A new broad specificity alkaline metalloprotease from a Pseudomonas sp. isolated from refrigerated milk: role of calcium in improving enzyme productivity
Metalloproteases represent the largest fraction of the global enzyme market. For biotechnological purposes 31 the accumulation of product (i.e. productivity) provides the best measure of assessing enzyme performance 32 because it takes into account the interplay between activity, stability, activa...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier B.V.
2015
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| Subjects: | |
| Online Access: | http://irep.iium.edu.my/44658/ http://irep.iium.edu.my/44658/4/Ertan_et_al._%282015%29_Pseudomonas_alkaline_metalloprotease.pdf |
| _version_ | 1848782641362370560 |
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| author | Ertan, Haluk Cassel, Camilo Verma, Arjun Poljak, Anne Charlton, Tim Aldrich-Wright, Janice Mohd Omar, Suhaila Siddiqui, Khawar Sohail Cavicchioli, Richardo |
| author_facet | Ertan, Haluk Cassel, Camilo Verma, Arjun Poljak, Anne Charlton, Tim Aldrich-Wright, Janice Mohd Omar, Suhaila Siddiqui, Khawar Sohail Cavicchioli, Richardo |
| author_sort | Ertan, Haluk |
| building | IIUM Repository |
| collection | Online Access |
| description | Metalloproteases represent the largest fraction of the global enzyme market. For biotechnological purposes 31
the accumulation of product (i.e. productivity) provides the best measure of assessing enzyme performance 32
because it takes into account the interplay between activity, stability, activation and inhibition. Studies 33
assessing the productivity of alkaline metalloproteases and chemicals that improve their productivity have 34
not previously been reported. In this study we report the specificity, productivity, kinetic and 35
thermodynamic properties of an extracellular protease, purified from a new strain of Pseudomonas sp. 36
isolated from refrigerated milk. Mass spectrometry analysis revealed the enzyme is a serralysin-type alkaline 37
metalloprotease, with broad cleavage-site specificity. By studying the effects of Ca2+ ion removal (using a 38
chelator) and Ca2+ ion addition, conditions were identified that led to an increase in productivity by 300 % 39
(6.3 vs 1.9 mg azopeptide μg-1 enzyme at 40C). The basis for the enhanced productivity was linked to 40
elevated melting temperatures of secondary (Tm 47 vs 38C) and tertiary structure (Tm 50 vs 44C), 41
increased half-life of inactivation (t1/2 30 vs 4.9 min), increased optimum temperature (44 vs 36C), and 42
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). 43
Thermodynamic data were indicative of Ca2+-binding causing the transition-state to be more ordered (less 44
entropy) relative to the folded-state, thereby resisting a transition to an unfolded state. The specificity, 45
kinetics and response to calcium of this AMP illustrate its potential usefulness for industrial applications, 46
and the research highlights the broader potential for using calcium to enhance the productivity of proteases |
| first_indexed | 2025-11-14T16:08:41Z |
| format | Article |
| id | iium-44658 |
| institution | International Islamic University Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T16:08:41Z |
| publishDate | 2015 |
| publisher | Elsevier B.V. |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | iium-446582021-05-25T04:35:25Z http://irep.iium.edu.my/44658/ A new broad specificity alkaline metalloprotease from a Pseudomonas sp. isolated from refrigerated milk: role of calcium in improving enzyme productivity Ertan, Haluk Cassel, Camilo Verma, Arjun Poljak, Anne Charlton, Tim Aldrich-Wright, Janice Mohd Omar, Suhaila Siddiqui, Khawar Sohail Cavicchioli, Richardo Q Science (General) QD Chemistry Metalloproteases represent the largest fraction of the global enzyme market. For biotechnological purposes 31 the accumulation of product (i.e. productivity) provides the best measure of assessing enzyme performance 32 because it takes into account the interplay between activity, stability, activation and inhibition. Studies 33 assessing the productivity of alkaline metalloproteases and chemicals that improve their productivity have 34 not previously been reported. In this study we report the specificity, productivity, kinetic and 35 thermodynamic properties of an extracellular protease, purified from a new strain of Pseudomonas sp. 36 isolated from refrigerated milk. Mass spectrometry analysis revealed the enzyme is a serralysin-type alkaline 37 metalloprotease, with broad cleavage-site specificity. By studying the effects of Ca2+ ion removal (using a 38 chelator) and Ca2+ ion addition, conditions were identified that led to an increase in productivity by 300 % 39 (6.3 vs 1.9 mg azopeptide μg-1 enzyme at 40C). The basis for the enhanced productivity was linked to 40 elevated melting temperatures of secondary (Tm 47 vs 38C) and tertiary structure (Tm 50 vs 44C), 41 increased half-life of inactivation (t1/2 30 vs 4.9 min), increased optimum temperature (44 vs 36C), and 42 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). 43 Thermodynamic data were indicative of Ca2+-binding causing the transition-state to be more ordered (less 44 entropy) relative to the folded-state, thereby resisting a transition to an unfolded state. The specificity, 45 kinetics and response to calcium of this AMP illustrate its potential usefulness for industrial applications, 46 and the research highlights the broader potential for using calcium to enhance the productivity of proteases Elsevier B.V. 2015-03 Article PeerReviewed application/pdf en http://irep.iium.edu.my/44658/4/Ertan_et_al._%282015%29_Pseudomonas_alkaline_metalloprotease.pdf Ertan, Haluk and Cassel, Camilo and Verma, Arjun and Poljak, Anne and Charlton, Tim and Aldrich-Wright, Janice and Mohd Omar, Suhaila and Siddiqui, Khawar Sohail and Cavicchioli, Richardo (2015) A new broad specificity alkaline metalloprotease from a Pseudomonas sp. isolated from refrigerated milk: role of calcium in improving enzyme productivity. Journal of Molecular Catalysis B: Enzymatic (113). pp. 1-8. ISSN 1381-1177 http://www.sciencedirect.com/science/article/pii/S1381117714003294 10.1016/j.molcatb.2014.12.010 |
| spellingShingle | Q Science (General) QD Chemistry Ertan, Haluk Cassel, Camilo Verma, Arjun Poljak, Anne Charlton, Tim Aldrich-Wright, Janice Mohd Omar, Suhaila Siddiqui, Khawar Sohail Cavicchioli, Richardo A new broad specificity alkaline metalloprotease from a Pseudomonas sp. isolated from refrigerated milk: role of calcium in improving enzyme productivity |
| title | A new broad specificity alkaline metalloprotease from a Pseudomonas sp. isolated from refrigerated milk: role of calcium in improving enzyme productivity |
| title_full | A new broad specificity alkaline metalloprotease from a Pseudomonas sp. isolated from refrigerated milk: role of calcium in improving enzyme productivity |
| title_fullStr | A new broad specificity alkaline metalloprotease from a Pseudomonas sp. isolated from refrigerated milk: role of calcium in improving enzyme productivity |
| title_full_unstemmed | A new broad specificity alkaline metalloprotease from a Pseudomonas sp. isolated from refrigerated milk: role of calcium in improving enzyme productivity |
| title_short | A new broad specificity alkaline metalloprotease from a Pseudomonas sp. isolated from refrigerated milk: role of calcium in improving enzyme productivity |
| title_sort | new broad specificity alkaline metalloprotease from a pseudomonas sp. isolated from refrigerated milk: role of calcium in improving enzyme productivity |
| topic | Q Science (General) QD Chemistry |
| url | http://irep.iium.edu.my/44658/ http://irep.iium.edu.my/44658/ http://irep.iium.edu.my/44658/ http://irep.iium.edu.my/44658/4/Ertan_et_al._%282015%29_Pseudomonas_alkaline_metalloprotease.pdf |