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

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

Efficient conversion of chitosan into chitooligosaccharides by a chitosanolytic activity from Bacillus thuringiensis

Starting from a commercial preparation of Bacillus thuringiensis var. aizawai, widely employed as biopesticide in agriculture, we obtained a chitosanolytic activity by washing the cells with concentrated sodium acetate. The optimum pH and temperature values for this enzyme were 6.0 and 60 °C, respectively. The enzymatic extract efficiently hydrolyzed various chitosans with different deacetylation degrees (DD) into small oligosaccharides. Combining mass spectrometry (ESI-Q-TOF) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), we observed the formation of fully deacetylated chitooligosaccharides [fdCOS, (GlcN)2-5] and, to a lesser extent, partially acetylated chitooligosaccharides [paCOS, GlcNAc-(GlcN)1-3]. A 10 g/L solution of chitosan with 600–800 kDa and DD ≥ 90% was fully converted into oligosaccharides in 55 h. Most of the products were fdCOS: 1.6 g/L of chitobiose, 1.7 g/L of chitotriose, 5 g/L of chitotetraose and 1.4 g/L of chitopentaose.Sin financiación2.883 JCR (2018) Q2, 148/299 Biochemistry & Molecular Biology, 59/162 Biotechnology & Applied Microbiology, 47/138 Engineering, Chemical0.754 SJR (2018) Q2, 213/462 Biochemistry, 57/188 Bioengineering, 37/114 Applied Microbiology and BiotechnologyNo data IDR 2018UE

Tailored Enzymatic Synthesis of Chitooligosaccharides with Different Deacetylation Degrees and Their Anti-Inflammatory Activity

By controlled hydrolysis of chitosan or chitin with different enzymes, three types of chitooligosaccharides (COS) with MW between 0.2 and 1.2 kDa were obtained: fully deacetylated (fdCOS), partially acetylated (paCOS), and fully acetylated (faCOS). The chemical composition of the samples was analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and MALDI-TOF mass spectrometry. The synthesized fdCOS was basically formed by GlcN, (GlcN)2, (GlcN)3, and (GlcN)4. On the contrary, faCOS contained mostly GlcNAc, (GlcNAc)2 and (GlcNAc)3, while paCOS corresponded to a mixture of at least 11 oligosaccharides with different proportions of GlcNAc and GlcN. The anti-inflammatory activity of the three COS mixtures was studied by measuring their ability to reduce the level of TNF-α (tumor necrosis factor) in murine macrophages (RAW 264.7) after stimulation with a mixture of lipopolysaccharides (LPS). Only fdCOS and faCOS were able to significantly reduce the production of tumor necrosis factor (TNF)-α at 6 h after stimulation with lipopolysaccharides.Fundación Ramón Areces (XIX Convocatoria de Ayudas a la Investigación en Vida y Ciencias de los Materiales) y el Ministerio de Economía y Competitividad de España (Becas BIO2016-76601-C3-1-R y BIO2016-76601-C3-3-R).El programa Horizonte 2020 de la Unión Europea también financió este trabajo (Blue Growth: Unlocking the potencial of Seas and Oceans; acuerdo de subvención No. 634486; INMARE).EU COST-Action CM1303.3.520 JCR (2019) Q2, 65/159 Chemistry, Physical0.722 SJR (2019) Q2, 51/174 Physical and Theoretical ChemistryNo data IDR 2019UE

Synthesis of prebiotic isomaltooligosaccharides from starch by a combination of amylolytic enzymes

Amylolytic enzymes have important applications in the food, pharmaceutical and biofuel industries. The synthesis of prebiotic isomaltooligosaccharides (IMOS) from starch is one of the biotransformations of greatest interest. It is a multi-enzyme process based on a first hydrolytic step (liquefaction), for which we are testing -amylases obtained from non-conventional yeasts, in particular Schwanniomyces occidentalis. To obtain syrups enriched in maltose or maltotriose, different β-amylases or maltotriose-forming enzymes are being investigated in our laboratory. The final step is a transglucosylation process catalyzed by α-glucosidases, which selectively form α(1→6) linkages between glucosyl residues. This transfer reaction is being studied with commercially available enzymes or those obtained from the yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma). Immobilization of the above enzymes (in the same support or compartmentalized in different carriers) allows a better control of the multi-step process and may help to reduce the costs.Peer Reviewe