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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Bibliographic Details
Published in:Catalysts
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, Doris Quay Huai Xia, Nor Muhammad Mahadi, Abdul Munir Abdul Murad, Farah Diba Abu Bakar
Format: Article in Journal/Newspaper
Language:English
Published: MDPI AG 2022
Subjects:
microbial proteases
proline specific protease
cold-active enzyme
gluten hydrolysis
protein structure
Chemical technology
TP1-1185
Chemistry
QD1-999
Antarc*
Antarctica
Online Access:https://doi.org/10.3390/catal12070722
https://doaj.org/article/5f50c563bcda4acfac678d3b318152e8
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Summary: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 Ga PIP 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 Ga PIP showed a specific activity of 3561 Umg −1 towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. Ga PIP 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 Ga PIP 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 Ga PIP was determined at a resolution of 2.4 Å. To date, Ga PIP is the first characterised PIP from yeasts and the structure of Ga PIP is the first structure for PIP from eukaryotes.

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