Activity-Based Protein Profiling for the Identification of Novel Carbohydrate-Active Enzymes Involved in Xylan Degradation in the Hyperthermophilic Euryarchaeon Thermococcus sp. Strain 2319x1E

Activity-based protein profiling (ABPP) has so far scarcely been applied in Archaea in general and, especially, in extremophilic organisms. We herein isolated a novel Thermococcus strain designated sp. strain 2319x1E derived from the same enrichment culture as the recently reported Thermococcus sp. strain 2319x1. Both strains are able to grow with xylan as the sole carbon and energy source, and for Thermococcus sp. strain 2319x1E (optimal growth at 85°C, pH 6–7), the induction of xylanolytic activity in the presence of xylan was demonstrated. Since the solely sequence-based identification of xylanolytic enzymes is hardly possible, we established a complementary approach by conducting comparative full proteome analysis in combination with ABPP using α- or β-glycosidase selective probes and subsequent mass spectrometry (MS)-based analysis. This complementary proteomics approach in combination with recombinant protein expression and classical enzyme characterization enabled the identification of a novel bifunctional maltose-forming α-amylase and deacetylase (EGDIFPOO_00674) belonging to the GH57 family and a promiscuous β-glycosidase (EGIDFPOO_00532) with β-xylosidase activity. We thereby further substantiated the general applicability of ABPP in archaea and expanded the ABPP repertoire for the identification of glycoside hydrolases in hyperthermophiles.

Accelerated discovery of novel glycoside hydrolases using targeted functional profiling and selective pressure on the rumen microbiome

Background Carbohydrate-active enzymes (CAZymes) form the most widespread and structurally diverse set of enzymes involved in the breakdown, biosynthesis, or modification of lignocellulose that can be found in living organisms. However, the structural diversity of CAZymes has rendered the targeted discovery of novel enzymes extremely challenging, as these proteins catalyze many different chemical reactions and are sourced by a vast array of microbes. Consequently, many uncharacterized members of CAZyme families of interest have been overlooked by current methodologies (e.g., metagenomic screening) used to discover lignocellulolytic enzymes. Results In the present study, we combined phenotype-based selective pressure on the rumen microbiota with targeted functional profiling to guide the discovery of unknown CAZymes. In this study, we found 61 families of glycoside hydrolases (GH) (out of 182 CAZymes) from protein sequences deposited in the CAZy database—currently associated with more than 20,324 microbial genomes. Phenotype-based selective pressure on the rumen microbiome showed that lignocellulolytic bacteria (e.g., Fibrobacter succinogenes, Butyrivibrio proteoclasticus) and three GH families (e.g., GH11, GH13, GH45) exhibited an increased relative abundance in the rumen of feed efficient cattle when compared to their inefficient counterparts. These results paved the way for the application of targeted functional profiling to screen members of the GH11 and GH45 families against a de novo protein reference database comprised of 1184 uncharacterized enzymes, which led to the identification of 18 putative xylanases (GH11) and three putative endoglucanases (GH45). The biochemical proof of the xylanolytic activity of the newly discovered enzyme validated the computational simulations and demonstrated the stability of the most abundant xylanase. Conclusions These findings contribute to the discovery of novel enzymes for the breakdown, biosynthesis, or modification of lignocellulose and demonstrate that the rumen microbiome is a source of promising enzyme candidates for the biotechnology industry. The combined approaches conceptualized in this study can be adapted to any microbial environment, provided that the targeted microbiome is easy to manipulate and facilitates enrichment for the microbes of interest.

Silage quality and biogas production from Spartina pectinata L. fermented with a novel xylan-degrading strain of Lactobacillus buchneri M B/00077

The aim of the current study was to determine the ability of the Lactobacillus buchneri M B/00077 strain to degrade xylan, its impact on the quality of silage made from the lignocellulosic biomass of Spartina pectinata L., as well as the efficiency of biogas production. In the model in vitro conditions the L. buchneri M B/00077 strain was able to grow in a medium using xylan as the sole source of carbon, and xylanolytic activity was detected in the post-culture medium. In the L. buchneri M B/00077 genome, genes encoding endo-1,4-xylanase and β-xylosidase were identified. The silages prepared using L. buchneri M B/00077 were characterized by a higher concentration of acetic and propionic acids compared to the controls or the silages prepared with the addition of commercial xylanase. The addition of bacteria increased the efficiency of biogas production. From the silages treated with L. buchneri M B/00077, 10% and 20% more biogas was obtained than from the controls and the silages treated with commercial xylanase, respectively. The results of the current study indicated the strain L. buchneri M B/00077 as being a promising candidate for further application in the field of pretreatment of lignocellulosic biomass.

Optimisation of xylanases production by two Cellulomonas strains and their use for biomass deconstruction

One of the main distinguishing features of bacteria belonging to the Cellulomonas genus is their ability to secrete multiple polysaccharide degrading enzymes. However, their application in biomass deconstruction still constitutes a challenge. We addressed the optimisation of the xylanolytic activities in extracellular enzymatic extracts of Cellulomonas sp. B6 and Cellulomonas fimi B-402 for their subsequent application in lignocellulosic biomass hydrolysis by culture in several substrates. As demonstrated by secretomic profiling, wheat bran and waste paper resulted to be suitable inducers for the secretion of xylanases of Cellulomonas sp. B6 and C. fimi B-402, respectively. Both strains showed high xylanolytic activity in culture supernatant although Cellulomonas sp. B6 was the most efficient xylanolytic strain. Upscaling from flasks to fermentation in a bench scale bioreactor resulted in equivalent production of extracellular xylanolytic enzymatic extracts and freeze drying was a successful method for concentration and conservation of the extracellular enzymes, retaining 80% activity. Moreover, enzymatic cocktails composed of combined extra and intracellular extracts effectively hydrolysed the hemicellulose fraction of extruded barley straw into xylose and xylooligosaccharides. Key points • Secreted xylanase activity of Cellulomonas sp. B6 and C. fimi was maximised. • Biomass-induced extracellular enzymes were identified by proteomic profiling. • Combinations of extra and intracellular extracts were used for barley straw hydrolysis.

In silico screening and experimental analysis of family GH11 xylanases for applications under conditions of alkaline pH and high temperature

Background Xylanases are one of the most extensively used enzymes for biomass digestion. However, in many instances, their use is limited by poor performance under the conditions of pH and temperature required by the industry. Therefore, the search for xylanases able to function efficiently at alkaline pH and high temperature is an important objective for different processes that use lignocellulosic substrates, such as the production of paper pulp and biofuels. Results A comprehensive in silico analysis of family GH11 sequences from the CAZY database allowed their phylogenetic classification in a radial cladogram in which sequences of known or presumptive thermophilic and alkalophilic xylanases appeared in three clusters. Eight sequences from these clusters were selected for experimental analysis. The coding DNA was synthesized, cloned and the enzymes were produced in E. coli. Some of these showed high xylanolytic activity at pH values > 8.0 and temperature > 80 °C. The best enzymes corresponding to sequences from Dictyoglomus thermophilum (Xyn5) and Thermobifida fusca (Xyn8). The addition of a carbohydrate-binding module (CBM9) to Xyn5 increased 4 times its activity at 90 °C and pH > 9.0. The combination of Xyn5 and Xyn8 was proved to be efficient for the saccharification of alkali pretreated rice straw, yielding xylose and xylooligosaccharides. Conclusions This study provides a fruitful approach for the selection of enzymes with suitable properties from the information contained in extensive databases. We have characterized two xylanases able to hydrolyze xylan with high efficiency at pH > 8.0 and temperature > 80 °C.

New Arctic Bacterial Isolates with Relevant Enzymatic Potential

Fragments of wood drifting in the vicinity of Spitzbergen were used for the isolation of microorganisms, carried out using atypical carbon sources: colloidal chitin, cellulose and carboxymethylcellulose, xylan, casein, tributrin and olive oil. Purified cultures were subjected to a three-step identification: with classical methods, using MALDI-TOF MS Biotyper whole-cell protein fingerprinting, and molecular analysis of 16S rDNA. Subsequently, a preliminary assessment of the enzymatic potential of isolates was carried out. As a result, cellulolytic activity was observed in more than 50% of the bacterial strains, exhibiting activity of 0.30–0.40 U/mL. Over 53% of the isolates demonstrated xylanolytic activity, of which the highest reached from 0.40 to 0.90 U. Polygalacturonase activity of 0.003–1.6 was also demonstrated in half of the bacterial strains studied. Proteolytic activity of isolates did not exceed 0.3 U. An important highlight was the ability of fluorescent dye production by certain strains, grown on skim milk-agar, but also on pure meat extract.

A quantum biochemistry investigation of the protein–protein interactions for the description of allosteric modulation on biomass-degrading chimera

Detailed binding energies features of interdomain allosteric modulation caused by xylose binding for enhanced xylanolytic activity on xylanase-XBP chimera.

Xylan-degrading ability of thermophilic Actinobacteria from soil in a geothermal area

Rachmania MK, Ningsih F, Setyaningsih PP, Syafitri WA, Sari DCAF, Yabe S, Yokota A, Oetari A, Sjamsuridjal W. 2020. Xylan-degrading ability of thermophilic Actinobacteria isolated from soil in a geothermal area. Biodiversitas 21: 144-154. This study was conducted to obtain the potential isolates of xylan-degrading thermophilic Actinobacteria. Seventeen isolates were obtained from the soil samples collected from Cisolok geysers in West Java, Indonesia. These isolates were screened for their xylan-degrading ability on minimal medium with the addition of 0.5% xylan as substrate, and were incubated at various temperatures for 7 days. A total of 15, 14, 4, and 3 isolates showed a xylan-degrading ability at 45°C, 50°C, 55°C, and 60°C, respectively. The three isolates (SL1-2-R-2, SL1-2-R-3, and SL1-2-R-4) that showed xylan-degrading ability at 60°C on 0.5% xylan were further tested by using minimal medium with the addition of 0.1% RBB-xylan as a substrate. The results showed that these isolates were also able to utilized RBB-xylan at 45 to 60°C. The xylanolytic activity of the crude enzymes from the three isolates on minimal medium containing 0.5% xylan or 0.1% RBB-xylan as substrates, indicated that these isolates were able to produce extracellular xylanase. This study revealed that the soil of Cisolok geysers served as an ideal source for finding the thermophilic Actinobacteria which produced xylanase at high temperatures.

The effect of cultivation technique on enzymes production from sugar beet pulp by Neurospora crassa

This study is concerned with the effect of different cultivation techniques on enzymes production from sugar beet pulp by strain Neurospora crassa isolated from the environment. Cultivation of selected producing microorganism was carried out under the same process conditions using five techniques. Bioprocess efficacy was estimated based on amylolytic, cellulolytic and xylanolytic activity of prepared enzymes mixtures. The obtained results indicate that the selection of cultivation technique had a statistically significant effect on the production of examined hydrolytic enzymes. It was confirmed that solid state cultivation with spontaneous aeration is the best cultivation technique for the production of amylolytic, cellulolytic and xylanolytic enzymes from sugar beet pulp by Neurospora crassa. Submerged cultivation of producing strain with spontaneous aeration resulted in the lowest production of all investigated enzymes under applied experimental conditions. The obtained results are the basis for further research aimed to increase the enzymes yield and activity of their mixture.