Characterization and Immunological Properties of Pseudomonas aeruginosa Hemolytic Phospholipase C | ||||
| Egyptian Journal of Botany | ||||
| Article 5, Volume 54, Issue 1, June 2014, Page 49-70 PDF (763.09 K) | ||||
| Document Type: Regular issue (Original Article) | ||||
| DOI: 10.21608/ejbo.2014.479 | ||||
 View on SCiNiTO | ||||
| Abstract | ||||
| PHOSPHOLIPASE C (PLC) produced from 4 day age culture of ……….P. aeruginosa strain in inorganic phosphate starved liquid medium (pH7.2) at 35C. The enzyme was purified to electrophoretic homogeneity using gel filtration chromatography. Purified PLC had 6.2 purification fold and 28.2% yield. The purified enzyme had a molecular mass of 58.7 kDa. Alanine was the dominant amino acid with 13.1%. DNA sequence analysis of the PCR products amplified from P. aeruginosa confirmed the presence putative plcH gene (1.6 kb). The enzyme was completely inactive above 65C with Tm 42C. The enzyme activity was approximately stable at pH 8.5 to 10. The purified enzyme was characterized as a Zn2+-metallophospholipase C and also divalent ions Ca2+, Ba2+ and Co2+ were stimulators for its activity. The catalytic affinity of PLC enzyme towards phosphatidylcholine (PC) gave Vmax = 1.85 Umg-1 min-1; Km = 0.12 M and Kcat = 1.96 x 10-5 S-1. Purified PLC enzyme was detoxified by heating and completely lost its lethal activity on mice after 60 min at 65C. Immunologically, the titer of IgG antibodies in blood serum of New Zealand rabbits was appeared after 15 days of the last injection dose of fresh or heat detoxified enzyme by 1.15 and 1.21 fold respectively higher as compared to control followed by a decrease in the titer up to 45 days. The hematological parameters reflected a decrease in the RBCs, WBCs and hemoglobin in rabbits injected with fresh enzyme and there was significant change in chemistry of liver and kidney. In contrarily detoxified enzyme had no obvious changes in these parameters. | ||||
| Keywords | ||||
| Pseudomonas aeruginosa; Phospholipase C, Antigenicity, | ||||
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