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

Enzymatic synthesis and characterization of different families of chitooligosaccharides and their bioactive properties

Chitooligosaccharides (COS) are homo- or hetero-oligomers of D-glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) that can be obtained by chitosan or chitin hydrolysis. Their enzymatic production is preferred over other methodologies (physical, chemical, etc.) due to the mild conditions required, the fewer amounts of waste and its efficiency to control product composition. By properly selecting the enzyme (chitinase, chitosanase or nonspecific enzymes) and the substrate properties (degree of deacetylation, molecular weight, etc.), it is possible to direct the synthesis towards any of the three COS types: fully acetylated (faCOS), partially acetylated (paCOS) and fully deacetylated (fdCOS). In this article, we review the main strategies to steer the COS production towards a specific group. The chemical characterization of COS by advanced techniques, e.g., high-performance anion-exchange chromatography with pulsed amperometric detection (HPAECPAD) and MALDI-TOF mass spectrometry, is critical for structure-function studies. The scaling of processes to synthesize specific COS mixtures is difficult due to the low solubility of chitin/chitosan, the heterogeneity of the reaction mixtures, and high amounts of salts. Enzyme immobilization can help to minimize such hurdles. The main bioactive properties of COS are herein reviewed. Finally, the anti-inflammatory activity of three COS mixtures was assayed in murine macrophages after stimulation with lipopolysaccharidesThis work was supported by grants from the EU EMFF-Blue Economy-2018 (FISH4FISH- 863697 project), the Spanish Ministry of Economy and Competitiveness (Grants BIO2016-76601- C3-1,2-R), the Spanish Ministry of Science and Innovation (Grants PID2019-105838RB-C31/C32), 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

Overproduction of a Trichoderma harzianum chitinase and analysis of its biotechnological potential to produce chitooligosaccharides

Trabajo presentado en la 7ª ed. del congreso internacional "FEMS" organizado por la Sociedad Española de Microbiología y la Federación Europea de Sociedades Microbiológicas en el Centro de Convenciones Feria Valencia (Valencia, España) durante los días 9 al 13 de julio de 2017.BACKGROUNDS: Chitooligosaccharides (COS) are β-(1,4)-linked oligomers of N-acetyl-glucosamine (GlcNAc) and glucosamine (GlcN) formed by chemical or enzymatic hydrolysis of chitosan or chitin. The growing biotechnological interest of COS in fields such as food or health increases the demand of the producing enzymes as well as their characterization and functional improvement. | OBJETIVES: Express a chitinase of 42 kDa from Trichoderma harzianum in a heterologous system, obtain protein levels compatible with its crystallization for the future protein structural resolution and evaluate the ability of the recombinant protein to produce COS. | METHODS: The chitinase gene cDNA from T. harzianum was expressed in Pichia pastoris using a restriction-free cloning strategy, production of heterologous protein was analysed and escalated up to a 5 L fermenter level. Recombinant protein was purified and some crystals were obtained which allows undertake the protein structural resolution. Synthesis of oligosaccharides from different substrates were evaluated and optimized using the recombinant enzyme. HPAEC-PAD on a Dionex ICS3000 system and Mass Spectrometry were used in the reaction studies and product characterization. | CONCLUSIONS: A chitinase of 42 kDa from T. harzianum was overexpressed in P. pastoris, the recombinant protein was purified, characterized and crystallized for the protein structural resolution. Production of COS mediated by this enzyme was evaluated and some of the molecules formed were characterized.N

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

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

Structural study of two glycoside hydrolase family 18 chitinases and their role in the production of chitooligosaccharides

Joint 12th EBSA congress / 10th ICBP – IUPAP congress: Biophysics for life and technology, Madrid, Spain, July 20-24, 2019. -- http://www.ebsa2019.org/Peer reviewe

Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism

13 pags., 5 figs., 3 tabs.Chitinases degrade chitin into low molecular weight chitooligomers, which have a broad range of industrial, agricultural, and medical functions. Understanding the relationship between the diverse characteristics of chitinases and their functions is necessary for the improvement of functional enzymes that meet specific requirements. We report here a full crystallographic analysis of three complexes obtained from the chitinase Chit42 from Trichoderma harzianum, which represent different states along the enzymatic mechanism. The inactive double mutant D169A/E171A was submitted to soaking/crystallization experiments with hexa-N-acetyl-glucosamine (NAG6) or tetra-N-acetyl-glucosamine (NAG4), trapping the enzyme-substrate complex (Chit42-NAG6), the enzyme-products complex (Chit42-NAG4-NAG2) and a someway intermediate state. Structural comparison among the different complexes depicts the determinants defining the different subsites and revealed a previously unobserved dynamic on-off ligand binding process associated with a motion of its insertion domain, which might be accompanying the role or aromatics in processivity. An ensemble refinement performed to extract dynamic details from the diffraction data elucidates the implication of some highly flexible residues in the productive sliding of the substrate and the product release event. These positions were submitted to mutagenesis and the activity of the variants was investigated in the hydrolysis of NAG6, colloidal chitin and two chitosans with different polymerization and acetylation degree. All the changes affected the Chit42 hydrolytic activity therefore confirming the involvement of these positions in catalysis. Furthermore, we found the variants R295S and E316S improving the apparent catalytic efficiency of chitin and NAG6 and, together with E316A, enhancing the specific activity on chitosan. Therefore, our results provide novel insight into the molecular mechanisms underlying the hydrolysis of chitinous material by fungal chitinases, and suggest new targets to address engineering of these biotechnologically important enzymes.This work was supported by grants from the Spanish Ministryof Economy and Competitiveness through grants BIO2016-76601-C3-3-R/-C3-2-R, PID2019-105838RB-C33/-C32, Fundación Ramón Areces [XIX Call of Research Grants in Life and Material Sciences]and EU EMFF-Blue Economy-2018 [Fish4Fish-863697]. We aregrateful to the staff of the Synchrotron Radiation Sources at Alba(Barcelona, Spain) for providing access and for technical assistanceat BL13-XALOC beamline and to the Fundación Ramón Areces foran institutional grant to the Centre of Molecular Biology SeveroOcho

Endo-chitinase Chit33 specificity on different chitinolytic materials allows the production of unexplored chitooligosaccharides with antioxidant activity

The biological activity of chitooligosaccharides (COS) has made them targets for industrial and medical sectors. In this work, endo-chitinase Chit33 from Trichoderma harzianum CECT 2413 was expressed in Pichia pastoris GS115 to levels never achieved before (630 mg/L; 3.3 U/mL), without its biochemical characteristics being substantially affected. Chit33 produced a mixture of fully and partially acetylated COS from different chitin derivatives. HPAEC-PAD Chromatography and mass spectrometry analyses showed that (GlcNAc) and GlcN-(GlcNAc) were mainly produced from colloidal chitin and chitosan, respectively. COS in reaction mixtures were fragmented according to their size and their antioxidant activity analyzed by reducing power and free radical scavenging activity essays. The highest antioxidant activity was achieved with COS in the range of 0.5−2 and 2−10 kDa produced from colloidal chitin and chitosan, respectively, which gives biotechnological potential to both the chitin derivatives of 0.5−10 kDa and the biocatalyst producing them.Spanish Ministry of Economy and Competitiveness [BIO2016-76601-C3-1/-2], Fundacio’n Ramo’n Areces [XIX Call of Research Grants in Life and Material Sciences], EU EMFF-Blue Economy-2018 [Fish4Fish-863697] and by an institutional grant from Fundacio’n Ramo’n Areces to the Centro de Biologi’a Molecular Severo Ochoa

Structure–Function Insights into the Fungal Endo-Chitinase Chit33 Depict its Mechanism on Chitinous Material

15 pags., 6 figs., 2 tabs.Chitin is the most widespread amino renewable carbohydrate polymer in nature and the second most abundant polysaccharide. Therefore, chitin and chitinolytic enzymes are becoming more importance for biotechnological applications in food, health and agricultural fields, the design of effective enzymes being a paramount issue. We report the crystal structure of the plant-type endo-chitinase Chit33 from Trichoderma harzianum and its D165A/E167A-Chit33-(NAG) complex, which showed an extended catalytic cleft with six binding subsites lined with many polar interactions. The major trait of Chit33 is the location of the non-conserved Asp117 and Arg274 acting as a clamp, fixing the distorted conformation of the sugar at subsite –1 and the bent shape of the substrate, which occupies the full catalytic groove. Relevant residues were selected for mutagenesis experiments, the variants being biochemically characterized through their hydrolytic activity against colloidal chitin and other polymeric substrates with different molecular weights and deacetylation percentages. The mutant S118Y stands out, showing a superior performance in all the substrates tested, as well as detectable transglycosylation capacity, with this variant providing a promising platform for generation of novel Chit33 variants with adjusted performance by further design of rational mutants’. The putative role of Tyr in binding was extrapolated from molecular dynamics simulation.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness through grants PID2019-105838RB-C33/-C32, Fundacioón Ramón Areces (XIX Call of Research Grants in Life and Material Sciences, and EU EMFF-Blue Economy-2018 (Fish4Fish-863697). We are grateful to the staff of the Synchrotron Radiation Sources at Alba (Barcelona, Spain) for providing access and for technical assistance with BL13-XALOC beamline and to the Fundacioón Ramón Areces for an institutional grant to the Centre of Molecular Biology Severo Ochoa

Production and characterization of chitooligosaccharides by the fungal chitinase Chit42 immobilized on magnetic nanoparticles and chitosan beads: selectivity, specificity and improved operational utility

Chitin-active enzymes are of great biotechnological interest due to the wide industrial application of chitinolytic materials. Non-stability and high cost are among limitations that hinder industrial application of soluble enzymes. Here we report the production and characterization of chitooligosaccharides (COS) using the fungal exo-chitinase Chit42 immobilized on magnetic nanoparticles and food-grade chitosan beads with an immobilization yield of about 60% using glutaraldehyde and genipin linkers. The immobilized enzyme gained operational stability with increasing temperature and acidic pH values, especially when using chitosan beads-genipin that retained more than 80% activity at pH 3. Biocatalysts generated COS from colloidal chitin and different chitosan types. The immobilized enzyme showed higher hydrolytic activity than free enzyme on chitosan, and produced COS mixtures with higher variability of size and acetylation degree. In addition, biocatalysts were reusable, easy to handle and to separate from the reaction mixture