Bioprospecting bacterial diversity for thermostable hydrolytic enzymes and characterization of a thermostable gh9 cellulase from Sabah Hot Spring

Bak Zaibah Fazal (2023) Bioprospecting bacterial diversity for thermostable hydrolytic enzymes and characterization of a thermostable gh9 cellulase from Sabah Hot Spring. Masters thesis, University Malaysia Sabah.

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Abstract

Hydrolytic enzymes are often required to catalyse reactions under harsh conditions, including high-temperature environments. Consequently, thermostable hydrolytic enzymes hold significant industrial importance due to their stability in high temperature working conditions. The Poring hot spring (PHS) in Sabah shows promise as a source for prospecting thermophilic microorganisms and their hydrolytic enzymes. However, the bacterial diversity and availability of thermostable hydrolytic enzymes at this site have not been explored to date. Therefore, this study aims to determine the bacterial diversity of Sabah hot springs and their hydrolytic enzyme production. To achieve this goal, water and sediment samples were collected from the site for physicochemical analysis, genome extraction and hydrolytic enzyme screening. During sampling, the recorded temperature ranged from 42 °C to 59 °C and the pH ranged from 6.5 to 7.0. The V3-V4 region of the 16S rRNA gene was amplified using the total genomic DNA (gDNA) from both water and sediment samples as templates. Subsequently, the amplicons were sequenced using the Illumina paired-end platform and subjected to analysis. The results showed that 99 % of the 1,127 OTUs obtained from water samples were bacteria, followed by 0.3 % archaea and 0.01 % unclassified microorganisms. In sediment samples, the majority of 1,559 OTUs were classified as 93 % bacteria, 0.03 % archaea and 7 % unclassified microorganisms. The most abundant phylum in water samples was Proteobacteria (84 %), while Cyanobacteria (49 %) dominated in sediment samples. Furthermore, hydrolytic enzyme screening, targeting viable bacteria yielded positive colonies producing proteases (11 colonies), amylases (9), lipases (8) and cellulases (2). As cellulases were considered the most relevant hydrolytic enzymes for Malaysia due to advanced oil palm plantations, the best colony exhibiting thermostable cellulases, was identified as Thermoflavifilum aggregans SP1 and selected for further studies. This strain was found to possess a gene encoding cellulase under glycoside hydrolase family 9 (GH9), which is less studied compared to other GH families. Accordingly, the GH9 cellulase of the SP1 strain (SP1-GH9) was chosen for further studies. For this purpose, an expression system for this gene was constructed in pET28a(+) for overexpression in Escherichia coli BL21(DE3). The protein was successfully expressed with an apparent size of 66 kDa and could be purified using Ni2+-NTA affinity chromatography coupled with size exclusion chromatography (SEC). Interestingly, under SEC, this protein eluted at an apparent size of 154.2 kDa, indicating that it forms a dimeric structure. Far-UV circular dichroism (CD) spectroscopy confirmed that the pure protein was correctly folded and exhibited a dominant helical structure, consistent with the structural model of this protein, characterized by an (α-α)6-barrel structural motif. The purified protein exhibited a specific activity of 0.53 x 10-2 U/mg against a p-Nitrophenyl β-D-Cellobioside (pNPC) substrate, with optimum activity observed at 60 °C and pH 4.0. Interestingly, the activity of SP1-GH9 was found to be enhanced in the presence of various metal ions (Zn, Mn, Ca, Li, Mg, K, Na), EDTA, Tween 20 and Triton X-100. This study highlights the potential development of biocatalysts with multifunctional thermostable glycoside hydrolases from local hot springs for various industrial applications.

Item Type:	Thesis (Masters)
Keyword:	Hydrolytic enzymes, Poring hot spring, Bacterial diversity
Subjects:	Q Science > QH Natural history > QH1-278.5 Natural history (General) > QH1-(199.5) General Including nature conservation, geographical distribution
Department:	INSTITUTE > Biotechnology Research Institute (BRI)
Depositing User:	DG MASNIAH AHMAD -
Date Deposited:	10 Sep 2024 10:23
Last Modified:	10 Sep 2024 10:23
URI:	https://eprints.ums.edu.my/id/eprint/40795

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