Nurshafrina Aida Yahya (2023) Characterization of a thermostable cellulases from thermoflavifilum aggregans sp1 isolated from Poring Hot Spring Sabah, Malaysia. Masters thesis, Universiti Malaysia Sabah.
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Abstract
Thermophilic microorganisms and their enzymes have been utilized in various industrial applications. Cellulases are essential enzymes in various industries and have great significance in present-day biotechnology. Currently, thermostable cellulases have been steadily increasing in demand due to their versatile applications under harsh conditions. Nevertheless, the availability of thermostable cellulases remains limited. Sabah houses hot springs and mud volcanoes that are promising as the sources of indigenous thermophilic bacteria producing thermostable cellulases. This study is aimed to isolate and characterize thermophilic cellulose-degrading bacteria from Sabah hot springs and mud volcanoes. To address, the samples were collected from Poring hot spring and Tawau mud volacanoes and spread onto the carboxymethylcellulose (CMC) agar medium for the screening of cellulose-degrading bacteria. Based on the biocehmical test, SP1 was chosen and undergone whole genome sequencing, assembly and annotation. The gene was then cloned into pET-28a (+) and transformed into Escherichia coli BL21(DE3). The expression of this protein was successfully performed by induction of 0.2 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 25 °C for overnight. The expressed protein was then successfully purified using Ni-NTA affinity chromatography and size-exclusion chromatography. As results, out of 6 isolated colonies exhibiting cellulose degradation activities, colony SP1 from Poring hot spring displayed the highest cellulolytic activity on crude enzyme at 60 °C. Further, 16S rRNA sequence analysis of SP1 showed closest similarity to Thermoflavifilum aggregans (accession no: AM749771), a thermostable bacterium isolated from New Zealand hot spring, with 99.74 % homology. Accordingly, SP1 is designated as T. aggregans SP1 strain. To note, the strain SP1 is the first strain from Poring hot spring known to exhibit thermostable cellulolytic activity. Whole genome sequence (WGS) of SP1 strain was then decoded using the Pacific Biosciences Single Molecule, Real-Time sequencing platform which revealed the genome size is 2,874,051 bp with the presence of three (3) genes encoding cellulose-degrading enzymes which might responsible for its celluloytic activity. From the three genes, one gene is from glycosyl hydrolase (GH)-5 and another two genes from GH-9 family members. Due to the unique properties of GH-5, one gene of GH-5 (designated as CePH4) was selected for further characterization. CePH4 is 1053 bp in size, which encodes a 347-residue polypeptide with theoretical molecular mass of 38 kDa. The sequence analysis of CePH4 indicated that this protein is organized into a catalytic domain of GH5 and an aryl-phospho-beta-D-glucosidase (BglC), with canonical active sites of Glu151 and Glu271. CePH4 was successfully expressed and purified with the yield of 24.22 mg per 1000 mL culture, with apparent size of 38 kDa on SDS. Size exclusion chromatography showed that CePH4 is a monomeric protein. Circular dichroism spectroscopy revealed that CePH4 has melting temperature (Tm) of 80 °C, indicating that this protein is a thermostable enzyme. Further, the specific activity of purified CePH4 against CMC substrate was 7.46 U/mg with the optimum activity at 70 °C and pH 7. Interestingly, up to 80°C, the activity of CePH4 decreased by up to 40 % only. Our findings suggest promising applications of these thermoaerobic bacteria and their potent enzymes for industrial purposes.
| Item Type: | Thesis (Masters) |
|---|---|
| Keyword: | Thermostable, Cellulases, Hot springs, Whole genome sequence |
| Subjects: | Q Science > QR Microbiology > QR1-502 Microbiology > QR100-130 Microbial ecology |
| Department: | INSTITUTE > Biotechnology Research Institute (BRI) |
| Depositing User: | DG MASNIAH AHMAD - |
| Date Deposited: | 25 Jul 2024 09:29 |
| Last Modified: | 25 Jul 2024 09:29 |
| URI: | https://eprints.ums.edu.my/id/eprint/39128 |
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