Functional metagenomics approach for discovery of novel cold-active protease from the antarctic region

The structural complexity of bacterial life makes most of it impossible to culture. Functional metagenomics approaches overcome the limitations of a culture-based approach in exploring and assessing the genetic materials of uncultured microbes. The objective of this study was to identify clones with...

Full description

Saved in:
Bibliographic Details
Main Authors: Abd Latip, Muhammad Asyraf, Nordin, Noor Faizul Hadry, Alias, Siti Aisyah, Smykla, Jerzy, Yusof, Faridah, Mohamad, Mohd Azrul Naim
Format: Article
Language:English
English
Published: IIUM Press, International Islamic University Malaysia 2024
Subjects:
Online Access:http://irep.iium.edu.my/118026/1/118026_Functional%20Metagenomics%20Approach.pdf
http://irep.iium.edu.my/118026/2/118026_Functional%20Metagenomics%20Approach_SCOPUS.pdf
http://irep.iium.edu.my/118026/
https://journals.iium.edu.my/ejournal/index.php/iiumej/article/view/3090
https://doi.org/10.31436/iiumej.v25i2.3090
Tags: Add Tag
No Tags, Be the first to tag this record!
Institution: Universiti Islam Antarabangsa Malaysia
Language: English
English
id my.iium.irep.118026
record_format dspace
spelling my.iium.irep.1180262025-01-04T14:43:19Z http://irep.iium.edu.my/118026/ Functional metagenomics approach for discovery of novel cold-active protease from the antarctic region Abd Latip, Muhammad Asyraf Nordin, Noor Faizul Hadry Alias, Siti Aisyah Smykla, Jerzy Yusof, Faridah Mohamad, Mohd Azrul Naim QR Microbiology TP248.13 Biotechnology The structural complexity of bacterial life makes most of it impossible to culture. Functional metagenomics approaches overcome the limitations of a culture-based approach in exploring and assessing the genetic materials of uncultured microbes. The objective of this study was to identify clones with cold-active proteases through functional metagenomics. In this work, the environmental DNA (eDNA) isolated directly from Antarctic soils was ligated into the pCC1FOS fosmid vector, transformed into EPI300-T1R E. coli host cells, and screened for proteolytic enzymes. Positive protease-producing clones were identified and isolated on skim milk agar supplemented with chloramphenicol and arabinose. This clone harbored a fosmid, pCC1FOS, which has a 48.5 kb insert that has been completely sequenced in both directions. Further analysis of the insert showed 70 NODEs. The NODE_42 encoded hypothetical protein of 297 amino acids showed a significant match to Peptidase M23 and PG-binding 1 proteins families. A three-dimensional model of the predicted protease was generated based on the known mesophilic protease of Neisseria meningitides (PDB: 3SLU). The structural alignment showed 27.07 % similarity with RMSD value of 0.402 Å based on 58 aligned residues. The active site residues were identical, but major deletions were observed in the predicted proteases. This predicted protease showed higher activity at -20 °C and 20 °C than the positive control (protease from bovine pancreas). Functional metagenomics is a promising approach in the discovery of cold-active protease with low homology to the known sequences and expressed in the host cell that has the potential for bioprospecting in low-temperature applications. IIUM Press, International Islamic University Malaysia 2024-07-14 Article PeerReviewed application/pdf en http://irep.iium.edu.my/118026/1/118026_Functional%20Metagenomics%20Approach.pdf application/pdf en http://irep.iium.edu.my/118026/2/118026_Functional%20Metagenomics%20Approach_SCOPUS.pdf Abd Latip, Muhammad Asyraf and Nordin, Noor Faizul Hadry and Alias, Siti Aisyah and Smykla, Jerzy and Yusof, Faridah and Mohamad, Mohd Azrul Naim (2024) Functional metagenomics approach for discovery of novel cold-active protease from the antarctic region. IIUM Engineering Journal, 25 (2). pp. 69-82. ISSN 1511-788X E-ISSN 2289-7860 https://journals.iium.edu.my/ejournal/index.php/iiumej/article/view/3090 https://doi.org/10.31436/iiumej.v25i2.3090
institution Universiti Islam Antarabangsa Malaysia
building IIUM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider International Islamic University Malaysia
content_source IIUM Repository (IREP)
url_provider http://irep.iium.edu.my/
language English
English
topic QR Microbiology
TP248.13 Biotechnology
spellingShingle QR Microbiology
TP248.13 Biotechnology
Abd Latip, Muhammad Asyraf
Nordin, Noor Faizul Hadry
Alias, Siti Aisyah
Smykla, Jerzy
Yusof, Faridah
Mohamad, Mohd Azrul Naim
Functional metagenomics approach for discovery of novel cold-active protease from the antarctic region
description The structural complexity of bacterial life makes most of it impossible to culture. Functional metagenomics approaches overcome the limitations of a culture-based approach in exploring and assessing the genetic materials of uncultured microbes. The objective of this study was to identify clones with cold-active proteases through functional metagenomics. In this work, the environmental DNA (eDNA) isolated directly from Antarctic soils was ligated into the pCC1FOS fosmid vector, transformed into EPI300-T1R E. coli host cells, and screened for proteolytic enzymes. Positive protease-producing clones were identified and isolated on skim milk agar supplemented with chloramphenicol and arabinose. This clone harbored a fosmid, pCC1FOS, which has a 48.5 kb insert that has been completely sequenced in both directions. Further analysis of the insert showed 70 NODEs. The NODE_42 encoded hypothetical protein of 297 amino acids showed a significant match to Peptidase M23 and PG-binding 1 proteins families. A three-dimensional model of the predicted protease was generated based on the known mesophilic protease of Neisseria meningitides (PDB: 3SLU). The structural alignment showed 27.07 % similarity with RMSD value of 0.402 Å based on 58 aligned residues. The active site residues were identical, but major deletions were observed in the predicted proteases. This predicted protease showed higher activity at -20 °C and 20 °C than the positive control (protease from bovine pancreas). Functional metagenomics is a promising approach in the discovery of cold-active protease with low homology to the known sequences and expressed in the host cell that has the potential for bioprospecting in low-temperature applications.
format Article
author Abd Latip, Muhammad Asyraf
Nordin, Noor Faizul Hadry
Alias, Siti Aisyah
Smykla, Jerzy
Yusof, Faridah
Mohamad, Mohd Azrul Naim
author_facet Abd Latip, Muhammad Asyraf
Nordin, Noor Faizul Hadry
Alias, Siti Aisyah
Smykla, Jerzy
Yusof, Faridah
Mohamad, Mohd Azrul Naim
author_sort Abd Latip, Muhammad Asyraf
title Functional metagenomics approach for discovery of novel cold-active protease from the antarctic region
title_short Functional metagenomics approach for discovery of novel cold-active protease from the antarctic region
title_full Functional metagenomics approach for discovery of novel cold-active protease from the antarctic region
title_fullStr Functional metagenomics approach for discovery of novel cold-active protease from the antarctic region
title_full_unstemmed Functional metagenomics approach for discovery of novel cold-active protease from the antarctic region
title_sort functional metagenomics approach for discovery of novel cold-active protease from the antarctic region
publisher IIUM Press, International Islamic University Malaysia
publishDate 2024
url http://irep.iium.edu.my/118026/1/118026_Functional%20Metagenomics%20Approach.pdf
http://irep.iium.edu.my/118026/2/118026_Functional%20Metagenomics%20Approach_SCOPUS.pdf
http://irep.iium.edu.my/118026/
https://journals.iium.edu.my/ejournal/index.php/iiumej/article/view/3090
https://doi.org/10.31436/iiumej.v25i2.3090
_version_ 1821105174860529664