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Biotechnological Application of Carbohydrate Active Enzymes

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (31 January 2022) | Viewed by 36512

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Prof. Dr. Marco Moracci

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1. Institute of Biosciences and BioResources - National Research Council of Italy, Via P. Castellino 111, 80131 Naples, Italy
2. Department of Biology, University of Naples “Federico II”, Complesso Universitario di Monte S. Angelo, Via Cintia 21, 80126 Napoli, Italy
3. Task Force on Microbiome Studies, University of Naples Federico II, 80126 Naples, Italy
Interests: enzymes from extremophiles; carbohydrate active enzymes; biotechnology of extremophiles

Dr. Beatrice Cobucci-Ponzano

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Institute of Biosciences and BioResources, National Research Council of Italy - CNR, Via P. Castellino, 111 - 80131 Naples, Italy
Interests: structure/function relationship of proteins and enzymes; mechanistic aspects related to hydrolysis and enzymatic synthesis of glycosides; CAZymes

Dr. Andrea Strazzulli

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Department of Biology - University of Naples “Federico II” (UNINA), Edificio 7, Complesso Universitario di Monte S. Angelo, Strada Vicinale Cupa Cintia, 21, 80126 Napoli, Italy
Interests: carbohydrate active enzymes; CAZymes; identification of novel glycosidases

Special Issue Information

Dear Colleagues,

Carbohydrate-active enzymes (CAZymes) are classified as enzymes involved in the assembly, modification, and breakdown of carbohydrate substrates. They are grouped into five different classes which comprise glycoside hydrolases (GH), glycosyl transferases (GT), carbohydrate esterases (CE), polysaccharide lyases (PL), and auxiliary activities (AA).

Among enzymatic activities, CAZymes play a key role and show great application potential in different biotechnological processes, including, for example, the hydrolysis of polysaccharides to obtain fermentable sugars in second-generation biorefineries, the development of biosensors, the synthesis of oligosaccharides of nutraceutical interest, as well as the synthesis and modification of bioactive molecules of industrial and pharmacological interest.

In this scenario, this Special Issue aims to collect new research results focused on the biotechnological application of CAZymes, including their characterization, modification, and engineering of their catalytic machinery, and physicochemical properties.

Authors are encouraged to submit their original research and review articles.

The following topics are relevant to this Special Issue:

Discovery of novel CAZymes;
Characterization of CAZymes of biotechnological interest;
Optimization of enzyme production;
Engineering of CAZymes;
Catalytic property optimization;
Enzymatic cocktails for polysaccharide hydrolysis;
Chemoenzymatic approaches for the synthesis of glycoconjugates.

Prof. Dr. Marco Moracci
Dr. Beatrice Cobucci-Ponzano
Dr. Andrea Strazzulli
Guest Editors

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Published Papers (10 papers)

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Research

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17 pages, 3445 KiB

Open AccessArticle

Valorization of Biomasses from Energy Crops for the Discovery of Novel Thermophilic Glycoside Hydrolases through Metagenomic Analysis

by Roberta Iacono, Andrea Strazzulli, Rosa Giglio, Federica Bitetti, Beatrice Cobucci-Ponzano and Marco Moracci

Int. J. Mol. Sci. 2022, 23(18), 10505; https://doi.org/10.3390/ijms231810505 - 10 Sep 2022

Cited by 5 | Viewed by 2127

Abstract

The increasing interest for environmentally friendly technologies is driving the transition from fossil-based economy to bioeconomy. A key enabler for circular bioeconomy is to valorize renewable biomasses as feedstock to extract high value-added chemicals. Within this transition the discovery and the use of robust biocatalysts to replace toxic chemical catalysts play a significant role as technology drivers. To meet both the demands, we performed microbial enrichments on two energy crops, used as low-cost feed for extremophilic consortia. A culture-dependent approach coupled to metagenomic analysis led to the discovery of more than 300 glycoside hydrolases and to characterize a new

α

-glucosidase from an unknown hyperthermophilic archaeon. Aglu1 demonstrated to be the most active archaeal GH31 on 4Np-α-Glc and it showed unexpected specificity vs. kojibiose, revealing to be a promising candidate for biotechnological applications such as the liquefaction/saccharification of starch. Full article

(This article belongs to the Special Issue Biotechnological Application of Carbohydrate Active Enzymes)

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15 pages, 2286 KiB

Open AccessArticle

Arabinoxylan and Pectin Metabolism in Crohn’s Disease Microbiota: An In Silico Study

by Carlos Sabater, Inés Calvete-Torre, Lorena Ruiz and Abelardo Margolles

Int. J. Mol. Sci. 2022, 23(13), 7093; https://doi.org/10.3390/ijms23137093 - 25 Jun 2022

Cited by 14 | Viewed by 2625

Abstract

Inflammatory bowel disease is a chronic disorder including ulcerative colitis and Crohn’s disease (CD). Gut dysbiosis is often associated with CD, and metagenomics allows a better understanding of the microbial communities involved. The objective of this study was to reconstruct in silico carbohydrate metabolic capabilities from metagenome-assembled genomes (MAGs) obtained from healthy and CD individuals. This computational method was developed as a mean to aid rationally designed prebiotic interventions to rebalance CD dysbiosis, with a focus on metabolism of emergent prebiotics derived from arabinoxylan and pectin. Up to 1196 and 1577 MAGs were recovered from CD and healthy people, respectively. MAGs of Akkermansia muciniphila, Barnesiella viscericola DSM 18177 and Paraprevotella xylaniphila YIT 11841 showed a wide range of unique and specific enzymes acting on arabinoxylan and pectin. These glycosidases were also found in MAGs recovered from CD patients. Interestingly, these arabinoxylan and pectin degraders are predicted to exhibit metabolic interactions with other gut microbes reduced in CD. Thus, administration of arabinoxylan and pectin may ameliorate dysbiosis in CD by promoting species with key metabolic functions, capable of cross-feeding other beneficial species. These computational methods may be of special interest for the rational design of prebiotic ingredients targeting at CD. Full article

(This article belongs to the Special Issue Biotechnological Application of Carbohydrate Active Enzymes)

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13 pages, 930 KiB

Open AccessArticle

Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides

by Pavlína Nekvasilová, Michaela Hovorková, Zuzana Mészáros, Lucie Petrásková, Helena Pelantová, Vladimír Křen, Kristýna Slámová and Pavla Bojarová

Int. J. Mol. Sci. 2022, 23(8), 4106; https://doi.org/10.3390/ijms23084106 - 7 Apr 2022

Cited by 8 | Viewed by 2448

Abstract

Enzymatic synthesis is an elegant biocompatible approach to complex compounds such as human milk oligosaccharides (HMOs). These compounds are vital for healthy neonatal development with a positive impact on the immune system. Although HMOs may be prepared by glycosyltransferases, this pathway is often complicated by the high price of sugar nucleotides, stringent substrate specificity, and low enzyme stability. Engineered glycosidases (EC 3.2.1) represent a good synthetic alternative, especially if variations in the substrate structure are desired. Site-directed mutagenesis can improve the synthetic process with higher yields and/or increased reaction selectivity. So far, the synthesis of human milk oligosaccharides by glycosidases has mostly been limited to analytical reactions with mass spectrometry detection. The present work reveals the potential of a library of engineered glycosidases in the preparative synthesis of three tetrasaccharides derived from lacto-N-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade reactions catalyzed by β3-N-acetylhexosaminidase BbhI from Bifidobacterium bifidum, β4-galactosidase BgaD-B from Bacillus circulans, β4-N-acetylgalactosaminidase from Talaromyces flavus, and β3-galactosynthase BgaC from B. circulans. The reaction products were isolated and structurally characterized. This work expands the insight into the multi-step catalysis by glycosidases and shows the path to modified derivatives of complex carbohydrates that cannot be prepared by standard glycosyltransferase methods. Full article

(This article belongs to the Special Issue Biotechnological Application of Carbohydrate Active Enzymes)

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16 pages, 1745 KiB

Open AccessArticle

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction

by Vânia Cardoso, Joana L. A. Brás, Inês F. Costa, Luís M. A. Ferreira, Luís T. Gama, Renaud Vincentelli, Bernard Henrissat and Carlos M. G. A. Fontes

Int. J. Mol. Sci. 2022, 23(7), 4024; https://doi.org/10.3390/ijms23074024 - 5 Apr 2022

Cited by 4 | Viewed by 2336

Abstract

In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources. Full article

(This article belongs to the Special Issue Biotechnological Application of Carbohydrate Active Enzymes)

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14 pages, 4139 KiB

Open AccessArticle

The Emergence of New Catalytic Abilities in an Endoxylanase from Family GH10 by Removing an Intrinsically Disordered Region

by Carlos Gil-Durán, Romina V. Sepúlveda, Maximiliano Rojas, Víctor Castro-Fernández, Victoria Guixé, Inmaculada Vaca, Gloria Levicán, Fernando D. González-Nilo, María-Cristina Ravanal and Renato Chávez

Int. J. Mol. Sci. 2022, 23(4), 2315; https://doi.org/10.3390/ijms23042315 - 19 Feb 2022

Cited by 2 | Viewed by 2063

Abstract

Endoxylanases belonging to family 10 of the glycoside hydrolases (GH10) are versatile in the use of different substrates. Thus, an understanding of the molecular mechanisms underlying substrate specificities could be very useful in the engineering of GH10 endoxylanases for biotechnological purposes. Herein, we analyzed XynA, an endoxylanase that contains a (β/α)₈-barrel domain and an intrinsically disordered region (IDR) of 29 amino acids at its amino end. Enzyme activity assays revealed that the elimination of the IDR resulted in a mutant enzyme (XynAΔ29) in which two new activities emerged: the ability to release xylose from xylan, and the ability to hydrolyze p-nitrophenyl-β-d-xylopyranoside (pNPXyl), a substrate that wild-type enzyme cannot hydrolyze. Circular dichroism and tryptophan fluorescence quenching by acrylamide showed changes in secondary structure and increased flexibility of XynAΔ29. Molecular dynamics simulations revealed that the emergence of the pNPXyl-hydrolyzing activity correlated with a dynamic behavior not previously observed in GH10 endoxylanases: a hinge-bending motion of two symmetric regions within the (β/α)₈-barrel domain, whose hinge point is the active cleft. The hinge-bending motion is more intense in XynAΔ29 than in XynA and promotes the formation of a wider active site that allows the accommodation and hydrolysis of pNPXyl. Our results open new avenues for the study of the relationship between IDRs, dynamics and activity of endoxylanases, and other enzymes containing (β/α)₈-barrel domain. Full article

(This article belongs to the Special Issue Biotechnological Application of Carbohydrate Active Enzymes)

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20 pages, 1625 KiB

Open AccessArticle

A Fungal Versatile GH10 Endoxylanase and Its Glycosynthase Variant: Synthesis of Xylooligosaccharides and Glycosides of Bioactive Phenolic Compounds

by Ana Pozo-Rodríguez, Juan A. Méndez-Líter, Laura I. de Eugenio, Manuel Nieto-Domínguez, Eva Calviño, Francisco Javier Cañada, Andrés G. Santana, Jaime Díez, Juan L. Asensio, Jorge Barriuso, Alicia Prieto and María Jesús Martínez

Int. J. Mol. Sci. 2022, 23(3), 1383; https://doi.org/10.3390/ijms23031383 - 26 Jan 2022

Cited by 5 | Viewed by 3248

Abstract

The study of endoxylanases as catalysts to valorize hemicellulosic residues and to obtain glycosides with improved properties is a topic of great industrial interest. In this work, a GH10 β-1,4-endoxylanase (XynSOS), from the ascomycetous fungus Talaromyces amestolkiae, has been heterologously produced in Pichia pastoris, purified, and characterized. rXynSOS is a highly glycosylated monomeric enzyme of 53 kDa that contains a functional CBM1 domain and shows its optimal activity on azurine cross-linked (AZCL)–beechwood xylan at 70 °C and pH 5. Substrate specificity and kinetic studies confirmed its versatility and high affinity for beechwood xylan and wheat arabinoxylan. Moreover, rXynSOS was capable of transglycosylating phenolic compounds, although with low efficiencies. For expanding its synthetic capacity, a glycosynthase variant of rXynSOS was developed by directed mutagenesis, replacing its nucleophile catalytic residue E236 by a glycine (rXynSOS-E236G). This novel glycosynthase was able to synthesize β-1,4-xylooligosaccharides (XOS) of different lengths (four, six, eight, and ten xylose units), which are known to be emerging prebiotics. rXynSOS-E236G was also much more active than the native enzyme in the glycosylation of a broad range of phenolic compounds with antioxidant properties. The interesting capabilities of rXynSOS and its glycosynthase variant make them promising tools for biotechnological applications. Full article

(This article belongs to the Special Issue Biotechnological Application of Carbohydrate Active Enzymes)

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20 pages, 3696 KiB

Open AccessArticle

Lytic Polysaccharide Monooxygenase from Talaromyces amestolkiae with an Enigmatic Linker-like Region: The Role of This Enzyme on Cellulose Saccharification

by Juan Antonio Méndez-Líter, Iván Ayuso-Fernández, Florian Csarman, Laura Isabel de Eugenio, Noa Míguez, Francisco J. Plou, Alicia Prieto, Roland Ludwig and María Jesús Martínez

Int. J. Mol. Sci. 2021, 22(24), 13611; https://doi.org/10.3390/ijms222413611 - 19 Dec 2021

Cited by 6 | Viewed by 3545

Abstract

The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete Talaromyces amestolkiae (TamAA9A) has been successfully expressed in Pichia pastoris and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera Penicillium or Talaromyces have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC. To ascertain the function of a C-terminal linker-like region present in the wild-type sequence of the LPMO, two variants of the wild-type enzyme, one without this sequence and one with an additional C-terminal carbohydrate binding domain (CBM), were designed. The three enzymes (native, without linker and chimeric variant with a CBM) were purified in two chromatographic steps and were thermostable and active in the presence of H₂O₂. The transition midpoint temperature of the wild-type LPMO (Tm = 67.7 °C) and its variant with only the catalytic domain (Tm = 67.6 °C) showed the highest thermostability, whereas the presence of a CBM reduced it (Tm = 57.8 °C) and indicates an adverse effect on the enzyme structure. Besides, the potential of the different T. amestolkiae LPMO variants for their application in the saccharification of cellulosic and lignocellulosic materials was corroborated. Full article

(This article belongs to the Special Issue Biotechnological Application of Carbohydrate Active Enzymes)

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12 pages, 1638 KiB

Open AccessArticle

Improved Production of Recombinant Myrosinase in Pichia pastoris

by Zuzana Rosenbergová, Zuzana Hegyi, Miroslav Ferko, Natália Andelová and Martin Rebroš

Int. J. Mol. Sci. 2021, 22(21), 11889; https://doi.org/10.3390/ijms222111889 - 2 Nov 2021

Cited by 6 | Viewed by 2411

Abstract

The effect of the deletion of a 57 bp native signal sequence, which transports the nascent protein through the endoplasmic reticulum membrane in plants, on improved AtTGG1 plant myrosinase production in Pichia pastoris was studied. Myrosinase was extracellularly produced in a 3-liter laboratory fermenter using α-mating factor as the secretion signal. After the deletion of the native signal sequence, both the specific productivity (164.8 U/L/h) and volumetric activity (27 U/mL) increased more than 40-fold compared to the expression of myrosinase containing its native signal sequence in combination with α-mating factor. The deletion of the native signal sequence resulted in slight changes in myrosinase properties: the optimum pH shifted from 6.5 to 7.0 and the maximal activating concentration of ascorbic acid increased from 1 mM to 1.5 mM. Kinetic parameters toward sinigrin were determined: 0.249 mM (K_m) and 435.7 U/mg (V_max). These results could be applied to the expression of other plant enzymes. Full article

(This article belongs to the Special Issue Biotechnological Application of Carbohydrate Active Enzymes)

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Review

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35 pages, 4505 KiB

Open AccessReview

From Cancer Therapy to Winemaking: The Molecular Structure and Applications of β-Glucans and β-1, 3-Glucanases

by Catarina Caseiro, Joana Nunes Ribeiro Dias, Carlos Mendes Godinho de Andrade Fontes and Pedro Bule

Int. J. Mol. Sci. 2022, 23(6), 3156; https://doi.org/10.3390/ijms23063156 - 15 Mar 2022

Cited by 36 | Viewed by 8771

Abstract

β-glucans are a diverse group of polysaccharides composed of β-1,3 or β-(1,3-1,4) linked glucose monomers. They are mainly synthesized by fungi, plants, seaweed and bacteria, where they carry out structural, protective and energy storage roles. Because of their unique physicochemical properties, they have important applications in several industrial, biomedical and biotechnological processes. β-glucans are also major bioactive molecules with marked immunomodulatory and metabolic properties. As such, they have been the focus of many studies attesting to their ability to, among other roles, fight cancer, reduce the risk of cardiovascular diseases and control diabetes. The physicochemical and functional profiles of β-glucans are deeply influenced by their molecular structure. This structure governs β-glucan interaction with multiple β-glucan binding proteins, triggering myriad biological responses. It is then imperative to understand the structural properties of β-glucans to fully reveal their biological roles and potential applications. The deconstruction of β-glucans is a result of β-glucanase activity. In addition to being invaluable tools for the study of β-glucans, these enzymes have applications in numerous biotechnological and industrial processes, both alone and in conjunction with their natural substrates. Here, we review potential applications for β-glucans and β-glucanases, and explore how their functionalities are dictated by their structure. Full article

(This article belongs to the Special Issue Biotechnological Application of Carbohydrate Active Enzymes)

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56 pages, 5160 KiB

Open AccessReview

Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases

by Ao Li, Mounir Benkoulouche, Simon Ladeveze, Julien Durand, Gianluca Cioci, Elisabeth Laville and Gabrielle Potocki-Veronese

Int. J. Mol. Sci. 2022, 23(6), 3043; https://doi.org/10.3390/ijms23063043 - 11 Mar 2022

Cited by 10 | Viewed by 4811

Abstract

Among carbohydrate active enzymes, glycoside phosphorylases (GPs) are valuable catalysts for white biotechnologies, due to their exquisite capacity to efficiently re-modulate oligo- and poly-saccharides, without the need for costly activated sugars as substrates. The reversibility of the phosphorolysis reaction, indeed, makes them attractive tools for glycodiversification. However, discovery of new GP functions is hindered by the difficulty in identifying them in sequence databases, and, rather, relies on extensive and tedious biochemical characterization studies. Nevertheless, recent advances in automated tools have led to major improvements in GP mining, activity predictions, and functional screening. Implementation of GPs into innovative in vitro and in cellulo bioproduction strategies has also made substantial advances. Herein, we propose to discuss the latest developments in the strategies employed to efficiently discover GPs and make the best use of their exceptional catalytic properties for glycoside bioproduction. Full article

(This article belongs to the Special Issue Biotechnological Application of Carbohydrate Active Enzymes)

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