Molecular verification and bioinformatics analysis of a metalloprotease gene from acinetobacter baumannII
The accumulation of eschar, dead tissue, microbes on the surface of wound lead to delay the wound healing particularly in diabetic wounds and foot ulcers. The process of removing these dead tissues to enhance the healing process is called debridement. A variety of approaches can be used for debridem...
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| Format: | Thesis |
| Language: | English |
| Published: |
2020
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| Subjects: | |
| Online Access: | http://eprints.utm.my/id/eprint/102471/1/DianaJalalMohammedMFS2020.pdf.pdf |
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| Summary: | The accumulation of eschar, dead tissue, microbes on the surface of wound lead to delay the wound healing particularly in diabetic wounds and foot ulcers. The process of removing these dead tissues to enhance the healing process is called debridement. A variety of approaches can be used for debridement including surgical, mechanical, enzymatic and maggot therapy. Recently enzymatic debridement is gaining more attention especially in situations where surgical debridement may not be suitable. The current available enzymatic debridement agents are limited, and they can also cause side effects to patients. Hence, there is a need to search for a new debridement agent that have high efficiency and specificity that can cause lesser side effects, one of the alternative methods is via protease enzymatic debridement. This research focused on verification of a cloned metalloprotease gene via PCR and sequencing as well as characterisation of the metalloprotease gene product via bioinformatic tools. The plasmid containing the cloned metalloprotease gene from Acinetobacter baumannii was successfully extracted from E.coli HSTO8 and amplified via PCR. The metalloprotease gene amplicon showed the correct size of approximately 717 bp on agarose gel. The concentration of the amplified gene was measured by nanodrop spectrophotometer showing that it has a concentration of 96.8 ng/µl, as well as 260/280 and 260/230 ratios of 1.93 and 2.18 respectively. The sequencing analysis result illustrated that the cloned gene is 100% identical to metalloprotease from Acinetobacter baumannii (accession number WP_000722324.1). The result of the in silico study showed that, the metalloprotease from Acinetobacter baumannii is a membrane protein, consisting of 238 amino acids with estimated molecular weight of 27.2 kDa. The nonpolar amino acids content is higher than the polar amino acids which illustrated that our metalloprotease is hydrophobic in nature, and it is stable with instability index of 39.58. The metalloprotease from Acinetobacter baumannii has Zinc-dependent metalloprotease domain Phe162 to Asn235 which characterized by the presence of zinc binding motive (H180, E181, H184, G187, H190). The generated model consists of five beta sheets and four alpha helixes, alpha helix number three (a 3), alpha helix number four (a 4) and beta sheet number five (ß 5) are located in the Zinc-dependent metalloprotease domain. The active site of metalloproteinases group of protein contains a catalytic divalent metal ion which is usually zinc atom, the zinc atom in the generated model attached to the three histidine residues of the active site (H180,H184 and H190) with distance 2Å, 2.3Å and 2Å respectively. This metalloprotease belonged in the same M12 family as well as having the same catalytic motif as a fibrinolytic enzyme isolated from snake venom which may indicate that this metalloprotease has the potential ability to have fibrinolytic activity. |
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