Utilizing Antarctic Metagenomic Resources for the Identification of Hydrolases
Permanently cold environments are populated by a diversity of microorganisms that possess cold-adapted enzymes with potential biotechnological applications. Advances in molecular techniques have enabled bioprospecting of novel enzymes from extreme environments without tedious cultivation efforts. Cl...
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Format: | Doctoral or Postdoctoral Thesis |
Language: | English |
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UNSW, Sydney
2014
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ftunswworks:oai:unsworks.library.unsw.edu.au:1959.4/53553 2023-05-15T13:55:10+02:00 Utilizing Antarctic Metagenomic Resources for the Identification of Hydrolases Mohd Omar, Suhaila 2014 application/pdf http://hdl.handle.net/1959.4/53553 https://unsworks.unsw.edu.au/bitstreams/0480a36c-8e08-4d5f-97e2-607fb3c74cac/download https://doi.org/10.26190/unsworks/16866 EN eng UNSW, Sydney http://hdl.handle.net/1959.4/53553 https://unsworks.unsw.edu.au/bitstreams/0480a36c-8e08-4d5f-97e2-607fb3c74cac/download https://doi.org/10.26190/unsworks/16866 open access https://purl.org/coar/access_right/c_abf2 CC BY-NC-ND 3.0 https://creativecommons.org/licenses/by-nc-nd/3.0/au/ free_to_read CC-BY-NC-ND Hydrolase Metagenomics Antarctic doctoral thesis http://purl.org/coar/resource_type/c_db06 2014 ftunswworks https://doi.org/10.26190/unsworks/16866 2022-08-09T07:43:59Z Permanently cold environments are populated by a diversity of microorganisms that possess cold-adapted enzymes with potential biotechnological applications. Advances in molecular techniques have enabled bioprospecting of novel enzymes from extreme environments without tedious cultivation efforts. Cloning and sequencing of the metagenomic DNA from the water column of the meromictic Antarctic lake, Ace Lake, enabled enzyme identification by sequence-based searches and functional screening for enzyme activity. The sequence-based search for hydrolases using profile Hidden Markov Models indicated the presence of uncharacterized subtilases, lipases and glycoside hydrolases in the metagenomic dataset. Information from the bioinformatic analysis was utilized to perform targeted functional screening of clones containing the candidate hydrolase genes. Alongside this targeted approach, ~20000 clones from the Ace Lake metagenomic library were randomly screened via agar-based assay. As both functional screening approaches yielded very low positive identifications, the capacity of the sequence-based screening to detect bona fide hydrolases was further tested by cloning selected subtilase genes and overexpressing them in Escherichia coli. One of the overexpressed subtilase genes showed proteolytic activity when tested with N-succinyl-Ala-Ala-Pro-Phe-ρ-nitroanilide. Finally, in order to get a bigger picture of the abundance of subtilase and other peptidase genes in Ace Lake, taxonomic affiliation and relative abundance of all candidate peptidases were assessed. The Ace Lake peptidases composition was also compared to peptidases in Organic Lake and Southern Ocean metagenomic datasets. The analysis indicated an abundance of diverse metallopeptidases and serine peptidases in all three datasets. Peptidases related to protein and membrane regulatory control and chaperones with peptidase activity were the most abundant groups, which are linked to microbial survival as a response to environmental stress. In conclusion, this study ... Doctoral or Postdoctoral Thesis Antarc* Antarctic Southern Ocean UNSW Sydney (The University of New South Wales): UNSWorks Ace Lake ENVELOPE(78.188,78.188,-68.472,-68.472) Antarctic Organic Lake ENVELOPE(78.190,78.190,-68.457,-68.457) Southern Ocean |
institution |
Open Polar |
collection |
UNSW Sydney (The University of New South Wales): UNSWorks |
op_collection_id |
ftunswworks |
language |
English |
topic |
Hydrolase Metagenomics Antarctic |
spellingShingle |
Hydrolase Metagenomics Antarctic Mohd Omar, Suhaila Utilizing Antarctic Metagenomic Resources for the Identification of Hydrolases |
topic_facet |
Hydrolase Metagenomics Antarctic |
description |
Permanently cold environments are populated by a diversity of microorganisms that possess cold-adapted enzymes with potential biotechnological applications. Advances in molecular techniques have enabled bioprospecting of novel enzymes from extreme environments without tedious cultivation efforts. Cloning and sequencing of the metagenomic DNA from the water column of the meromictic Antarctic lake, Ace Lake, enabled enzyme identification by sequence-based searches and functional screening for enzyme activity. The sequence-based search for hydrolases using profile Hidden Markov Models indicated the presence of uncharacterized subtilases, lipases and glycoside hydrolases in the metagenomic dataset. Information from the bioinformatic analysis was utilized to perform targeted functional screening of clones containing the candidate hydrolase genes. Alongside this targeted approach, ~20000 clones from the Ace Lake metagenomic library were randomly screened via agar-based assay. As both functional screening approaches yielded very low positive identifications, the capacity of the sequence-based screening to detect bona fide hydrolases was further tested by cloning selected subtilase genes and overexpressing them in Escherichia coli. One of the overexpressed subtilase genes showed proteolytic activity when tested with N-succinyl-Ala-Ala-Pro-Phe-ρ-nitroanilide. Finally, in order to get a bigger picture of the abundance of subtilase and other peptidase genes in Ace Lake, taxonomic affiliation and relative abundance of all candidate peptidases were assessed. The Ace Lake peptidases composition was also compared to peptidases in Organic Lake and Southern Ocean metagenomic datasets. The analysis indicated an abundance of diverse metallopeptidases and serine peptidases in all three datasets. Peptidases related to protein and membrane regulatory control and chaperones with peptidase activity were the most abundant groups, which are linked to microbial survival as a response to environmental stress. In conclusion, this study ... |
format |
Doctoral or Postdoctoral Thesis |
author |
Mohd Omar, Suhaila |
author_facet |
Mohd Omar, Suhaila |
author_sort |
Mohd Omar, Suhaila |
title |
Utilizing Antarctic Metagenomic Resources for the Identification of Hydrolases |
title_short |
Utilizing Antarctic Metagenomic Resources for the Identification of Hydrolases |
title_full |
Utilizing Antarctic Metagenomic Resources for the Identification of Hydrolases |
title_fullStr |
Utilizing Antarctic Metagenomic Resources for the Identification of Hydrolases |
title_full_unstemmed |
Utilizing Antarctic Metagenomic Resources for the Identification of Hydrolases |
title_sort |
utilizing antarctic metagenomic resources for the identification of hydrolases |
publisher |
UNSW, Sydney |
publishDate |
2014 |
url |
http://hdl.handle.net/1959.4/53553 https://unsworks.unsw.edu.au/bitstreams/0480a36c-8e08-4d5f-97e2-607fb3c74cac/download https://doi.org/10.26190/unsworks/16866 |
long_lat |
ENVELOPE(78.188,78.188,-68.472,-68.472) ENVELOPE(78.190,78.190,-68.457,-68.457) |
geographic |
Ace Lake Antarctic Organic Lake Southern Ocean |
geographic_facet |
Ace Lake Antarctic Organic Lake Southern Ocean |
genre |
Antarc* Antarctic Southern Ocean |
genre_facet |
Antarc* Antarctic Southern Ocean |
op_relation |
http://hdl.handle.net/1959.4/53553 https://unsworks.unsw.edu.au/bitstreams/0480a36c-8e08-4d5f-97e2-607fb3c74cac/download https://doi.org/10.26190/unsworks/16866 |
op_rights |
open access https://purl.org/coar/access_right/c_abf2 CC BY-NC-ND 3.0 https://creativecommons.org/licenses/by-nc-nd/3.0/au/ free_to_read |
op_rightsnorm |
CC-BY-NC-ND |
op_doi |
https://doi.org/10.26190/unsworks/16866 |
_version_ |
1766261448595472384 |