Truncation of a novel C‐terminal domain of a β‐glucanase improves its thermal stability and specific activity
Abstract Enzymes that degrade β‐glucan play important roles in various industries, including those related to brewing, animal feed, and health care. Csph16A, an endo‐β‐1,3(4)‐glucanase encoded by a gene from the halotolerant, xerotolerant, and radiotrophic black fungus Cladosporium sphaerospermum ,...
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Online Access: | http://dx.doi.org/10.1002/biot.202400245 |
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crwiley:10.1002/biot.202400245 2024-10-13T14:02:27+00:00 Truncation of a novel C‐terminal domain of a β‐glucanase improves its thermal stability and specific activity Klemanska, Anastasia Dwyer, Kelly Walsh, Gary Irish Research Council 2024 http://dx.doi.org/10.1002/biot.202400245 en eng Wiley http://creativecommons.org/licenses/by-nc/4.0/ Biotechnology Journal volume 19, issue 8 ISSN 1860-6768 1860-7314 journal-article 2024 crwiley https://doi.org/10.1002/biot.202400245 2024-09-17T04:49:11Z Abstract Enzymes that degrade β‐glucan play important roles in various industries, including those related to brewing, animal feed, and health care. Csph16A, an endo‐β‐1,3(4)‐glucanase encoded by a gene from the halotolerant, xerotolerant, and radiotrophic black fungus Cladosporium sphaerospermum , was cloned and expressed in Pichia pastoris . Two isoforms (Csph16A.1 and Csph16A.2) are produced, arising from differential glycosylation. The proteins were predicted to contain a catalytic Lam16A domain, along with a C‐terminal domain (CTD) of unknown function which exhibits minimal secondary structure. Employing PCR‐mediated gene truncation, the CTD of Csph16A was excised to assess its functional impact on the enzyme and determine potential alterations in biotechnologically relevant characteristics. The truncated mutant, Csph16A‐ΔC, exhibited significantly enhanced thermal stability at 50°C, with D‐values 14.8 and 23.5 times greater than those of Csph16A.1 and Csph16A.2, respectively. Moreover, Csph16A‐ΔC demonstrated a 20%–25% increase in halotolerance at 1.25 and 1.5 M NaCl, respectively, compared to the full‐length enzymes. Notably, specific activity against cereal β‐glucan, lichenan, and curdlan was increased by up to 238%. This study represents the first characterization of a glucanase from the stress‐tolerant fungus C. sphaerospermum and the first report of a halotolerant and engineered endo‐β‐1,3(4)‐glucanase. Additionally, it sheds light on a group of endo‐β‐1,3(4)‐glucanases from Antarctic rock‐inhabiting black fungi harboring a Lam16A catalytic domain and a novel CTD of unknown function. Article in Journal/Newspaper Antarc* Antarctic Wiley Online Library Antarctic Biotechnology Journal 19 8 |
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Wiley Online Library |
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crwiley |
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English |
description |
Abstract Enzymes that degrade β‐glucan play important roles in various industries, including those related to brewing, animal feed, and health care. Csph16A, an endo‐β‐1,3(4)‐glucanase encoded by a gene from the halotolerant, xerotolerant, and radiotrophic black fungus Cladosporium sphaerospermum , was cloned and expressed in Pichia pastoris . Two isoforms (Csph16A.1 and Csph16A.2) are produced, arising from differential glycosylation. The proteins were predicted to contain a catalytic Lam16A domain, along with a C‐terminal domain (CTD) of unknown function which exhibits minimal secondary structure. Employing PCR‐mediated gene truncation, the CTD of Csph16A was excised to assess its functional impact on the enzyme and determine potential alterations in biotechnologically relevant characteristics. The truncated mutant, Csph16A‐ΔC, exhibited significantly enhanced thermal stability at 50°C, with D‐values 14.8 and 23.5 times greater than those of Csph16A.1 and Csph16A.2, respectively. Moreover, Csph16A‐ΔC demonstrated a 20%–25% increase in halotolerance at 1.25 and 1.5 M NaCl, respectively, compared to the full‐length enzymes. Notably, specific activity against cereal β‐glucan, lichenan, and curdlan was increased by up to 238%. This study represents the first characterization of a glucanase from the stress‐tolerant fungus C. sphaerospermum and the first report of a halotolerant and engineered endo‐β‐1,3(4)‐glucanase. Additionally, it sheds light on a group of endo‐β‐1,3(4)‐glucanases from Antarctic rock‐inhabiting black fungi harboring a Lam16A catalytic domain and a novel CTD of unknown function. |
author2 |
Irish Research Council |
format |
Article in Journal/Newspaper |
author |
Klemanska, Anastasia Dwyer, Kelly Walsh, Gary |
spellingShingle |
Klemanska, Anastasia Dwyer, Kelly Walsh, Gary Truncation of a novel C‐terminal domain of a β‐glucanase improves its thermal stability and specific activity |
author_facet |
Klemanska, Anastasia Dwyer, Kelly Walsh, Gary |
author_sort |
Klemanska, Anastasia |
title |
Truncation of a novel C‐terminal domain of a β‐glucanase improves its thermal stability and specific activity |
title_short |
Truncation of a novel C‐terminal domain of a β‐glucanase improves its thermal stability and specific activity |
title_full |
Truncation of a novel C‐terminal domain of a β‐glucanase improves its thermal stability and specific activity |
title_fullStr |
Truncation of a novel C‐terminal domain of a β‐glucanase improves its thermal stability and specific activity |
title_full_unstemmed |
Truncation of a novel C‐terminal domain of a β‐glucanase improves its thermal stability and specific activity |
title_sort |
truncation of a novel c‐terminal domain of a β‐glucanase improves its thermal stability and specific activity |
publisher |
Wiley |
publishDate |
2024 |
url |
http://dx.doi.org/10.1002/biot.202400245 |
geographic |
Antarctic |
geographic_facet |
Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
Biotechnology Journal volume 19, issue 8 ISSN 1860-6768 1860-7314 |
op_rights |
http://creativecommons.org/licenses/by-nc/4.0/ |
op_doi |
https://doi.org/10.1002/biot.202400245 |
container_title |
Biotechnology Journal |
container_volume |
19 |
container_issue |
8 |
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1812817358315585536 |