Characterization of ligninolytic agrobacterium sp. Strain s2 and identification of the lignin depolymerization enzymes involved
The abundant availability of lignocellulosic biomass has contributed to an interest in the conversion of biomass into bioethanol or biochemical products via a sustainable pre-treatment process to break down the recalcitrant lignin structure. This study attempts to characterize the ligninolytic Agrob...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2022
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| Subjects: | |
| Online Access: | http://eprints.utm.my/id/eprint/99718/1/UmmuHabibahFaisalMMJIIT2022.pdf |
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| Summary: | The abundant availability of lignocellulosic biomass has contributed to an interest in the conversion of biomass into bioethanol or biochemical products via a sustainable pre-treatment process to break down the recalcitrant lignin structure. This study attempts to characterize the ligninolytic Agrobacterium sp. strain S2 isolated from empty fruit bunch (EFB) and subsequently to identify the lignin depolymerization enzymes involved. The strain was previously isolated from decaying EFB that was left for more than six months at a plantation. Biochemical characterization and quantitate study were administered to study the lignin depolymerization and degrading ability of the strain. The strain grew in minimal media with alkali lignin as the sole carbon source and it reached maximum growth on day three. Several known ligninolytic enzyme assays were performed lignin peroxidase (LiP), laccase (Lac) and manganese peroxidase (MnP) activities had been detected. Gel permeation chromatography (GPC) analysis was administered to confirm the strain’s ability to depolymerize or degrade lignin macromolecules. The alkali lignin treated with strain S2 was depolymerized to 2,263 Da on day three and further degraded to 1,004 Da on day 7, achieving 70.48% lignin degradation. After confirming the lignin-degrading ability of the respective strain, whole-genome sequencing was administered. The strain was identified as species that belongs to the Agrobacterium genus. The draft genome revealed genes encoding enzymes responsible for degradation of high and low molecular lignins. Catalase peroxidase (CP), superoxide dismutase (SD), non-heme chloroperoxidase (NCP) and cytochrome P450 (CP450) were selected for molecular study because these enzymes were believed to be involved in lignin depolymerization. The enzymes were selected based on the inferred homology via Basic Local Alignment Search Tool (BLAST), a sequence alignment of the target proteins against the databases. Genes encoding the metabolism of peripheral pathways for catabolism of aromatic compounds and central aromatic intermediates were also predicted. TA cloning and heterologous expression of genes encoding enzymes of interest in E.coli (JMP109) were administered via recombinant DNA technology and a recombinant CP450 was successfully expressed. The expression of CP450 confirmed the ease of genetic modification and recombinant efficiency in bacteria of smaller genome. CP450 is known to catalyse aromatic O-demethylation, a rate-limiting step in the conversion of aromatic compounds to valuable chemicals. In conclusion, the Agrobacterium sp. strain studied has demonstrated promising ligninolytic potential and a variety of enzyme candidates for future study. |
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