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Biochemical characterisation and structure determination of a novel cold-active Proline iminopeptidase from the Psychrophilic yeast, Glaciozyma antarctica PI12

Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively ch...

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Main Authors: Shazilah Kamaruddin, Rohaiza Ahmad Redzuan, Nurulermila Minor, Wan Mohd Khairulikhsan Wan Seman, Mahzan Md Tab, Nardiah Rizwana Jaafar, Nazahiyah Ahmad Rodzli, Mohd Anuar Jonet, Izwan Bharudin, Nur Athirah Yusof, Quay, Doris Huai Xia, Nor Muhammad Mahadi, Abdul Munir Abdul Murad, Farah Diba Abu Bakar
Format: Article
Language:English
English
Published: Multidisciplinary Digital Publishing Institute (MDPI) 2022
Subjects:
QR1-502 Microbiology
Online Access:https://eprints.ums.edu.my/id/eprint/33921/1/Biochemical%20characterisation%20and%20structure%20determination%20of%20a%20novel%20cold-active%20proline%20iminopeptidase%20from%20the%20Psychrophilic%20yeast%2C%20Glaciozyma%20antarctica%20PI12%20_ABSTRACT.pdf
https://eprints.ums.edu.my/id/eprint/33921/3/Biochemical%20characterisation%20and%20structure%20determination%20of%20a%20novel%20cold-active%20proline%20iminopeptidase%20from%20the%20Psychrophilic%20yeast%2C%20Glaciozyma%20antarctica%20PI12.pdf
https://eprints.ums.edu.my/id/eprint/33921/
https://www.mdpi.com/2073-4344/12/7/722/htm
https://doi.org/10.3390/catal12070722
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spelling my.ums.eprints.339212022-08-24T06:48:52Z https://eprints.ums.edu.my/id/eprint/33921/ Biochemical characterisation and structure determination of a novel cold-active Proline iminopeptidase from the Psychrophilic yeast, Glaciozyma antarctica PI12 Shazilah Kamaruddin Rohaiza Ahmad Redzuan Nurulermila Minor Wan Mohd Khairulikhsan Wan Seman Mahzan Md Tab Nardiah Rizwana Jaafar Nazahiyah Ahmad Rodzli Mohd Anuar Jonet Izwan Bharudin Nur Athirah Yusof Quay, Doris Huai Xia Nor Muhammad Mahadi Abdul Munir Abdul Murad Farah Diba Abu Bakar QR1-502 Microbiology Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively characterised in bacteria and filamentous fungi. However, no similar reports exist for yeasts. In this study, a protease gene from Glaciozyma antarctica designated as GaPIP was cloned and overexpressed in Escherichia coli. Sequence analyses of the gene revealed a 960 bp open reading frame encoding a 319 amino acid protein (35,406 Da). The purified recombinant GaPIP showed a specific activity of 3561 Umg−1 towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. GaPIP is a cold-active enzyme with an optimum activity of 30◦ C at pH 7.0. The enzyme is stable between pH 7.0 and 8.0 and able to retain its activity at 10–30◦ C. Although GaPIP is a serine protease, only 25% inhibition by the serine protease inhibitor, phenylmethanesulfonylfluoride (PMSF) was recorded. This enzyme is strongly inhibited by the presence of EDTA, suggesting that it is a metalloenzyme. The dimeric structure of GaPIP was determined at a resolution of 2.4 Å. To date, GaPIP is the first characterised PIP from yeasts and the structure of GaPIP is the first structure for PIP from eukaryotes. Multidisciplinary Digital Publishing Institute (MDPI) 2022 Article PeerReviewed text en https://eprints.ums.edu.my/id/eprint/33921/1/Biochemical%20characterisation%20and%20structure%20determination%20of%20a%20novel%20cold-active%20proline%20iminopeptidase%20from%20the%20Psychrophilic%20yeast%2C%20Glaciozyma%20antarctica%20PI12%20_ABSTRACT.pdf text en https://eprints.ums.edu.my/id/eprint/33921/3/Biochemical%20characterisation%20and%20structure%20determination%20of%20a%20novel%20cold-active%20proline%20iminopeptidase%20from%20the%20Psychrophilic%20yeast%2C%20Glaciozyma%20antarctica%20PI12.pdf Shazilah Kamaruddin and Rohaiza Ahmad Redzuan and Nurulermila Minor and Wan Mohd Khairulikhsan Wan Seman and Mahzan Md Tab and Nardiah Rizwana Jaafar and Nazahiyah Ahmad Rodzli and Mohd Anuar Jonet and Izwan Bharudin and Nur Athirah Yusof and Quay, Doris Huai Xia and Nor Muhammad Mahadi and Abdul Munir Abdul Murad and Farah Diba Abu Bakar (2022) Biochemical characterisation and structure determination of a novel cold-active Proline iminopeptidase from the Psychrophilic yeast, Glaciozyma antarctica PI12. Catalysts, 12 (722). pp. 1-14. ISSN 2073-4344 https://www.mdpi.com/2073-4344/12/7/722/htm https://doi.org/10.3390/catal12070722
institution Universiti Malaysia Sabah
building UMS Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Malaysia Sabah
content_source UMS Institutional Repository
url_provider http://eprints.ums.edu.my/
language English
English
topic QR1-502 Microbiology
spellingShingle QR1-502 Microbiology
Shazilah Kamaruddin
Rohaiza Ahmad Redzuan
Nurulermila Minor
Wan Mohd Khairulikhsan Wan Seman
Mahzan Md Tab
Nardiah Rizwana Jaafar
Nazahiyah Ahmad Rodzli
Mohd Anuar Jonet
Izwan Bharudin
Nur Athirah Yusof
Quay, Doris Huai Xia
Nor Muhammad Mahadi
Abdul Munir Abdul Murad
Farah Diba Abu Bakar
Biochemical characterisation and structure determination of a novel cold-active Proline iminopeptidase from the Psychrophilic yeast, Glaciozyma antarctica PI12
description Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively characterised in bacteria and filamentous fungi. However, no similar reports exist for yeasts. In this study, a protease gene from Glaciozyma antarctica designated as GaPIP was cloned and overexpressed in Escherichia coli. Sequence analyses of the gene revealed a 960 bp open reading frame encoding a 319 amino acid protein (35,406 Da). The purified recombinant GaPIP showed a specific activity of 3561 Umg−1 towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. GaPIP is a cold-active enzyme with an optimum activity of 30◦ C at pH 7.0. The enzyme is stable between pH 7.0 and 8.0 and able to retain its activity at 10–30◦ C. Although GaPIP is a serine protease, only 25% inhibition by the serine protease inhibitor, phenylmethanesulfonylfluoride (PMSF) was recorded. This enzyme is strongly inhibited by the presence of EDTA, suggesting that it is a metalloenzyme. The dimeric structure of GaPIP was determined at a resolution of 2.4 Å. To date, GaPIP is the first characterised PIP from yeasts and the structure of GaPIP is the first structure for PIP from eukaryotes.
format Article
author Shazilah Kamaruddin
Rohaiza Ahmad Redzuan
Nurulermila Minor
Wan Mohd Khairulikhsan Wan Seman
Mahzan Md Tab
Nardiah Rizwana Jaafar
Nazahiyah Ahmad Rodzli
Mohd Anuar Jonet
Izwan Bharudin
Nur Athirah Yusof
Quay, Doris Huai Xia
Nor Muhammad Mahadi
Abdul Munir Abdul Murad
Farah Diba Abu Bakar
author_facet Shazilah Kamaruddin
Rohaiza Ahmad Redzuan
Nurulermila Minor
Wan Mohd Khairulikhsan Wan Seman
Mahzan Md Tab
Nardiah Rizwana Jaafar
Nazahiyah Ahmad Rodzli
Mohd Anuar Jonet
Izwan Bharudin
Nur Athirah Yusof
Quay, Doris Huai Xia
Nor Muhammad Mahadi
Abdul Munir Abdul Murad
Farah Diba Abu Bakar
author_sort Shazilah Kamaruddin
title Biochemical characterisation and structure determination of a novel cold-active Proline iminopeptidase from the Psychrophilic yeast, Glaciozyma antarctica PI12
title_short Biochemical characterisation and structure determination of a novel cold-active Proline iminopeptidase from the Psychrophilic yeast, Glaciozyma antarctica PI12
title_full Biochemical characterisation and structure determination of a novel cold-active Proline iminopeptidase from the Psychrophilic yeast, Glaciozyma antarctica PI12
title_fullStr Biochemical characterisation and structure determination of a novel cold-active Proline iminopeptidase from the Psychrophilic yeast, Glaciozyma antarctica PI12
title_full_unstemmed Biochemical characterisation and structure determination of a novel cold-active Proline iminopeptidase from the Psychrophilic yeast, Glaciozyma antarctica PI12
title_sort biochemical characterisation and structure determination of a novel cold-active proline iminopeptidase from the psychrophilic yeast, glaciozyma antarctica pi12
publisher Multidisciplinary Digital Publishing Institute (MDPI)
publishDate 2022
url https://eprints.ums.edu.my/id/eprint/33921/1/Biochemical%20characterisation%20and%20structure%20determination%20of%20a%20novel%20cold-active%20proline%20iminopeptidase%20from%20the%20Psychrophilic%20yeast%2C%20Glaciozyma%20antarctica%20PI12%20_ABSTRACT.pdf
https://eprints.ums.edu.my/id/eprint/33921/3/Biochemical%20characterisation%20and%20structure%20determination%20of%20a%20novel%20cold-active%20proline%20iminopeptidase%20from%20the%20Psychrophilic%20yeast%2C%20Glaciozyma%20antarctica%20PI12.pdf
https://eprints.ums.edu.my/id/eprint/33921/
https://www.mdpi.com/2073-4344/12/7/722/htm
https://doi.org/10.3390/catal12070722
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score 13.211869

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