Agar is a marine heteropolysaccharide with repeating units consisting of 3,6-α-anhydro-L-galactopyranose and D-galactopyranose linked by α-(1,3) and β-(1,4) linkages. It has been promoted as a prospective replacement for petroleum-based feedstocks and other applications. Enzymatic biotransformation of agar generates high specific products: It is also more environmentally friendly than chemical hydrolysis. In particular, agarolytic bacteria and their agarases are preferred for the processing of agar into sugar derivatives.
Agar-producing macroalgae are one of Indonesia's national commodities. However, agar-based products and technology are rarely developed in Indonesia. This research is aimed to explore the potential of an Indonesian marine bacterium and its agarases as bioagents for agar bioprocessing. The research objectives are to identify the novelty of the isolate among known agarolytic bacteria using microbiology and molecular biology approaches, to elucidate the agarolytic system of the bacterium using in silico genome analysis, to express and characterize the recombinant agarases, and to elucidate their potential for producing agar-derived saccharides from Indonesian natural agar.
Microbulbifer elongatus PORT2 is a gram-negative marine bacterium that had been isolated from Batu Karas seawater, Pangandaran, West Java Indonesia. PORT2 shows potential as biocatalysts for agar saccharides conversion by showing remarkable agar liquefaction. The annotation of the draft genome identifies six putative β-agarases consist of three GH50, two GH86, and one GH16 in M. elongatus PORT2. Those agarases are clustered at two different contigs. Besides agarases, other genes for D-galactose and 3,6 anhydro-L galactose metabolism, sugar transports and regulatory system are found in the vicinity of the agarases clusters. Despite the ability to utilize agar as a sole carbon sole, PORT2 lacks any putative α-agarase GH117 or GH96. Both are responsible for the cleavage of α-glycosidic bonds in agar. Indeed, several hypothetical proteins are in the neighborhood of the agarase gene clusters in M. elongatus PORT2. They probably could have a function as the alternative machinery or pathway for agar monomerization that needs clarification in future research work.
Four recombinant β-agarases from PORT2; AgaA50, AgaB50, AgaC50, and AgaF16A have been successfully overexpressed in E.coli and characterized. The AgaA50 and AgaC50 exhibit metal-dependent activity. They perform exo-agarolytic modes and generates neoagarobiose (NA2). The AgaB50 can act as endo-and exo-β-agarase without any additional activator and produces neoagarohexaose (NA6), neoagarotetraose (NA4), and NA2. AgaF16 produces NA6 and NA4. The enzyme shows pure endo-catalytic action which thiol agents positively affect its activity. The synergetic reaction of AgaF16A and AgaA50 converts Indonesian Gelidium agar into NA2 and Gracilaria agar into modified NA2. The modified NA2 from Gracilaria agar could promise new potential bioactivity that is different from agarose-derived NA2 due to the presence of additional side chains on the saccharide backbone. The NA6, NA4, and NA2 products from agarose have shown potential pharmaceutical applications such as immunomodulator, anti-tumor, antioxidant, anti-diabetic, and moisturizer.
Despite being isolated from a mesophilic marine bacterium, the recombinant agarases from M. elongatus PORT2 are active at 50 °C and pH between 6.5 to 8. They maintain more than 75% of their activities even after 1 h preincubation at 50 °C, except for AgaC50. Their thermostability gives advantages for the effective biocatalytic conversion of agar because the substrate is more accessible at mild pH and the temperature above the sol-gel condition (> 40 °C).:Contents
1. Introduction 1
1.1. Motivation and Scientific Goals 1
1.2. Literature Review 3
2. Materials and Methods 12
2.1. Materials 12
2.2. Methods 13
3. Agarolytic Bacterium Microbulbifer elongatus PORT2 22
3.1. Results 22
3.2. Discussion 28
4. Genome Profiling for In Silico Elucidation of the Agarolytic System 32
4.1. Results 32
4.2. Discussion 41
5. Recombinant Agarases from Microbulbifer elongatus PORT2 44
5.1. Results 44
5.2. Discussion 71
6. Conclusions and Outlooks 78
References 81
Appendices 97
Acknowledgements 110
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:73107 |
| Date | 09 December 2020 |
| Creators | Anggraeni, Santi Rukminita |
| Contributors | Ansorge Schumacher, Marion B., Czjzek, Mirjam, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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