4-Nitrophenyl-α-L-arabinofuranoside

Horizontal gene transfer (HGT) is defined as the acquisition by an organism of hereditary material from a phylogenetically unrelated organism. This process is mostly observed among bacteria and archaea, and considered less likely between microbes and multicellular eukaryotes. However, recent studies provide compelling evidence of the evolutionary importance of HGT in eukaryotes, driving functional innovation. Here, we study an HGT event in Folsomia candida (Collembola, Hexapoda) of a carbohydrate-active enzyme homologous to glycosyl hydrase group 43 (GH43). The gene encodes an N-terminal signal peptide, targeting the product for excretion, which suggests that it contributes to the diversity of digestive capacities of the detritivore host. The predicted α-L-arabinofuranosidase shows high similarity to genes in two other Collembola, an insect and a tardigrade. The gene was cloned and expressed in Escherichia coli using a cell-free protein expression system. The expressed protein showed activity against p-nitrophenyl-α-L-arabinofuranoside. Our work provides evidence for functional activity of an HGT gene in a soil-living detritivore, most likely from a bacterial donor, with genuine eukaryotic properties, such as a signal peptide. Co-evolution of metazoan GH43 genes with the Panarthropoda phylogeny suggests the HGT event took place early in the evolution of this ecdysozoan lineage.

Protein nanoparticles from chickpea protein isolates were prepared by heat treatment and their properties were evaluated. Protein was extracted from defatted chickpea seed under alkaline conditions after pretreatment with single arabinofuranosidase or combination of cellulase and xylanase, or without it. Both enzymatic pretreatments delivered protein isolates with enhanced ratio between α-helices and β-sheets/β-strands in their secondary structure comparing to alkaline isolate. Applied heat treatment was performed for 10 min or 20 min at 90 °C, and at pH 7 or pH 9.3. Particle size of prepared nanoparticles varied from 28 to 290 nm, with the smallest particles fabricated from isolate from (cellulase + xylanase)-assisted alkaline extraction at pH 9.3. Protein isolate extracted with the assistance of arabinofuranosidase enabled preparation of nanoparticles with the highest linoleic acid binding capacity - 58% at pH 7, and 69% at pH 9.3. Generally, nanoparticles with smaller particle sizes, and higher linoleic acid binding capacity and ABTS scavenging activity were fabricated from protein extracted by enzyme-assisted alkali protocols and at higher investigated pH. Chickpea protein isolates from enzyme-assisted extractions can be great source for preparation of nanoparticles with advanced properties for the application in food sector.

A study was carried out to investigate the characterization of a novel Aspergillus sulphureus JCM01963 xylanase (AS-xyn10A) with a carbohydrate binding module (CBM) and its application in degrading alkali pretreated corncob, rapeseed meal and corn stover alone and in combination with a commercial cellulase. In this study, the 3D structure of AS-xyn10A, which contained a CBM at C-terminal. AS-xyn10A and its CBM-truncated variant (AS-xyn10A-dC) was codon-optimized and over-expressed in Komagaella phaffii X-33 (syn. Pichia pastoris) and characterized with optimal condition at 70°C and pH 5.0, respectively. AS-xyn10A displayed high activity to xylan extracted from corn stover, corncob, and rapeseed meal. The concentration of hydrolyzed xylo-oligosaccharides (XOSs) reached 1592.26 μg/mL, 1149.92 μg/mL, and 621.86 μg/mL, respectively. Xylobiose was the main product (~70%) in the hydrolysis mixture. AS-xyn10A significantly synergized with cellulase to improve the hydrolysis efficiency of corn stover, corncob, and rapeseed meal to glucose. The degree of synergy (DS) was 1.32, 1.31, and 1.30, respectively. Simultaneously, XOSs hydrolyzed with AS-xyn10A and cellulase was improved by 46.48%, 66.13% and 141.45%, respectively. In addition, CBM variant decreased the yields of xylo-oligosaccharide and glucose in rapeseed meal degradation. This study provided a novel GH10 endo-xylanase, which has potential applications in hydrolysis of biomass.

Arabinofuranosidase plays an essential role in the process of hydrolysis of arabinoxylan (AX). Thermostable, versatile, and efficient arabinofuranosidase is thus of great interest for the biorefinery industry. A GH51 arabinofuranosidase, Abf51, from Hungateiclostridium clariflavum DSM 19732 was heterogeneously expressed in Escherichia coli. Abf51 was found to have an optimal pH and temperature of 6.5 and 60°C, respectively, with very high thermostability. At the optimal working temperature (60°C), Abf51 retained over 90% activity after a 2-day incubation and over 60% activity after a 6-day incubation. Abf51 could effectively remove the arabinofuranosyls from three kinds of AX oligosaccharides [2³-α-L-arabinofuranosyl-xylotriose (A²XX), 3²-α-L-arabinofuranosyl-xylobiose (A³X), and 2³3³-di-α-L-arabinofuranosyl-xylotriose (A^{2 + 3}XX)], which characterized as either single substitution or double substitution by arabinofuranosyls on terminal xylopyranosyl units. The maximal catalytic efficiency (K_cat/K_m) was observed using p-nitrophenyl-α-L-arabinofuranoside (pNPAF) as a substrate (205.0 s⁻¹ mM⁻¹), followed by using A³X (22.8 s⁻¹ mM⁻¹), A²XX (6.9 s⁻¹ mM⁻¹), and A^{2 + 3}XX (0.5 s⁻¹ mM⁻¹) as substrates. Moreover, the presence of Abf51 significantly stimulated the saccharification level of AX (18.5 g L⁻¹) up to six times along with a β-xylanase as well as a β-xylosidase. Interestingly, in our survey of top thermostable arabinofuranosidases, most members were found from GH51, probably due to their owning of (β/α)₈-barrel architectures. Our results suggested the great importance of GH51s as candidates for thermostable, versatile, and efficient arabinofuranosidases toward industry application.

Many glycoside hydrolases involved in deconstruction of cellulose and xylan from the excellent plant cell wall polysaccharides-degrader Caldicellulosiruptor bescii have been cloned and analyzed. However, far less is known about the enzymatic breakdown of mannan, an important component of hemicellulose. We herein cloned, expressed and purified the first β-mannosidase CbMan2A from C. bescii. CbMan2A is thermophilic, with an optimal temperature of 80°C. CbMan2A hydrolyzes mannooligosaccharides with degrees of polymerization from 2 to 6 mainly into mannose and shows strong synergy with CbMan5A, an endo-mannanase from the same bacterium, in releasing mannose from β-1,4-mannan. Thus CbMan2A forms the missing link in enzymatic conversion of mannan into the ready-to-use mannose by C. bescii. Based on these observations, a model illustrating how CbMan2A may assist C. bescii in mannan utilization is presented. In addition, CbMan2A appeared to bind to insoluble galactomannan in a pH-dependent fashion. Although the relation of this feature to mannan utilization remains elusive, CbMan2A represents an excellent model for investigation of the binding of GH2 β-mannosidases to galactomannan.

The enzymatic depolymerization of xylans into their monomeric sugars by hemicellulases is of great interest from both the ecological and the economical point of view; however, the high costs of these enzymes impede their employment on industrial scales. The utilization of whole cells displaying the enzymes on their surface could reduce costs by allowing a direct employment of the cells after cultivation and their reuse in multiple reaction cycles. Here, we present the surface display of an endo‐1,4‐β‐xylanase (XynA), a 1,4‐β‐xylosidase (XynB), and two α‐L‐arabinofuranosidases (Abf2 and AbfCelf) in the gram‐negative soil bacterium Pseudomonas putida KT2440 by fusing the enzymes to the EhaA autotransporter unit from Escherichia coli. The surface display of the enzymes was verified by flow cytometry. All four enzymes retained their functionality with hydrolytic activities of 48.5 mU mL⁻¹ for XynA towards beechwood xylan, 6 mU mL⁻¹ for XynB towards 4‐nitrophenyl‐β‐D‐xylopyranoside, and 8.6 mU mL⁻¹ and 6.2 mU mL⁻¹ for the two α‐L‐arabinofuranosidases Abf2 and AbfCelf towards 4‐nitrophenyl‐α‐L‐arabinofuranoside, respectively. Measurements were done with cell suspensions of an OD₅₇₈=1. A mixture of strains displaying the three types of hemicellulases could degrade 2.5 % (w/v) raw arabinoxylan from rye bran to D‐xylose with a yield of 133.5 mg L⁻¹ cell suspension after 24 h.

A novel, family GH10 enzyme, Xyn10B from Acidothermus cellulolyticus 11B was cloned and expressed in Escherichia coli. This enzyme was purified to homogeneity by binding to regenerated amorphous cellulose. It had higher binding on Avicel as compared to insoluble xylan due to the presence of cellulose-binding domains, CBM3 and CBM2. This enzyme was optimally active at 70°C and pH 6.0. It was stable up to 70°C while the CD spectroscopy analysis showed thermal unfolding at 80°C. Xyn10B was found to be a trifunctional enzyme having endo-xylanase, arabinofuranosidase and acetyl xylan esterase activities. Its activities against beechwood xylan, p-Nitrophenyl arabinofuranoside and p-Nitrophenyl acetate were found to be 126,480, 10,350 and 17,250 U µmol^-1, respectively. Xyn10B was highly active producing xylobiose and xylose as the major end products, as well as debranching the substrates by removing arabinose and acetyl side chains. Due to its specific characteristics, this enzyme seems to be of importance for industrial applications such as pretreatment of poultry cereals, bio-bleaching of wood pulp and degradation of plant biomass.