Wan Nur Shuhaida Wan Mahadi (2020) Cold-adapted mannan-degrading enzymes and genes from the antarctic isolates arthrobacter sp. EM1 and ES1. Masters thesis, Universiti Malaysia Sabah.
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Abstract
Mannan-degrading enzymes refer to a group of enzymes that catalyze the hydrolysis of glyosidic bonds in mannan-based polysaccharides. Among mannan-degrading enzymes, mannanases are considered as major mannan-degrading enzymes that cleave the mannan-backbone chain. Mannanases are divided into endo-mannanases and exomannanases (mannosidases), which cleaves the internal and external linkages of the mannan-backbone, respectively. The enzymes are widely applicable in various industries, including food, feed and stock, detergent production, pharmaceutical, and in pulp and paper industries. Mannanases were found in various organisms; yet, mannanases produced by microorganisms are most promising to be investigated because of its low cost, high generation rate and promptly controlled conditions. To date no reports on the heterologous study of cold-adapted mannanases, so far. Coldadapted enzymes are enzymes having specific enzymatic activity relatively higher at lower temperature (near 0 ° C). Cold adapted enzymes are gaining wide interest in industrial applications conducted at low temperature. Previously, Arthrobacter sp. ESl and EM1, which were isolated from Schirmacher Oasis, Antarctic, were found to exhibit mannan-degrading activities. These strains were found to be cold adapted bacteria which lead to an assumption that the enzymes involved in the mannan-degrading activity might belong to cold adapted enzymes. Whole genome sequence of ES1 strain demonstrated the presence of endo-a-mannanase (AmanESl), which might be responsible for the mannan-degradation. Nevertheless, the gene responsible of mannan-degrading activity of EMl strain remains unknown. This study is aimed to firstly determine the enzymatic properties AmanES1 and secondly identify and characterize the gene responsible for mannan-degradation of EM1 strain. To address, the gene encoding AmanESl was cloned into pET100/TOPO and transformed into Escherichia coli BL21 (DE3) CodonPlus and was successfully overexpressed in a soluble form at 15°C for 16 hours. The expressed AmanESl was then successfully purified under a single purification step of Ni-NTA chromatography yielding 17.5 mg of protein from 1 L culture. Enzymatic activity of AmanESl was measured using 3,5-dinitrosalicylic acid (ONS-assay), which showed that the optimum activity of this enzyme was found to be at 15°C with the specific activity of 0. 75 U/ml and catalytic efficiency U<caJ KM) of 3.83 x 10·9 μM·1s·1. This indicated that AmanESl is typically a cold adapted enzyme which is also supported by the amino acid profile of this enzyme. Further, structural homology modelling indicated that AmanESl forms a canonical structure of GH76 family with (a/a)5 barrel motif representing a steady fold for presenting active-site residues in a cleft at the N-terminal ends of the inner a helices. Further in silica followed by mutagenesis analysis also suggested that two consecutive aspartic acid residues, D94 and D95, were predicted to be the catalytic residues. In addition, EM1 strain was proven to be a cold adapted bacterium as the optimum growth temperature for this strain was found to be at 15°C. Meanwhile, mannan-degrading activity of EMl strain as observed under guar gum plate assay showed that the activity is only observed at 15°C, but not at 37°C. Further, whole genome sequence analysis of EM1 strain using PacBio RSII sequencing system indicated that the presence of the genes encoding cold shcok protein A (CspA) and and cold shock protein C (CspC), which might be the important genes for cold adaptation of the EM1 strain. More interestingly, the genome sequence of EM1 strain also indicated the existence of a gene encoding amannosidase (EC 3.2.1.24). The presence of a-mannosidase, instead of a-mannanse, suggested that the mannan-degrading activity in Arthobacter sp. does not necessarily require a-mannanase.
| Item Type: | Thesis (Masters) |
|---|---|
| Keyword: | Mannan-degrading enzymes, Cold-adapted enzymes, Mannanases, Exomannanases, Endo-mannanases, Cold shock proteins, Enzyme activity |
| Subjects: | Q Science > QR Microbiology > QR1-502 Microbiology > QR75-99.5 Bacteria |
| Department: | INSTITUTE > Institute for Tropical Biology and Conservation |
| Depositing User: | DG MASNIAH AHMAD - |
| Date Deposited: | 17 Jan 2025 14:20 |
| Last Modified: | 17 Jan 2025 14:20 |
| URI: | https://eprints.ums.edu.my/id/eprint/42633 |
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