Staff View: Biochemical characterisation and structure determination of a novel cold-active proline iminopeptidase from the psychrophilic yeast, Glaciozyma antarctica PI12

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:	Kamaruddin, Shazilah, Ahmad Redzuan, Rohaiza, Minor, Nurulermila, Wan Seman, Wan Mohd. Khairulikhsan, Md. Tab, Mahzan, Jaafar, Nardiah Rizwana, Ahmad Rodzli, Nazahiyah, Jonet, Mohd. Anuar, Bharudin, Izwan, Yusof, Nur Athirah, Quay, Doris Huai Xia
Format:	Article
Language:	English
Published:	MDPI 2022
Subjects:	TP Chemical technology
Online Access:	http://eprints.utm.my/id/eprint/101345/1/NardiahRizwanaJaafar2022_BiochemicalCharacterisationandStructureDeterminationofaNovel.pdf http://eprints.utm.my/id/eprint/101345/ http://dx.doi.org/10.3390/catal12070722
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spelling	my.utm.1013452023-06-08T09:35:10Z http://eprints.utm.my/id/eprint/101345/ Biochemical characterisation and structure determination of a novel cold-active proline iminopeptidase from the psychrophilic yeast, Glaciozyma antarctica PI12 Kamaruddin, Shazilah Ahmad Redzuan, Rohaiza Minor, Nurulermila Wan Seman, Wan Mohd. Khairulikhsan Md. Tab, Mahzan Jaafar, Nardiah Rizwana Ahmad Rodzli, Nazahiyah Jonet, Mohd. Anuar Bharudin, Izwan Yusof, Nur Athirah Quay, Doris Huai Xia TP Chemical technology 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. MDPI 2022 Article PeerReviewed application/pdf en http://eprints.utm.my/id/eprint/101345/1/NardiahRizwanaJaafar2022_BiochemicalCharacterisationandStructureDeterminationofaNovel.pdf Kamaruddin, Shazilah and Ahmad Redzuan, Rohaiza and Minor, Nurulermila and Wan Seman, Wan Mohd. Khairulikhsan and Md. Tab, Mahzan and Jaafar, Nardiah Rizwana and Ahmad Rodzli, Nazahiyah and Jonet, Mohd. Anuar and Bharudin, Izwan and Yusof, Nur Athirah and Quay, Doris Huai Xia (2022) Biochemical characterisation and structure determination of a novel cold-active proline iminopeptidase from the psychrophilic yeast, Glaciozyma antarctica PI12. Catalysts, 12 (7). pp. 1-14. ISSN 2073-4344 http://dx.doi.org/10.3390/catal12070722 DOI : 10.3390/catal12070722
institution	Universiti Teknologi Malaysia
building	UTM Library
collection	Institutional Repository
continent	Asia
country	Malaysia
content_provider	Universiti Teknologi Malaysia
content_source	UTM Institutional Repository
url_provider	http://eprints.utm.my/
language	English
topic	TP Chemical technology
spellingShingle	TP Chemical technology Kamaruddin, Shazilah Ahmad Redzuan, Rohaiza Minor, Nurulermila Wan Seman, Wan Mohd. Khairulikhsan Md. Tab, Mahzan Jaafar, Nardiah Rizwana Ahmad Rodzli, Nazahiyah Jonet, Mohd. Anuar Bharudin, Izwan Yusof, Nur Athirah Quay, Doris Huai Xia 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	Kamaruddin, Shazilah Ahmad Redzuan, Rohaiza Minor, Nurulermila Wan Seman, Wan Mohd. Khairulikhsan Md. Tab, Mahzan Jaafar, Nardiah Rizwana Ahmad Rodzli, Nazahiyah Jonet, Mohd. Anuar Bharudin, Izwan Yusof, Nur Athirah Quay, Doris Huai Xia
author_facet	Kamaruddin, Shazilah Ahmad Redzuan, Rohaiza Minor, Nurulermila Wan Seman, Wan Mohd. Khairulikhsan Md. Tab, Mahzan Jaafar, Nardiah Rizwana Ahmad Rodzli, Nazahiyah Jonet, Mohd. Anuar Bharudin, Izwan Yusof, Nur Athirah Quay, Doris Huai Xia
author_sort	Kamaruddin, Shazilah
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	MDPI
publishDate	2022
url	http://eprints.utm.my/id/eprint/101345/1/NardiahRizwanaJaafar2022_BiochemicalCharacterisationandStructureDeterminationofaNovel.pdf http://eprints.utm.my/id/eprint/101345/ http://dx.doi.org/10.3390/catal12070722
_version_	1769842041068453888
score	13.211869

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

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