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 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 kcat/Km 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.España, MINECO BIO2013‑48779‑ C4‑1/‑2/‑4, BIO2016‑76601‑ C3‑1/‑2/‑3

Utilización de materiales quitinolíticos para la obtención de oligosacáridos bioactivos. Caracterización de biocatalizadores y productos

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-04-2021Esta tesis tiene embargado el acceso al texto completo hasta el 14-10-2022Chitin, the most abundant natural biopolymer in aquatic environment, is composed of repeating β-1-4 linked N-acetyl-β-D-glucosamine (GlcNAc) units, and constitutes the structural component of protective biological structures such as fungal cell walls, crustaceans and insect exoskeletons. Recently, enzymes acting on chitin and chitosan (a deacetylation product of chitin) have gained strong interest due to wide range of biotechnological applications of these polymer derivatives. The biological activity of chitin, chitosan and/ particularly chitooligosaccharides (COS) has made these compounds the targets for the industrial and medical sectors. In this context chitinases and chitosanases are essential glycosyl hydrolases (GH) for the biotransformation of chitin and chitosan into COS by hydrolyzing the β-1,4-linkages. In this work, two fungal chitinases, the endo-chitinase Chit33 and the exo-chitinase Chit42, both from Trichoderma harzianum were expressed in Pichia pastoris, using fed-batch fermentation, to 0.63 and 3 g/L, respectively. The heterologous proteins were purified, characterized biochemically and their kinetic parameters on different types of chitinolytic materials analyzed. Numerous variants of both enzymes were also obtained using rational targeted mutation based on their structural determinants. Utilization of the two enzymes (Chit42 also immobilized on magnetic nanoparticles and chitosan beads) as well as that of commercial preparations based on Bacillus spp cells extracts were analyzed for their application in the production of fully acetylated (faCOS), fully deacetylated (fdCOS) and partially acetylated (paCOS) COS mixtures. The product profiles were analyzed, when possible characterized using different chromatographic techniques and MALDI-TOF mass spectrometry, and their biological activity evaluated. COS mixtures in the range of 0.5-2 and 2-10 kDa produced from colloidal chitin and chitosan clearly exhibited antioxidant activity. The mixtures of fdCOS and faCOS apparently showed anti-inflammatory activity and some of them even a neuroprotective effect. The biological properties of COS obtained from chitin and chitosan gives biotechnological potential to both, the biocatalyst producing them and the products that they generateThis work was supported by the EU EMFF-Blue Economy-2018-FISH4FISH-863697 project, the Spanish Ministry of Economy and Competitiveness [BIO2016-76601-C3-1/-2], the Spanish Ministry of Science and Innovation PID2019-105838RB-C32/-31, Fundación Ramón Areces [XIX Call of Research Grants in Life and Material Sciences], and by an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecula

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

Abstract 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