Recombinant production and characterization of subtilisin-like serine protease from Acinetobacter baumannii
Acinetobacter genus specifically Acinetobacter baumannii is notoriously known in the past five decades as one of the most dangerous saprophytic organisms that cause broad arrays of diseases particularly nosocomial infections. One of the factors that may contribute to A. baumannii virulence are the s...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2020
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| Subjects: | |
| Online Access: | http://eprints.utm.my/id/eprint/101713/1/NurSyafiqahMuhammedMFS2020.pdf |
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| Summary: | Acinetobacter genus specifically Acinetobacter baumannii is notoriously known in the past five decades as one of the most dangerous saprophytic organisms that cause broad arrays of diseases particularly nosocomial infections. One of the factors that may contribute to A. baumannii virulence are the secretory proteases that it synthesizes. In order to formulate the effective antibiotics targeting secretory proteases from A. baumannii, this protease need to be characterized for functional and structural studies. Therefore, in the present study, a subtilisin-like serine protease isolated from A. baumannii designated as ―SPSFQ‖ was cloned, purified and characterized. The nucleotide sequences of spsfq revealed 1,104 bp open reading frame corresponding to 368 amino acid residues. Amino acid sequence comparison revealed that SPSFQ shared 38.6 - 60% sequences identity with those of other serine proteases reported to have broad substrate specificity; keratinolytic and collagenolytic properties. Homology model of mature SPSFQ revealed its structure composed of 10 β-strands, 8 α-helices with connecting loops resembling a typical architecture of subtilisin-like α/β motif. Expressed recombinant SPSFQ was purified using a combination of HiTrap HP and Q-Sepharose columns prior to characterization studies. The optimum temperature and pH for SPSFQ activity were achieved at 40°C and pH 9.0, respectively. SPSFQ activity was significantly reduced by phenylmethylsulfonyl fluoride (PMSF) while the presence of Ca2+ ion significantly enhanced the activity, suggesting that SPSFQ is a serine protease that require divalent metal ions, Ca2+ as a cofactor. Substrate specificity test concluded that purified SPSFQ has high catalytic activity for casein followed by gelatin (hydrolysed collagen) and keratin. As a conclusion, this study suggests that SPSFQ from A. baumannii is a potent hydrolytic protease. This data could serve as an impetus for further in-depth study on the function of extracellular proteases and their potential role in A. baumannii pathogenicity. |
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