Research - MS (Thesis)

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Title: Isolation, Purification and Characterization of Protease from Clinical Isolates of Pseudomonas aeruginosa and Demonstration of Its Elastase Properties

Author: Salma Akther

Supervisor: Md. Abdul Hakim

Co-Supervisor: Md. Nuruddin Mahmud

Research Field: Clinical Microbiology

Batch: 11th

Session: 2010-2011


Abstract

Pathogenic bacteria contribute to the most of the diseases in human. Infective process of these bacteria is intimately involved with some factors called virulence factors. One of these virulence factors is enzyme. Detection of the presence of specific extracellular enzyme having virulent properties in clinical specimens, collected from patients is of prime importance in the diagnosis and effective treatment of the diseases. In addition, it’ll not only show the way to discover drug against specific target molecules but also facilitate the disease recovery processes. Considering the importance of the above mentioned points, in our present research work, we targeted two specific enzymes of the Pseudomonas aeruginosa such as protease, elastase.
For the present study, blood, throat swab and pus samples of the patients from Chittagong Maa Shishu O General Hospital were collected. 15 isolates of Pseudomonas sp. were suspected by observing their growth characteristics on cetrimide agar medium. Primary screening of the isolates was done on the basis of their ability to hydrolyze the protein substance (casein). Secondary screening was done by quantitative method in three broth media. After primary and secondary screening for the protease producer, three isolates were finally selected for detail studies and these were coded as Ps(b), Ps(Ts) and Ps(P).
On the basis of morphological, physiological and biochemical characteristics it was found that, all the isolates belong to the genus Pseudomonas and provisionally identified as Pseudomonas aeruginosa when compared with the standard description given in “Bergey’s Manual of Determinative Bacteriology,” 9th edition (Baltimore: Williams & Wilkins, 1994).
Antibiotic sensitivity pattern of the selected isolates was determined to investigate the inhibitory effect of any antibiotics against them. It was observed that all the isolates were resistant to most of the antibiotics. Among the antibiotics, Ciprofloxacin showed largest zone of inhibition against all the isolates.
The incubation period (48 hrs), pH (8.0) and temperature (37ºC) were found to be optimum for maximum protease production by all the isolates.
The effect of carbon and nitrogen sources on protease production was evaluated and dextrose and yeast extract were found to be optimum as carbon and nitrogen source, respectively.
Optimum salt concentration (1.0%) was observed for Ps(Ts) and Ps(P) while Ps(b) showed 0.50% as optimum.
Maximum protease production was observed by all isolates when they were kept in shaking condition.
Reaction time, temperature, pH, substrate specificity, substrate concentration and the effect of metal salts were also evaluated during enzyme-substrate reaction. The crude enzymes of the isolates were studied to observe the efficiency towards various substrates such as Casein, Gelatin, Bovine serum albumin and Tryptone. The results showed that, the isolates Ps(Ts) and Ps(P) exhibited maximum protease activity when gelatin (0.50% and 1.5%, respectively) was used as substrate whereas the isolate Ps(b) showed highest activity with Bovine serum albumin (0.25%).
The optimum enzyme-substrate reaction temperature for crude enzymes of the isolates Ps(b) and Ps(P) were observed at 35°C but isolate Ps(Ts) showed maximum activity at 40°C.
Maximum protease activity by the isolates Ps(b) and Ps(P) was recorded at pH 6.0 while pH 7.0 was found as optimum for Ps(Ts).
The maximum enzyme activity was found for Ps(Ts) and Ps(P) when the reaction was carried out in water bath for 40 minutes whereas the isolate Ps(b) showed maximum activity when reaction was performed for 20 minutes.
CaCl2 and MgCl2 stimulated the protease activity but ZnSO4 and CuSO4 came out as less efficient in such stimulatory function. The protease activity was suppressed by SDS and glycerol, but not by urea. The crude protease of all isolates was found to be stable up to 75°C and pH 10.
Elastase properties of these proteases were tested indirectly by some in vitro experiments such as milk clotting test and lysozyme inhibition test. Coagulation of milk was observed rapidly when incubated with the crude protease of all isolates at 40°C. The elastolytic protease of the isolate Ps(b) was the most efficient than that of other isolates, which showed milk clot formation within 2 minutes. Lysozyme inhibition test was performed on Mac-conkey agar plate by using E. coli. The inhibitory effect of lysozyme on the growth of E. coli was reduced due to lysozyme inactivating capacity of the isolated elastolytic protease.
Finally, The crude enzyme of all isolates was partially purified by precipitation method using ammonium sulphate at 50% saturation. The molecular weight of the partially purified enzymes of the isolates Ps(b), Ps(Ts) and Ps(P) were found 24 kDa, 25 kDa and 24.5 kDa, respectively by using SDS-PAGE.
The elastolytic properties of protease are life threatening and can be acted as hazardous virulence factor. From our study, we tried to assay the virulence properties of proteases secreted from Pseudomonas aeruginosa and it may be suggested that Ciprofloxacin can be used as antibacterial agent to inhibit the growth of P. aeruginosa as the organism is resistant to most of the commercial antibiotics. Further study on P. aeruginosa protease is essential to improve antibacterial agent against it to control its virulent activity.