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Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity

BACKGROUND: Chitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is rela...

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Autores principales: Kidibule, Peter Elias, Santos-Moriano, Paloma, Jiménez-Ortega, Elena, Ramírez-Escudero, Mercedes, Limón, M. Carmen, Remacha, Miguel, Plou, Francisco José, Sanz-Aparicio, Julia, Fernández-Lobato, María
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5863366/
https://www.ncbi.nlm.nih.gov/pubmed/29566690
http://dx.doi.org/10.1186/s12934-018-0895-x
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author Kidibule, Peter Elias
Santos-Moriano, Paloma
Jiménez-Ortega, Elena
Ramírez-Escudero, Mercedes
Limón, M. Carmen
Remacha, Miguel
Plou, Francisco José
Sanz-Aparicio, Julia
Fernández-Lobato, María
author_facet Kidibule, Peter Elias
Santos-Moriano, Paloma
Jiménez-Ortega, Elena
Ramírez-Escudero, Mercedes
Limón, M. Carmen
Remacha, Miguel
Plou, Francisco José
Sanz-Aparicio, Julia
Fernández-Lobato, María
author_sort Kidibule, Peter Elias
collection PubMed
description BACKGROUND: Chitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and acetylation degree. Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-d-glucosamine (GlcNAc) units. Gene chit42 was previously characterized, and according to its sequence, the encoded protein included in the structural Glycoside Hydrolase family GH18. RESULTS: Chit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. Protein heterologously expressed showed similar biochemical properties to those expressed by the natural producer (42 kDa, optima pH 5.5–6.5 and 30–40 °C). In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Chit42 hydrolysed colloidal chitin at least 10-times more efficiently (defined by the k(cat)/K(m) ratio) than any of the assayed chitosan. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Masses corresponding to (d-glucosamine)(1–8)-GlcNAc were identified from the hydrolysis of different substrates. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates. CONCLUSIONS: Chitinase Chit42 was expressed in a heterologous system to levels never before achieved. The enzyme produced small partially acetylated chitooligosaccharides, which have enormous biotechnological potential in medicine and food. Chit42 3D structure was characterized and analysed. Production and understanding of how the enzymes generating bioactive chito-oligomers work is essential for their biotechnological application, and paves the way for future work to take advantage of chitinolytic activities. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-018-0895-x) contains supplementary material, which is available to authorized users.
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spelling pubmed-58633662018-03-27 Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity Kidibule, Peter Elias Santos-Moriano, Paloma Jiménez-Ortega, Elena Ramírez-Escudero, Mercedes Limón, M. Carmen Remacha, Miguel Plou, Francisco José Sanz-Aparicio, Julia Fernández-Lobato, María Microb Cell Fact Research BACKGROUND: Chitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and acetylation degree. Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-d-glucosamine (GlcNAc) units. Gene chit42 was previously characterized, and according to its sequence, the encoded protein included in the structural Glycoside Hydrolase family GH18. RESULTS: Chit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. Protein heterologously expressed showed similar biochemical properties to those expressed by the natural producer (42 kDa, optima pH 5.5–6.5 and 30–40 °C). In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Chit42 hydrolysed colloidal chitin at least 10-times more efficiently (defined by the k(cat)/K(m) ratio) than any of the assayed chitosan. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Masses corresponding to (d-glucosamine)(1–8)-GlcNAc were identified from the hydrolysis of different substrates. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates. CONCLUSIONS: Chitinase Chit42 was expressed in a heterologous system to levels never before achieved. The enzyme produced small partially acetylated chitooligosaccharides, which have enormous biotechnological potential in medicine and food. Chit42 3D structure was characterized and analysed. Production and understanding of how the enzymes generating bioactive chito-oligomers work is essential for their biotechnological application, and paves the way for future work to take advantage of chitinolytic activities. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-018-0895-x) contains supplementary material, which is available to authorized users. BioMed Central 2018-03-22 /pmc/articles/PMC5863366/ /pubmed/29566690 http://dx.doi.org/10.1186/s12934-018-0895-x Text en © The Author(s) 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Kidibule, Peter Elias
Santos-Moriano, Paloma
Jiménez-Ortega, Elena
Ramírez-Escudero, Mercedes
Limón, M. Carmen
Remacha, Miguel
Plou, Francisco José
Sanz-Aparicio, Julia
Fernández-Lobato, María
Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity
title Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity
title_full Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity
title_fullStr Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity
title_full_unstemmed Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity
title_short Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity
title_sort use of chitin and chitosan to produce new chitooligosaccharides by chitinase chit42: enzymatic activity and structural basis of protein specificity
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5863366/
https://www.ncbi.nlm.nih.gov/pubmed/29566690
http://dx.doi.org/10.1186/s12934-018-0895-x
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