Procesos biocatalíticos para la producción de carbohidratos bioactivos: fructooligosacáridos y quitooligosacáridos

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-07-2017Esta tesis tiene embargado el acceso al texto completo hasta el 14-01-201

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

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

Marine chitinolytic enzymes, a biotechnological treasure hidden in the ocean?

Chitinolytic enzymes are capable to catalyze the chitin hydrolysis. Due to their biomedical and biotechnological applications, nowadays chitinolytic enzymes have attracted worldwide attention. Chitinolytic enzymes have provided numerous useful materials in many different industries, such as food, pharmaceutical, cosmetic, or biomedical industry. Marine enzymes are commonly employed in industry because they display better operational properties than animal, plant, or bacterial homologs. In this mini-review, we want to describe marine chitinolytic enzymes as versatile enzymes in different biotechnological fields. In this regard, interesting comments about their biological role, reaction mechanism, production, functional characterization, immobilization, and biotechnological application are shown in this work.Sin financiación3.670 JCR (2018) Q2, 41/162 Biotechnology & Applied Microbiology1.127 SJR (2018) Q1, 53/342 Biotechnology, 22/114 Applied Microbiology and Biotechnology, 451/2844 Medicine (miscellaneous)No data IDR 2018UE

Structure-Guided Immobilization of an Evolved Unspecific Peroxygenase

Unspecific peroxygenases (UPOs) are highly promiscuous biocatalyst with self-sufficient mono(per)oxygenase activity. A laboratory-evolved UPO secreted by yeast was covalently immobilized in activated carriers through one-point attachment. In order to maintain the desired orientation without compromising the enzyme’s activity, the S221C mutation was introduced at the surface of the enzyme, enabling a single disulfide bridge to be established between the support and the protein. Fluorescence confocal microscopy demonstrated the homogeneous distribution of the enzyme, regardless of the chemical nature of the carrier. This immobilized biocatalyst was characterized biochemically opening an exciting avenue for research into applied synthetic chemistry.Repsol S.A and the EU project H2020-BBI-PPP2015-2-720297-ENZOX2.4.556 JCR (2019) Q1, 74/297 Biochemistry & Molecular Biology1.317 SJR (2019) Q1, 93/2331 Computer Science ApplicationsNo data IDR 2019UE

Bioplásticos: el reto del presente y del futuro

Sin financiaciónNo data WoSNo data Scopus911 SPI - ICEE (2018), 3/105 General – Editoriales españolasUE

From bench to market: A project based learning experience in the biotechnology degree

Nowadays, the efficient incorporation of recent biotechnology graduates into the market requires the development of new skills, such as communication, critical analysis, as well as the ability to solve challenges and implement real solutions. Focused on this purpose, the development of Project-Based Learning (PBL) methodologies have shown as promising alternative. This pedagogical method arises as an initiative for a more autonomous learning through stimulating challenges that motivates students to investigate, becoming experts in a subject before carrying it out in practice and being able to critically discuss the results obtained leading to a more solid learning.At Universidad Europea de Madrid (UEM), since the academic year 2017-2018, students belonging to the 4th year of the Biotechnology Degree, enrolled in the courses of Bioreactors, Business Management and Biotechnological Processes and Products, have participated in the development of the PBL project aimed at promoting entrepreneurship. With the goal of promoting the connection between the real needs of the biotechnology industry and the commercial study, our students faced a challenge posed by a fictitious biotechnology company. The students carried out the development of real proposals applied to a wide range of biotech markets and provided a solution with an original and innovative idea developing aptitudes such as planning, information integration, individual responsibility and teamwork. In addition to training their practical skills in the laboratory with fermentation experiments, they developed their written communication skills through the elaboration of an integrative report complementing their learning. Finally, the project ends with an oral presentation session and consequent critical review with the participation of all the students and teachers involved. This project has fostered the students' confidence and autonomy to face the world of work, corroborated thanks to surveys and questionnaires that evaluate their learning and satisfaction with the methodology.Sin financiaciónNo data 2020UE

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

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