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https://ptsldigital.ukm.my/jspui/handle/123456789/499449Full metadata record
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | Ainon Hamzah, Prof. Datin Dr. | - |
| dc.contributor.author | Arezoo Tavakoli (P40963) | - |
| dc.date.accessioned | 2023-10-13T09:32:05Z | - |
| dc.date.available | 2023-10-13T09:32:05Z | - |
| dc.date.issued | 2013-06-09 | - |
| dc.identifier.other | ukmvital:74697 | - |
| dc.identifier.uri | https://ptsldigital.ukm.my/jspui/handle/123456789/499449 | - |
| dc.description | Toluene is naturally presents in crude oil and widely exists in petroleum products. Thus, it is commonly found to pollute soil and groundwater in the vicinity of petroleum industries. Toluene removal from the environment using biological approach is preferred because microbes produce enzyme that can degrade toluene without producing toxic secondary metabolites. The objectives of this study were to clone and characterize several recombinant enzymes, benzyl alcohol dehydrogenase (BADH), benzaldehyde dehydrogenase (BZDH) and benzoate dioxygenase (BD), which have specific roles in toluene degradation. From a screening of toluene degraders on 12 bacterial isolates were selected and grown in minimal salt medium (MSM) with the addition 250 μM toluene. The supernatant from resuspended cells in phosphate buffer with toluene was used for assaying toluene oxygenase activities using Horseradish peroxidase (HRP) and indole method. The isolates showed different toluene oxygenase activity in the upper pathway of toluene degradation. The oxygenase activities assay using HRP and indole for all isolates ranged between 0.05-0.35 U/mL and 0.05-0.3 U/mL, respectively. Rhodococcus ruber UKMP-5M was selected for further study since it was able to degrade toluene by the xyl pathway. Three recombinant proteins BADH, BZDH and BD encoded by xylB, xylC and benA genes, respectively, were studied. The gene sequences were obtained from the draft genome of R. rubber UKMP-5. These genes were successfully amplified by PCR and cloned into pet 28b vector and transformed into E. coli BL21 (DE3). The recombinant protein of BADH, BZDH and BD were expressed with induction using 0.3 mM, 1 mM and 0.5 mM isopropyl-thio-β-D-galactopyranoside (IPTG) at 25°C, 37°C and 22°C, respectively. The results were confirmed by SDS-PAGE and western blotting. The BADH was purified by affinity chromatography, meanwhile the BZDH and the BD by ion exchange chromatography. The size of proteins for BADH, BZDH and BD were 38 kDa, 27 kDa and 25 kDa, respectively. BADH and BZDH used benzyl alcohol and benzaldehyde, respectively, and BA used sodium benzoate as a substrate. All the enzymes were assayed by using β-Nicotinamide dinucleotide (NAD) and NADH as cofactor. The enzyme activity for BADH, BZDH and BD were 0.7 U/mL, 9.4 U/mL and 5.3 U/mL, respectively. All proteins showed optimal pH and temperature at range from 8.5-9.5 and 25-30°C, respectively. The maximum velocity (Vmax) for BADH, BZDH and BD were 1.3 U/mL, 19.7 U/mL and 7.4 U/mL, respectively which was calculated using Lineweaver-Burk plot. The Km values were 705 μM, 4.2 mM and 5.57 μM for BADH, BZDH and BD, respectively. The end products of degradation using BADH, BZDH and BD were benzaldehyde, benzene carboxylic acid and cyclohexane-1, 3-dione, respectively which suggest the possible pathway capable by the bacteria to degrade toluene.,Ph.D | - |
| dc.language.iso | eng | - |
| dc.publisher | UKM, Bangi | - |
| dc.relation | Faculty of Science and Technology / Fakulti Sains dan Teknologi | - |
| dc.rights | UKM | - |
| dc.subject | Molecular and enzymatic | - |
| dc.subject | Oxygenase and dehydrogenase | - |
| dc.subject | Toluene | - |
| dc.subject | Rhodococcus ruber UKMP-5M | - |
| dc.subject | Molecular biology | - |
| dc.title | Molecular and enzymatic studies of oxygenase and dehydrogenase for toluene degradation by Rhodococcus ruber UKMP-5M | - |
| dc.type | Theses | - |
| dc.format.pages | 218 | - |
| dc.identifier.callno | QH506.T348 2013 | - |
| dc.identifier.barcode | 000726 | - |
| Appears in Collections: | Faculty of Science and Technology / Fakulti Sains dan Teknologi | |
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| File | Description | Size | Format | |
|---|---|---|---|---|
| ukmvital_74697+Source01+Source010.PDF Restricted Access | 4.04 MB | Adobe PDF |  View/Open |
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