A Step-by-Step Guide for the Production of Recombinant Fluorescent TAT-HA-Tagged Proteins and their Transduction into Mammalian Cells.

Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers.

Novel β-galactosidase activity and first crystal structure of Glycoside Hydrolase family 154.

Polysaccharide Utilization Loci (PULs) are physically linked gene clusters conserved in the Gram-negative phylum of Bacteroidota and are valuable sources for Carbohydrate Active enZyme (CAZyme) discovery. This study focuses on BD-β-Gal, an enzyme encoded in a metagenomic PUL and member of the Glycoside Hydrolase family 154 (GH154). BD-β-Gal showed exo-β-galactosidase activity with regiopreference for hydrolyzing β-d-(1,6) glycosidic linkages.

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans.

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xyl) backbone that can be substituted with an acetyl group at -2 and 3 positions, and α-(1→2)-linked 4--methylglucopyranosyluronic acid (MeGlcA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xyl are well characterized; however, the previously studied AcXE from (AcXE) was the first to remove the acetyl group from 2--MeGlcA-3--acetyl-substituted Xyl units, yet structural characteristics of these enzymes remain unspecified.

A tripartite carbohydrate-binding module to functionalize cellulose nanocrystals.

The development of protein and microorganism engineering have led to rising expectations of biotechnology in the design of emerging biomaterials, putatively of high interest to reduce our dependence on fossil carbon resources. In this way, cellulose, a renewable carbon based polysaccharide and derived products, displays unique properties used in many industrial applications. Although the functionalization of cellulose is common, it is however limited in terms of number and type of functions.

Enantioselective Reductive Oligomerization of Carbon Dioxide into l-Erythrulose via a Chemoenzymatic Catalysis.

A cell-free enantioselective transformation of the carbon atom of CO has never been reported. In the urgent context of transforming CO into products of high value, the enantiocontrolled synthesis of chiral compounds from CO would be highly desirable. Using an original hybrid chemoenzymatic catalytic process, we report herein the reductive oligomerization of CO into C (dihydroxyacetone, DHA) and C (l-erythrulose) carbohydrates, with perfect enantioselectivity of the latter chiral product.

Synthesis of α-l-Araf and β-d-Galf series furanobiosides using mutants of a GH51 α-l-arabinofuranosidase.

The GH-51 α-l-arabinofuranosidase from Thermobacillus xylanilyticus (TxAbf) possesses versatile catalytic properties, displaying not only the ability to hydrolyze glycosidic linkages but also to synthesize furanobiosides in α-l-Araf and β-d-Galf series. Herein, mutants are investigated to evaluate their ability to perform self-condensation, assessing both yield improvements and changes in regioselectivity. Overall yields of oligo-α-l-arabino- and oligo-β-d-galactofuranosides were increased up to 4.

Polysaccharide utilization loci-driven enzyme discovery reveals BD-FAE: a bifunctional feruloyl and acetyl xylan esterase active on complex natural xylans.

Background: Nowadays there is a strong trend towards a circular economy using lignocellulosic biowaste for the production of biofuels and other bio-based products. The use of enzymes at several stages of the production process (e.g.

Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Transglycosylases.

Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis.

Regioselective chemoenzymatic syntheses of ferulate conjugates as chromogenic substrates for feruloyl esterases.

Generally, carbohydrate-active enzymes are studied using chromogenic substrates that provide quick and easy color-based detection of enzyme-mediated hydrolysis. For feruloyl esterases, commercially available chromogenic ferulate derivatives are both costly and limited in terms of their experimental application. In this study, we describe solutions for these two issues, using a chemoenzymatic approach to synthesize different ferulate compounds.

Probing the determinants of the transglycosylation/hydrolysis partition in a retaining α-l-arabinofuranosidase.

The use of retaining glycoside hydrolases as synthetic tools for glycochemistry is highly topical and the focus of considerable research. However, due to the incomplete identification of the molecular determinants of the transglycosylation/hydrolysis partition (t/h), rational engineering of retaining glycoside hydrolases to create transglycosylases remains challenging. Therefore, to understand better the factors that underpin transglycosylation in a GH51 retaining α-l-arabinofuranosidase from Thermobacillus xylanilyticus, the investigation of this enzyme's active site was pursued.

Synthetic Derivatives of (+)--α-Bisabolol Are Formed by Mammalian Cytochromes P450 Expressed in a Yeast Reconstituted Pathway.

Identification of the enzyme(s) involved in complex biosynthetic pathways can be challenging. An alternative approach might be to deliberately diverge from the original natural enzyme source and use promiscuous enzymes from other organisms. In this paper, we have tested the ability of a series of human and animal cytochromes P450 involved in xenobiotic detoxification to generate derivatives of (+)--α-bisabolol and attempt to produce the direct precursor of hernandulcin, a sweetener from for which the last enzymatic steps are unknown.

Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides.

Combined with chemical synthesis, the use of glycoenzyme biocatalysts has shown great synthetic potential over recent decades owing to their remarkable versatility in terms of substrates and regio- and stereoselectivity that allow structurally controlled synthesis of carbohydrates and glycoconjugates. Nonetheless, the lack of appropriate enzymatic tools with requisite properties in the natural diversity has hampered extensive exploration of enzyme-based synthetic routes to access relevant bioactive oligosaccharides, such as cell-surface glycans or prebiotics. With the remarkable progress in enzyme engineering, it has become possible to improve catalytic efficiency and physico-chemical properties of enzymes but also considerably extend the repertoire of accessible catalytic reactions and tailor novel substrate specificities.

Directed evolution of the type C feruloyl esterase from Fusarium oxysporum FoFaeC and molecular docking analysis of its improved variants.

The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica.

Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis.

The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme.

Enzymatic Activity of Xyloglucan Xylosyltransferase 5.

Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a β-(1,4)-glucan backbone decorated with d-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated.

Biochemical identification of the catalytic residues of a glycoside hydrolase family 120 β-xylosidase, involved in xylooligosaccharide metabolisation by gut bacteria.

The β-xylosidase B from Bifidobacterium adolescentis ATCC15703 belongs to the newly characterized family 120 of glycoside hydrolases. In order to investigate its catalytic mechanism, an extensive kinetic study of the wild-type enzyme and mutants targeting the three highly conserved residues Asp(393), Glu(416) and Glu(364) was performed. NMR analysis of the xyloside hydrolysis products, the change of the reaction rate-limiting step for the Glu(416) mutants, the pH dependency of E416A activity and its chemical rescue allowed to demonstrate that this GH120 enzyme uses a retaining mechanism of glycoside hydrolysis, Glu(416) playing the role of acid/base catalyst and Asp(393) that of nucleophile.

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases.

Carbohydrates are ubiquitous in Nature and play vital roles in many biological systems. Therefore the synthesis of carbohydrate-based compounds is of considerable interest for both research and commercial purposes. However, carbohydrates are challenging, due to the large number of sugar subunits and the multiple ways in which these can be linked together.

Enhancing the chemoenzymatic synthesis of arabinosylated xylo-oligosaccharides by GH51 α-L-arabinofuranosidase.

Random mutagenesis was performed on the α-l-arabinofuranosidase of Thermobacillus xylanilyticus in order to enhance its ability to perform transarabinofuranosylation using natural xylo-oligosaccharides as acceptors. To achieve this goal, a two-step, high-throughput digital imaging protocol involving a colorimetric substrate was used to screen a library of 30,000 mutants. In the first step this screen selected for hydrolytically-impaired mutants, and in the second step the screen identified mutants whose global activity was improved in the presence of a xylo-oligosaccharide mixture.

A ¹H NMR study of the specificity of α-l-arabinofuranosidases on natural and unnatural substrates.

Background: The detailed characterization of arabinoxylan-active enzymes, such as double-substituted xylan arabinofuranosidase activity, is still a challenging topic. Ad hoc chromogenic substrates are useful tools and can reveal subtle differences in enzymatic behavior. In this study, enzyme selectivity on natural substrates has been compared with enzyme selectivity towards aryl-glycosides.

Mutation of a pH-modulating residue in a GH51 α-l-arabinofuranosidase leads to a severe reduction of the secondary hydrolysis of transfuranosylation products.

Background: The development of enzyme-mediated glycosynthesis using glycoside hydrolases is still an inexact science, because the underlying molecular determinants of transglycosylation are not well understood. In the framework of this challenge, this study focused on the family GH51 α-l-arabinofuranosidase from Thermobacillus xylanilyticus, with the aim to understand why the mutation of position 344 provokes a significant modification of the transglycosylation/hydrolysis partition.

Methods: Detailed kinetic analysis (kcat, KM, pKa determination and time-course NMR kinetics) and saturation transfer difference nuclear magnetic resonance spectroscopy was employed to determine the synthetic and hydrolytic ability modification induced by the redundant N344 mutation disclosed in libraries from directed evolution.

Mining for hemicellulases in the fungus-growing termite Pseudacanthotermes militaris using functional metagenomics.

Background: The metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris.

Results: Using whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes.

Engineering transglycosidase activity into a GH51 α-l-arabinofuranosidase.

Directed evolution was applied to the α-l-arabinofuranosidase from Thermobacillus xylanilyticus to confer better transglycosylation ability, particularly for the synthesis of benzyl α-l-arabinofuranosyl-(1,2)-α-d-xylopyranoside, starting from p-nitrophenyl α-l-arabinofuranoside (donor) and benzyl α-d-xylopyranoside (acceptor). The aim was to obtain mutants displaying both lower hydrolytic and greater transglycosylation activities to favour the stable production of the target disaccharide. The implementation of a simple chromogenic screen ultimately provided three mutant enzymes whose properties correspond to those sought after.

Xylosylation as an effective means for reducing yeast growth inhibition by 2-phenylethanol.

Microbial bioproduction processes of 2-phenylethanol, an important rose-like flavor and fragrance compound that occurs naturally in the essential oils of many flowers and plants, are hindered by the growth inhibition it exerts towards the producing microorganism, mainly yeast. We show here for the first time that glycosylation of 2-phenylethanol with xylose increased the inhibitory concentration inducing 50% decrease of the yeast Saccharomyces cerevisiae strain BY4741 growth rate (IC50 ) from 14 mM (1.71 g/L) 2-phenylethanol (2PE) to 100 mM (25.

A versatile and colorful screening tool for the identification of arabinofuranose-acting enzymes.

Selecting wall-nibblers: Three 4-nitrocatechol derivatives were designed to facilitate high-throughput screening of arabinofuranose hydrolases, enzymes that typically digest plant cell walls. The designed compounds can be used in solid and liquid media, and, importantly, one allows the specific detection of AXH-d, a specialized enzyme that only releases L-arabinose from disubstituted D-xylosyl moieties.

Functional roles of H98 and W99 and β2α2 loop dynamics in the α-l-arabinofuranosidase from Thermobacillus xylanilyticus.

This study is focused on the elucidation of the functional role of the mobile β2α2 loop in the α-L-arabinofuranosidase from Thermobacillus xylanilyticus, and particularly on the roles of loop residues H98 and W99. Using site-directed mutagenesis, coupled to characterization methods including isothermal titration calorimetry (ITC) and saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy, and molecular dynamics simulations, it has been possible to provide a molecular level view of interactions and the consequences of mutations. Binding of para-nitrophenyl α-L-arabinofuranoside (pNP-α-l-Araf) to the wild-type arabinofuranosidase was characterized by K(d) values (0.