Please use this identifier to cite or link to this item:
https://ptsldigital.ukm.my/jspui/handle/123456789/499633| Title: | Characterisation of recombinant β-glucanases from psychrophilic yeast, glaciozyma antarctica PI12 |
| Authors: | Salimeh Mohammadi (P58669) |
| Supervisor: | Abdul Munir Abdul Murad, Prof. Dr. |
| Keywords: | Universiti Kebangsaan Malaysia -- Dissertations Psychrophilic yeast Fungi Hydrolytic products Dissertations, Academic -- Malaysia |
| Issue Date: | 12-Jan-2015 |
| Description: | β-1,3-Glucanases are enzymes that cleave beta-1,3-glycosidic linkages of glucans which are mostly found in the cell wall of fungi, algae and plants. Fungi secrete β-1,3-glucanases to their surroundings to hydrolyse dead plant and algal cell walls to obtain sugars for cell metabolism and to inhibit the growth of other fungal cells by destroying their cell wall. The aim of this study is to isolate β-1,3-glucanase genes from a psychrophilic yeast, Glaciozyma antarctica, and to produce and characterise the biochemical properties of the recombinant β-1,3-glucanases. cDNA sequence encoding β-1,3-glucanases from G. antarctica PI12, GaEXG55, GaEXG52 and GaEG1, have been amplified by reverse-transcription polymerase chain reaction (RT-PCR). Domain analysis showed that both GaExg55 and GaExg52 belong to the glycosyl hydrolase family 5 while GaEgl belongs to the glycosyl hydrolase family 16. Subsequently, the recombinant β-glucanases were produced in Escherichia coli expression system and the production of all recombinant proteins were verified via western blotting analyses. The recombinant GaExg55 (rGaExg55), GaExg52 (rGaExg52) and GaEgl (rGaEg1) with an approximate molecular weight of 55, 52 and 44 kDa, respectively, were purified with nickel affinity chromatography and subjected to enzymatic characterisation. Biochemical characterisations such as determination of optimum temperature and pH, stability at different temperature and pH, effect of metal ions and substrate specificity had been carried out. The rGaExg52 and rGaEgl displayed an optimum pH of 7.0 while rGaExg55 at pH 8.0. The optimum temperature for rGaExg55 and rGaEgl activity was at 20°C while the optimum temperature for rGaExg52 was at 25οC. All of the recombinant β-glucanases were active between pH 4 to 10 and they were able to retain at least 40% of their enzyme activity at temperature between 15οC to 30°C. The activity of rGaExg55 was enhanced in the presence of metal ions Co2+, Mg2+, Fe2+, Ni2+, Li+, while Co2+, Fe2+ and Mn2+ enhanced the activity of rGaExg52. The activity of rGaEg1, on the other hand, was enhanced in the presence of Co2+ and Mn2+. The rGaEg1 showed the highest activity towards lichenan with the Km and Vmax value of 8.87 mgmL-1 and 37.45 Umg-1, respectively. In contrast, both rGaExg52 and rGaExg55 showed the highest activity towards laminarin with a Km value of 16.8 mg mL-1 and 10.04 mgmL-1, and Vmax value of 6.26 Umg-1 and 14.12 Umg-1, respectively. Analysis of the products released from enzymatic hydrolysis of laminarin using high performance liquid chromatography (HPLC) revealed that the main hydrolysis products for the rGaExg55 were monosaccharides, disaccharides and oligosaccharides. In contrast, the rGaExg52 only produced monosaccharides when laminarin was hydrolysed, while rGaEgl produced disaccharides and oligosaccharides when laminarin was hydrolysed. In conclusion, all three β-1,3-glucanases from G. antarctica are active at low temperature and at a broad pH range, display specificity towards different substrates and produced different forms of hydrolytic products when hydrolysing the substrates.,Ph.D. |
| Pages: | 190 |
| Publisher: | UKM, Bangi |
| Appears in Collections: | Faculty of Science and Technology / Fakulti Sains dan Teknologi |
Files in This Item:
| File | Description | Size | Format | |
|---|---|---|---|---|
| ukmvital_80378+SOURCE1+SOURCE1.0.PDF Restricted Access | 408.33 kB | Adobe PDF |  View/Open |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.