Exploring thermophilic metagenome for biodiversity comparisons and characterisation of a novel beta glucosidase
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Date
2018
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Universiti Teknologi Malaysia
Abstract
Malaysia consists of a significant number of hot springs, especially along the flank of Titiwangsa Range. The exploitation of hot springs in Malaysia for biological studies is scarce, attribute to the lack of comprehensive information on the microbial community. Without the knowledge of microbial diversity, harnessing thermophiles or thermozymes as biocatalysts for bioprocess industries is hampered. In this regard, this study aims to examine the diversity of microbial communities in six Malaysian hot springs, namely, Ulu Slim (US), Sungai Klah (SK), Dusun Tua (DT), Sungai Serai (SS), Semenyih (SE), and Ayer Hanyat (AH). To achieve this objective, the 16S rRNA V3–V4 region amplicon sequencing was performed for the aforementioned hot springs. Using Quantitative Insights Into Microbial Ecology (QIIME) pipeline analysis, the results suggested that archaeal communities mainly comprised of Crenarchaeota, Euryarchaeota, and Parvarchaeota. As the abiotic conditions of the six hot springs vary, the bacteria communities within each site are unique. A total of 59, 61, 72, 73, 65, and 52 bacteria phyla were identified in US, SK, DT, SS, SE, and AH, respectively. SS and AH exhibited lower community (<4% total phylum) of phylum Firmicutes; while the other sites have a higher abundance (>10%) of phyla Proteobacteria and Firmicutes. Beta-diversity analysis showed that, the hot springs microbial communities were clustered primarily on the basis of temperature and salinity. In a subsequent study, intensive shotgun metagenome sequencing was performed for the SK due to its unique physicochemicals and microbiome characteristics. The sulphur, nitrogen, and carbon metabolism within the SK community were evaluated, and the data revealed diversity in terms of metabolic activity and dynamics. It is also speculated that symbiotic relationships occur between the members of the community. Hidden within a wooded area, the SK hot spring is continually fed by fallen leaves, twigs, branches, and even tree trunks. Thus, the SK microbial community is likely to be able to acquire carbon sources from these surrounding natural organic matters. Gene mining on novel thermostable ß-glucosidases that are putatively important for biomass saccharification was performed and 282 sequences were found. Gene contig2844_15 (denoted as dt-bgl) with 99% identity closest to Anoxybacillus sp. DT3-1 was selected for characterisation, since the putative enzyme and its homologues have not been biochemically investigated. The dt-bgl with 1,359-bp long was synthesised by GenScript and cloned in the pET-28a expression vector. Sequence analysis indicated that DT-Bgl belonged to the GH family 1. DT-Bgl exhibited optimum activity at 70°C and pH 8.5. DT-Bgl was highly active on cello-oligosaccharides and p-nitrophenyl-ß-D-glucopyranoside and displayed high glucose tolerance by retaining 93% enzymatic activity in the presence of 10 M glucose. The enzyme may be used to improve the conversion rate of cellulosic biomass to glucose. The results of this work reveal the ecological aspect of thermophiles in Malaysian hot springs, and the potential of exploiting thermozymes for biotechnology application
Description
Thesis (PhD. (Biosciences))
Keywords
Enzymes—Research, Enzymes—Biotechnology, Microbial diversity—Malaysia, Thermophilic bacteria