipase production and of these three isolates,
identified as Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus
aureus, were selected and used to study the effect of different factors on the
PLC production using a chromogenic assay method.
PLC production by the test isolates was found to take place in phosphate starved media (phosphate-starved TMM). Studying the different factors affecting PLC production revealed that for Pseudomonas aeruginosa D183, phosphate-starved TMM was the best medium for PLC production. For
this isolate, replacing glucose with other carbon sources at different
concentrations, increasing glucose concentration, adding organic protein
sources (with the exception of peptone at 0.5% and BSA at 1.5%), or replacing metal salt components of phosphate-starved TMM, inhibited PLC production. Phosphate-starved TMM, to which BSA (1 or 1.5%) or sodium cholate (0.05%) was added, proved to be the media at which maximum PLC production by Bacillus cereus D101 occurred. PLC production by this isolate was increased with increasing the concentration of glucose in phosphatestarved TMM, while the replacement of glucose with other carbon sources either decreased or abolished PLC production by the isolate. The addition of BSA, triton X-100, sodium cholate (at 0.05 and 0.1%), tween 20, or tween 80 to phosphate starved TMM increased PLC production. Production was also increased upon the removal of CaCl2 from the culture medium. Production of PLC by the Staphylococcus isolate in phosphate-starved TMM occurred only when glucose concentration of phosphate-starved TMM was increased to 110 mM, when BSA was added or when sodium cholate, at 1.25 or 2.5%, was added.
PLC production by the test isolates was found to be growth associated and maximum PLC production was obtained after an incubation of 24 h(Pseudomonas isolate) and after 36 h (Bacillus and Staphylococcus isolates).
The optimum pH and temperature for PLC production by the test isolates
were found to be pH 7.5-8 and temperature 37°C (Pseudomonas aeruginosa
D183), pH 7.5 and temperature 30°C (Bacillus cereus D101) and pH 7.2-7.5
and temperature 25-37°C (Staphylococcus aureus D173).
PLC from Bacillus cereus D101 and Pseudomonas aeruginosa D183 were partially purified and used to study the effect of different factors on their catalytic activity.
For Pseudomonas aeruginosa PLC, maximum catalytic activity was recorded at pH 7 and 8 and at temperature 40°C, however, for Bacillus cereus PLC, maximum catalytic activity was recorded at pH 8-10 and temperature 37°C. Catalytic activity from both PLCs was inhibited by
inorganic phosphate at 5 mM or higher, whereas, the catalytic activity of PLC
from Bacillus cereus only was inhibited by EDTA. Activity of Pseudomonas
aeruginosa PLC was not affected by the removal of Zn2+ ions from the reaction mixture, or their replacement with Ca2+, Ba2+, Mg2+ or Mn2+ ions.
Nevertheless, the activity of this enzyme was inhibited by increasing the
concentration of Zn2+ ions (0.5 mM and higher) or its replacement by Co2+,
Ni2+, or Li+ ions. The activity of Bacillus cereus PLC was found to be metal ion dependent, where the activity was lost when Zn2+ was removed from the
reaction medium, but was restored, without a decrease in activity, when Zn2+ was replaced by Li+ or other divalent cations. PLC from both isolates was
relatively thermostable and showed maximum affinity toward phosphatidylcholine as a substrate.
Partially purified PLC from both isolates induced lysis of Vero cells,
in the presence and absence of the producing bacterial cells. Non-cytolytic
dilutions of the partially purified PLC from Pseudomonas aeruginosa increased adherence of the producing cells to Vero cells but did not affect
internalization. However, Bacillus cereus cells neither adhered to nor were
they internalized within the Vero cells in the presence or absence of noncytolytic dilutions of partially purified PLC from the isolate. Both PLC
preparations were hemolytic to human RBCs but did not induce human platelet aggregation. PLC production by the two isolates was found to be chromosomal-mediated rather than plasmid-mediated.