A new broad specificity alkaline metalloprotease from a Pseudomonas sp. isolated from refrigerated milk: Role of calcium in improving enzyme productivity

Ertan, H; Cassel, C; Verma, A; Poljak, A; Charlton, T; Aldrich-Wright, J; Omar, SM; Siddiqui, KS; Cavicchioli, R

A new broad specificity alkaline metalloprotease from a Pseudomonas sp. isolated from refrigerated milk: Role of calcium in improving enzyme productivity

Ertan, H Cassel, C Verma, A

Poljak, A Charlton, T Aldrich-Wright, J Omar, SM Siddiqui, KS Cavicchioli, R

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Publication Type:: Journal Article
Citation:: Journal of Molecular Catalysis B: Enzymatic, 2015, 113 pp. 1 - 8
Issue Date:: 2015-01-01

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Field	Value	Language
dc.contributor.author	Ertan, H	en_US
dc.contributor.author	Cassel, C	en_US
dc.contributor.author	Verma, A https://orcid.org/0000-0003-0119-9915	en_US
dc.contributor.author	Poljak, A	en_US
dc.contributor.author	Charlton, T	en_US
dc.contributor.author	Aldrich-Wright, J	en_US
dc.contributor.author	Omar, SM	en_US
dc.contributor.author	Siddiqui, KS	en_US
dc.contributor.author	Cavicchioli, R	en_US
dc.date.issued	2015-01-01	en_US
dc.identifier.citation	Journal of Molecular Catalysis B: Enzymatic, 2015, 113 pp. 1 - 8	en_US
dc.identifier.issn	1381-1177	en_US
dc.identifier.uri	http://hdl.handle.net/10453/139044
dc.description.abstract	© 2015 Published by Elsevier B.V. Metalloproteases represent the largest fraction of the global enzyme market. For biotechnological purposes the accumulation of product (i.e. productivity) provides the best measure of assessing enzyme performance because it takes into account the interplay between activity, stability, activation and inhibition. Studies assessing the productivity of alkaline metalloproteases and chemicals that improve their productivity have not previously been reported. In this study we report the specificity, productivity, kinetic and thermodynamic properties of an extracellular protease, purified from a new strain of Pseudomonas sp. isolated from refrigerated milk. Mass spectrometry analysis revealed the enzyme is a serralysin-type alkaline metalloprotease, with broad cleavage-site specificity. By studying the effects of Ca2+ ion removal (using a chelator) and Ca2+ ion addition, conditions were identified that led to an increase in productivity by 300% (6.3 vs 1.9 mg azopeptide μg-1 enzyme at 40 °C). The basis for the enhanced productivity was linked to elevated melting temperatures of secondary (Tm 47 vs 38°C) and tertiary structure (Tm 50 vs 44°C), increased half-life of inactivation (t1/2 30 vs 4.9 min), increased optimum temperature (44 vs 36°C), and changes in both catalytic activity (kcat 3.3 vs 2.2 min-1) and substrate affinity (Km 3.9 vs 2.5 mg mL-1). Thermodynamic data were indicative of Ca2+-binding causing the transition-state to be more ordered (less entropy) relative to the folded-state, thereby resisting a transition to an unfolded state. The specificity, kinetics and response to calcium of this AMP illustrate its potential usefulness for industrial applications, and the research highlights the broader potential for using calcium to enhance the productivity of proteases.	en_US
dc.relation.ispartof	Journal of Molecular Catalysis B: Enzymatic	en_US
dc.relation.isbasedon	10.1016/j.molcatb.2014.12.010	en_US
dc.subject.classification	Biotechnology	en_US
dc.title	A new broad specificity alkaline metalloprotease from a Pseudomonas sp. isolated from refrigerated milk: Role of calcium in improving enzyme productivity	en_US
dc.type	Journal Article
utslib.citation.volume	113	en_US
utslib.for	0601 Biochemistry and Cell Biology	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - C3 - Climate Change Cluster
utslib.copyright.status	closed_access
pubs.publication-status	Published	en_US
pubs.volume	113	en_US

Abstract:

© 2015 Published by Elsevier B.V. Metalloproteases represent the largest fraction of the global enzyme market. For biotechnological purposes the accumulation of product (i.e. productivity) provides the best measure of assessing enzyme performance because it takes into account the interplay between activity, stability, activation and inhibition. Studies assessing the productivity of alkaline metalloproteases and chemicals that improve their productivity have not previously been reported. In this study we report the specificity, productivity, kinetic and thermodynamic properties of an extracellular protease, purified from a new strain of Pseudomonas sp. isolated from refrigerated milk. Mass spectrometry analysis revealed the enzyme is a serralysin-type alkaline metalloprotease, with broad cleavage-site specificity. By studying the effects of Ca2+ ion removal (using a chelator) and Ca2+ ion addition, conditions were identified that led to an increase in productivity by 300% (6.3 vs 1.9 mg azopeptide μg-1 enzyme at 40 °C). The basis for the enhanced productivity was linked to elevated melting temperatures of secondary (Tm 47 vs 38°C) and tertiary structure (Tm 50 vs 44°C), increased half-life of inactivation (t1/2 30 vs 4.9 min), increased optimum temperature (44 vs 36°C), and changes in both catalytic activity (kcat 3.3 vs 2.2 min-1) and substrate affinity (Km 3.9 vs 2.5 mg mL-1). Thermodynamic data were indicative of Ca2+-binding causing the transition-state to be more ordered (less entropy) relative to the folded-state, thereby resisting a transition to an unfolded state. The specificity, kinetics and response to calcium of this AMP illustrate its potential usefulness for industrial applications, and the research highlights the broader potential for using calcium to enhance the productivity of proteases.

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http://hdl.handle.net/10453/139044