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Enhancing the Hydrolysis and Acyl Transfer Activity of Carboxylesterase DLFae4 by a Combinational Mutagenesis and In-Silico Method

In the present study, a feruloyl esterase DLFae4 identified in our previous research was modified by error-prone PCR and site-directed saturation mutation to enhance the catalytic efficiency and acyltransferase activity further. Five mutants with 6.9–118.9% enhanced catalytic activity toward methyl...

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Autores principales: Li, Longxiang, Ding, Liping, Shao, Yuting, Sun, Shengwei, Wang, Mengxi, Xiang, Jiahui, Zhou, Jingjie, Wu, Guojun, Song, Zhe, Xin, Zhihong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10048530/
https://www.ncbi.nlm.nih.gov/pubmed/36981096
http://dx.doi.org/10.3390/foods12061169
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author Li, Longxiang
Ding, Liping
Shao, Yuting
Sun, Shengwei
Wang, Mengxi
Xiang, Jiahui
Zhou, Jingjie
Wu, Guojun
Song, Zhe
Xin, Zhihong
author_facet Li, Longxiang
Ding, Liping
Shao, Yuting
Sun, Shengwei
Wang, Mengxi
Xiang, Jiahui
Zhou, Jingjie
Wu, Guojun
Song, Zhe
Xin, Zhihong
author_sort Li, Longxiang
collection PubMed
description In the present study, a feruloyl esterase DLFae4 identified in our previous research was modified by error-prone PCR and site-directed saturation mutation to enhance the catalytic efficiency and acyltransferase activity further. Five mutants with 6.9–118.9% enhanced catalytic activity toward methyl ferulate (MFA) were characterized under the optimum conditions. Double variant DLFae4-m5 exhibited the highest hydrolytic activity (270.97 U/mg), the Km value decreased by 83.91%, and the Kcat/Km value increased by 6.08-fold toward MFA. Molecular docking indicated that a complex hydrogen bond network in DLFae4-m5 was formed, with four of five bond lengths being shortened compared with DLFae4, which might account for the increase in catalytic activity. Acyl transfer activity assay revealed that the activity of DLFae4 was as high as 1550.796 U/mg and enhanced by 375.49% (5823.172 U/mg) toward 4-nitrophenyl acetate when residue Ala-341 was mutated to glycine (A341G), and the corresponding acyl transfer efficiency was increased by 7.7 times, representing the highest acyltransferase activity to date, and demonstrating that the WGG motif was pivotal for the acyltransferase activity in family VIII carboxylesterases. Further experiments indicated that DLFae4 and variant DLFae4 (A341G) could acylate cyanidin-3-O-glucoside effectively in aqueous solution. Taken together, our study suggested the effectiveness of error-prone PCR and site-directed saturation mutation to increase the specific activity of enzymes and may facilitate the practical application of this critical feruloyl esterase.
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spelling pubmed-100485302023-03-29 Enhancing the Hydrolysis and Acyl Transfer Activity of Carboxylesterase DLFae4 by a Combinational Mutagenesis and In-Silico Method Li, Longxiang Ding, Liping Shao, Yuting Sun, Shengwei Wang, Mengxi Xiang, Jiahui Zhou, Jingjie Wu, Guojun Song, Zhe Xin, Zhihong Foods Article In the present study, a feruloyl esterase DLFae4 identified in our previous research was modified by error-prone PCR and site-directed saturation mutation to enhance the catalytic efficiency and acyltransferase activity further. Five mutants with 6.9–118.9% enhanced catalytic activity toward methyl ferulate (MFA) were characterized under the optimum conditions. Double variant DLFae4-m5 exhibited the highest hydrolytic activity (270.97 U/mg), the Km value decreased by 83.91%, and the Kcat/Km value increased by 6.08-fold toward MFA. Molecular docking indicated that a complex hydrogen bond network in DLFae4-m5 was formed, with four of five bond lengths being shortened compared with DLFae4, which might account for the increase in catalytic activity. Acyl transfer activity assay revealed that the activity of DLFae4 was as high as 1550.796 U/mg and enhanced by 375.49% (5823.172 U/mg) toward 4-nitrophenyl acetate when residue Ala-341 was mutated to glycine (A341G), and the corresponding acyl transfer efficiency was increased by 7.7 times, representing the highest acyltransferase activity to date, and demonstrating that the WGG motif was pivotal for the acyltransferase activity in family VIII carboxylesterases. Further experiments indicated that DLFae4 and variant DLFae4 (A341G) could acylate cyanidin-3-O-glucoside effectively in aqueous solution. Taken together, our study suggested the effectiveness of error-prone PCR and site-directed saturation mutation to increase the specific activity of enzymes and may facilitate the practical application of this critical feruloyl esterase. MDPI 2023-03-10 /pmc/articles/PMC10048530/ /pubmed/36981096 http://dx.doi.org/10.3390/foods12061169 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Li, Longxiang
Ding, Liping
Shao, Yuting
Sun, Shengwei
Wang, Mengxi
Xiang, Jiahui
Zhou, Jingjie
Wu, Guojun
Song, Zhe
Xin, Zhihong
Enhancing the Hydrolysis and Acyl Transfer Activity of Carboxylesterase DLFae4 by a Combinational Mutagenesis and In-Silico Method
title Enhancing the Hydrolysis and Acyl Transfer Activity of Carboxylesterase DLFae4 by a Combinational Mutagenesis and In-Silico Method
title_full Enhancing the Hydrolysis and Acyl Transfer Activity of Carboxylesterase DLFae4 by a Combinational Mutagenesis and In-Silico Method
title_fullStr Enhancing the Hydrolysis and Acyl Transfer Activity of Carboxylesterase DLFae4 by a Combinational Mutagenesis and In-Silico Method
title_full_unstemmed Enhancing the Hydrolysis and Acyl Transfer Activity of Carboxylesterase DLFae4 by a Combinational Mutagenesis and In-Silico Method
title_short Enhancing the Hydrolysis and Acyl Transfer Activity of Carboxylesterase DLFae4 by a Combinational Mutagenesis and In-Silico Method
title_sort enhancing the hydrolysis and acyl transfer activity of carboxylesterase dlfae4 by a combinational mutagenesis and in-silico method
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10048530/
https://www.ncbi.nlm.nih.gov/pubmed/36981096
http://dx.doi.org/10.3390/foods12061169
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