Molecular characterisation of lignocellulose degradation of roseithermus sacchariphilus strain RA

• Main Author: Liew, Kok Jun
• Format: Thesis
• Language: English
• Published: 2020
• Subjects: Q Science (General)
• Online Access: http://eprints.utm.my/id/eprint/102446/1/LiewKokJunPFS2020.pdf.pdf; http://eprints.utm.my/id/eprint/102446/; http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:146026

An enormous amout of available scientific publications are experimental findings and knowledge of well-known prokaryotic strains. Very little attention has been given to uncommon prokaryotes. The current work focused on the molecular characteristic of strain RA, a minority bacterium of Ayer Hangat hot spring, to understand its strategies to survive the harsh environment. The work also aimed to investigate the ability of strain RA to hydrolyse lignocellulosic compounds and its role in microbial consortium of the hot spring. This work used methods of microbiology, genomic, transcriptomic, molecular biology, enzymology, and bioinformatics. Strain RA is the closest in similarity with Roseithermus sacchariphilus strain MEBiC09517T with slight differences in its genome rearrangement and sets of unique genes. Strain RA is a halo-thermophile which uses thermophilic lipids, has high genome GC, stable enzyme, and ‘salt-in’ strategy by different transporters to thrive in the salted and heated ecosystem. When strain RA was cultured in MB media added with xylan, the bacterium exhibited higher growth rate, better enzymatic activities, and greater degree of differentially expressed genes which was significantly higher than when other carbon sources (EFB, CMC, glucose, and xylose) were used. Genes encoding auxiliary activity enzymes (AA), proteins that act on lignin, were non-responsive in all of the experimental setups. Strain RA harbours 54 glycosyl hydrolase (GH) genes that are affiliated with 30 families. Majority of these genes, especially cellulose and hemicellulose-acting GHs, were upregulated when strain RA was grown in MB+xylan. Xylanase XynRA1 was one of the upregulated GHs. Recombinant protein of the enzyme was purified and biochemically characterised. XynRA1 achieved maximum activity at pH 8 and 60°C, and exhibited an activity half-life (t1/2) of at least one hour at 50-60°C. It can thus be concluded that strain RA is a subspecies of R. sacchariphilus. Findings accumulated in this work showed that strain RA adapts itself to a warm and salty environment by multiple strategies, instead of using a single approach. Even though strain RA is a minority in-situ, it plays a role in plant litter decomposition using, in particular the hemicellulose fraction. Strain RA most probably collaborates with other bacteria to form a multi-species biofilm that attach to the plant litter for complete degradation. The bacterium is a rare species in the hot spring because of its slow growth rate, and is less competitive compared to other prokaryotes due to its low preference for glucose. To better survive against ecological changes, strain RA generates non-homologous halo-thermostable enzymes with unique biochemical characteristics. The current work successfully elucidated the molecular features of R. sacchariphilus strain RA. It is proposed that strain RA can be an excellent candidate to be included in bacteria consortia for lignocellulosic biomass degradation or used in enzyme cocktail formulation.