Workshop April 25th 2018

A Workshop of the GLICOENZ Consorptium was held on April 25th 2018 at the Faculty of Sciences of the Autonomous University of Madrid. The students of the four laboratories involved in GLICOENZ presented their main results. These presentations served as a basis for planning  new experiments and initiating new sinergies.

Recombinant chitinase Chit42 from Trichoderma harzianum for the production of chitooligosaccharides

Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-D-glucosamine (GlcNAc) units. Chit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. 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.

Reference: “Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity”. P.E. Kidibule,  P. Santos-Moriano, E. Jiménez-Ortega, M. Ramírez-Escudero, M.C. Limón, M. Remacha, F.J. Plou, J. Sanz-Aparicio,  M. Fernández-Lobato. Microbial Cell Factories 17:47 (2018). doi:10.1186/s12934-018-0895-x

Production of deacetylated chitooligosaccharides

Several commercial enzymes were screened for chitosanolytic activity. The hydrolysis of different chitosans was followed by size exclusion chromatography (SEC-ELSD), mass spectrometry (ESI-Q-TOF), and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Neutrase 0.8L converted 10 g/L of various chitosans into mostly deacetylated oligosaccharides, yielding approximately 2.5 g/L of chitobiose, 4.5 g/L of chitotriose and 3 g/L of chitotetraose. In collaboration with the Institute of Parasitology and Biomedicine "Lopez-Neyra" (CSIC), the synthesized COS were tested in vitro for their neuroprotective and anti-inflammatory activities, and compared with other functional ingredients, namely fructooligosaccharides.

Reference: “Enzymatic production of fully deacetylated chitooligosaccharides and their neuroprotective and anti-inflammatory properties”. P. Santos-Moriano, L. Fernandez-Arrojo, M. Mengibar, E. Belmonte-Reche, P. Peñalver, F.N. Acosta, P. Kidibule, A.O. Ballesteros, J.C. Morales, M. Fernandez-Lobato and F.J. Plou. Biocatalysis and Biotransformation (2017), doi: 10.1080/10242422.2017.1295231

Production of isomaltooligosaccharides by engineered S. cerevisiae cells

The α-glucosidase gene of Aspergillus niger was expressed in Saccharomyces cerevisiae under control of a galactose-inducible promoter. Recombinant yeast cells expressing the aglA gene produced extracellular α-glucosidase activity. With maltose as the substrate, panose is the main transglycosylation product after 8 h of incubation, whereas isomaltose is predominant after 24 h. Isomaltose also becomes predominant at shorter times if a mixture of maltose and glucose is used instead of maltose. To facilitate IMO production, we have designed a procedure by which yeast cells can be used directly as the catalytic agent. For this purpose, we expressed in S. cerevisiae gene constructs in which the aglA gene is fused to glycosylphosphatidylinositol anchor sequences, from the yeast SED1 gene, that determine the covalent binding of the hybrid protein to the cell membrane. The resulting hybrid enzymes were stably attached to the cell surface. The cells from cultures of recombinant yeast strains expressing aglA-SED1 constructions can be used to produce IMOs in successive batches.

Reference: M. Casa-Villegas, J. Marín-Navarro, J. Polaina. "Synthesis of isomaltooligosaccharides by Saccharomyces cerevisiae cells expressing Aspergillus niger alpha-glucosidases". ACS Omega 2, 11, 8062-8068 (2017). doi:10.1021/acsomega.7b01189

Galactooligosaccharide production from a microorganism isolated from “Tejuino”, a Mexican traditional fermented beverage

The Enzymes

Francisco Plou has just published the book "What we know about ...The Enzymes", a disclosure book jointly edited by CSIC and Catarata Publishers. The aim of this book is bringing the world of enzymes to readers, and show them the importance of these biological catalysts in our health and in many aspects of our daily life (food, drugs, fuels, polymers, etc.). It includes many aspects related to the enzymes acting on carbohydrates.

Dissecting of the active-site of Thermotoga maritima β‑Galactosidase

β-galactosidases, can be used to synthesize galacto-oligosaccharides (GOS) due to the transglycosylating (secondary) activity of these enzymes. Site-directed mutagenesis of a thermoresistant β-galactosidase from Thermotoga maritima has been carried out to study the structural basis of transgalactosylation and to obtain enzymatic variants with better performance for GOS biosynthesis.
Reference: D. Talens-Perales, J. Polaina, and J. Marín-Navarro. "Structural Dissection of the Active Site of Thermotoga maritima β‑Galactosidase Identifies Key Residues for Transglycosylating Activity". J. Agric. Food Chem. 2016, 64, 2917−2924. DOI: 10.1021/acs.jafc.6b00222

Structure-function analysis of beta-fructofuranosidase from Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous β-fructofuranosidase (XdINV) is a highly glycosylated dimeric enzyme that hydrolyzes sucrose and releases fructose from various fructooligosaccharides (FOS) and fructans. It also catalyzes the synthesis of FOS, prebiotics that stimulate the growth of beneficial bacteria in human gut. In contrast to most fructosylating enzymes, XdINV produces neo-FOS, which makes it an interesting biotechnology target. We have just reported its three-dimensional structure, which shows the expected bimodular arrangement but, also, a long extension of its C-terminus that together with a N-linked glycan mediate the formation of an unusual dimer. This arrangement could be representative of a group of GH32 yeast enzymes having the traits observed in XdINV. The role of relevant residues has been investigated by mutagenesis and kinetics measurements and a model for the transfructosylating reaction has been proposed. The plasticity of its active site makes XdINV a valuable and flexible biocatalyst to produce novel bioconjugates.

Ramírez-Escudero, M., Gimeno-Pérez, M., González, B., Linde, D., Merzdo, Z. Fernández-Lobato, M., Sanz-Aparicio, J.,"Structural analysis of β-fructofuranosidase from Xanthophyllomyces dendrorhous reveals unique features and the crucial role of N-glycosylation in oligomerization and activity". J. Biol. Chem.  (2016). doi:10.1074/jbc.M115.708495

Structure-function studies on glucose oxidase, published in Plos ONE

Among the applications of glucose oxidase, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. In this work, we have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described. Link to the article

Heterologous expression of alpha-glucosidase

Basidiomycetous yeast Xanthophyllomyces dendrorhous expresses an α-glucosidase with strong transglycosylation activity producing prebiotic sugars such as panose and an unusual tetrasaccharides mixture including α–(1–6) bonds as major products. In this study, the gene Xd-AlphaGlu was characterized and expressed in Saccharomyces cerevisiae.

"Molecular characterization and heterologous expression of a Xanthophyllomyces dendrorhous α-glucosidase with potential for prebiotics production". Patricia Gutiérrez-Alonso, María Gimeno-Pérez, Mercedes Ramírez-Escudero, Francisco J. Plou, Julia Sanz-Aparicio, María Fernández-Lobato. Applied Microbiology and Biotechnolog,, first online: 28 November 2015. DOI: 10.1007/s00253-015-7171