CAZy Database Research Articles

Xây dựng Probe để khai thác và chọn gen mã hoá βeta-glucosidase GH3 từ dữ liệu giải trình tự DNA metagenome vi khuẩn dạ cỏ dê

Beta-glucosidase enzyme belongs to family GH3 is widely used in food, medicine and pharmaceutical industries. In order to quickly search for these enzyme coding sequences from DNA metagenomic data, in this study, bacterial-derived enzyme-coding sequences which was investigated empirically on the CAZy database was collected. By analogy, we have developed that a specific probe for beta-glucosidase GH3 had a length of 330 amino acids, which contains 26 conserved residues in all sequences, 37 residues similar in almost sequences, and 21 residues conserved in many sequences and homologous with all the reference sequences with the lowest coverage and identity of 79% and 38% respectively and max score of 122. Probe was used and extracted 59 coded sequences of beta-glucosidase GH3 from the metagenome DNA sequences of goat rumen bacteria. The most ORFs were annotated as beta-glucosidase GH3 by KEGG and CAZy. The sequences are estimated to have a tertiary structure similar to beta-glucosidase.

Proteiniphilum saccharofermentans str. M3/6T isolated from a laboratory biogas reactor is versatile in polysaccharide and oligopeptide utilization as deduced from genome-based metabolic reconstructions

Proteiniphilum saccharofermentans str. M3/6T is a recently described species within the family Porphyromonadaceae (phylum Bacteroidetes), which was isolated from a mesophilic laboratory-scale biogas reactor. The genome of the strain was completely sequenced and manually annotated to reconstruct its metabolic potential regarding biomass degradation and fermentation pathways. The P. saccharofermentans str. M3/6T genome consists of a 4,414,963 bp chromosome featuring an average GC-content of 43.63%. Genome analyses revealed that the strain possesses 3396 protein-coding sequences. Among them are 158 genes assigned to the carbohydrate-active-enzyme families as defined by the CAZy database, including 116 genes encoding glycosyl hydrolases (GHs) involved in pectin, arabinogalactan, hemicellulose (arabinan, xylan, mannan, β-glucans), starch, fructan and chitin degradation. The strain also features several transporter genes, some of which are located in polysaccharide utilization loci (PUL). PUL gene products are involved in glycan binding, transport and utilization at the cell surface. In the genome of strain M3/6T, 64 PUL are present and most of them in association with genes encoding carbohydrate-active enzymes. Accordingly, the strain was predicted to metabolize several sugars yielding carbon dioxide, hydrogen, acetate, formate, propionate and isovalerate as end-products of the fermentation process. Moreover, P. saccharofermentans str. M3/6T encodes extracellular and intracellular proteases and transporters predicted to be involved in protein and oligopeptide degradation. Comparative analyses between P. saccharofermentans str. M3/6T and its closest described relative P. acetatigenes str. DSM 18083T indicate that both strains share a similar metabolism regarding decomposition of complex carbohydrates and fermentation of sugars.

Probe design for exploiting gene encoding pectinesterase from dna metagenome data of bacteria in goat rumen and co-expression of gpecs1 gen with chaperone pg-kje8 in Escherichia coli

This article introduces the steps of constructing and using probe to exploit the gene encoding pectinesterase from metagenome DNA sequencing data by next generation gene sequencing tools. Probe was used to exploit and select the gene encoding for pectinesterase from the metagenome DNA sequences of bacteria in goat rumen and thereby select a sequence to express in E. coli. According to the CAZy classification system, pectinesterase belongs to the family of carbohydrates esterases CE8 is an enzyme that has many applications in the food processing industry, environmental treatment, animal feed processing and medicine. As the results, 3 sequences of CE8 was retrieved from CAZy database and one probe was designed, this probe length was 367 amino acids contained all the conserved amino acid residues: 200 conserved residues in all sequence, 72 residues similar in almost sequences and residues conserved in many sequences and homologus; choosed highest alkalinity index. Using the probe designed, we filtered four coding sequences for pectinesterase from metagenome DNA sequencing data of bacteria in goat rumen. Spatial structure estimation with Phyre2 has only one sequencing (code 46301) with 100% sequence identity and 90% query coverage with pectinesterase. A artificial gene were synthesized and inserted into the vector pET22b (+) at the NcoI, XhoI to co-express with chaperone pG-KJE8 in E. coli. The recombinant pectinesterase enzyme is expressed in soluble form and has a pectin substrate biodegradation activity. The results demonstrate that using probe for gene extraction is feasible.

Probe design for mining and selection of genes coding endo 1- 4 xylanase from dna metagenome data

According to the CAZY classification, endo 1- 4 xylanase belongs to GH 5, 8, 10, 11, 30, 51, 98. However only 03 sequences of GH8, 27 sequences of GH10, 18 sequence of GH11, only one sequence of each GH30 and GH51 from CAZy and NCBI database were thouroughly experimentally studied for biological activity and characteristics of the enzyme. Through the collected sequences, two probes for endo 1- 4 xylanase of GH10 and GH11 were designed, based on the sequence homology. The GH10 probe was 338 amino acids lenghth contained all the conserved amino acid residues (16 conserved residues in all sequences, 13 residues similar in almost sequences, 14 residues conserved in many sequences) with the lowest maxscore of 189, coverage of 88% and identity of 39%. The GH11 probe was 204 amino acids contained all the conserved amino acid residues (54 conserved residues were identity in all sequences, 25 residues similar in almost sequences, 24 residues conserved in many sequences) with the lowest maxscore of 165, coverage of 84% and identity of 50%. Using the two probes, we mined only one sequence (GL0018509) for endo 1- 4 xylanase from metagenomic DNA data of free-living bacteria in Coptotermes termite gut. Prediction of three-dimention structure of GL0018509 sequence by Phyre2 and Swiss Prot showed that this sequence was high similarity (95% by Phyre2 and 93,4% by Swiss Prot) with endo 1- 4 xylanase with the 100% confidence.

Carbohydrate Esterases: An Overview.

Carbohydrate esterases are a group of enzymes which release acyl or alkyl groups attached by ester linkage to carbohydrates. The CAZy database, which classifies enzymes that assemble, modify, and break down carbohydrates and glycoconjugates, classifies all carbohydrate esterases into 16 families. This chapter is an overview of the research for nearly 50years around the main groups of carbohydrate esterases dealing with the degradation of polysaccharides, their main biochemical and molecular traits, as well as its application for the synthesis of high added value esters.

Receptor protein of Lysinibacillus sphaericus mosquito-larvicidal toxin displays amylomaltase activity

The activated binary toxin (BinAB) from Lysinibacillus sphaericus binds to surface receptor protein (Cqm1) on the midgut cell membrane and kills Culex quinquefasciatus larvae on internalization. Cqm1 is attached to cells via a glycosyl-phosphatidylinositol (GPI) anchor. It has been classified as a member of glycoside hydrolase family 13 of the CAZy database. Here, we report characterization of the ordered domain (residues 23–560) of Cqm1. Gene expressing Cqm1 of BinAB susceptible mosquito was chemically synthesized and the protein was purified using E. coli expression system. Values for the Michaelis-Menten kinetics parameters towards 4-nitrophenyl α-D-glucopyranoside (α-pNPG) substrate were estimated to be 0.44 mM (Km) and 1.9 s−1 (kcat). Thin layer chromatography experiments established Cqm1 as α-glucosidase competent to cleave α-1,4-glycosidic bonds of maltose and maltotriose with high glycosyltransferase activity to form glucose-oligomers. The observed hydrolysis and synthesis of glucose-oligomers is consistent with open and accessible active-site in the structural model. The protein also hydrolyses glycogen and sucrose. These activities suggest that Cqm1 may be involved in carbohydrate metabolism in mosquitoes. Further, toxic BinA component does not inhibit α-glucosidase activity of Cqm1, while BinB reduced the activity by nearly 50%. The surface plasmon resonance study reveals strong binding of BinB with Cqm1 (Kd, 9.8 nM). BinA interaction with Cqm1 however, is 1000-fold weaker. Notably the estimated Kd values match well with dissociation constants reported earlier with larvae brush border membrane fractions. The Cqm1 protein forms a stable dimer that is consistent with its apical localization in lipid rafts. Its melting temperature (Tm) as observed by thermofluor-shift assay is 51.5 °C and Ca2+ provides structural stability to the protein.

Fungal glucuronoyl esterases: Genome mining based enzyme discovery and biochemical characterization

4-O-Methyl-d-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and therefore may play a significant role as auxiliary enzymes in biomass saccharification for the production of biofuels and biochemicals. GEs belong to a relatively new family of carbohydrate esterases (CE15) in the CAZy database (www.cazy.org), and so far around ten fungal GEs have been characterized. To explore additional GE enzymes, we used a genome mining strategy. BLAST analysis with characterized GEs against approximately 250 publicly accessible fungal genomes identified more than 150 putative fungal GEs, which were classified into eight phylogenetic sub-groups. To validate the genome mining strategy, 21 selected GEs from both ascomycete and basidiomycete fungi were heterologously produced in Pichia pastoris. Of these enzymes, 18 were active against benzyl d-glucuronate demonstrating the suitability of our genome mining strategy for enzyme discovery.

Carbohydrate-active enzymes in Trichoderma harzianum: a bioinformatic analysis bioprospecting for key enzymes for the biofuels industry

BackgroundTrichoderma harzianum is used in biotechnology applications due to its ability to produce powerful enzymes for the conversion of lignocellulosic substrates into soluble sugars. Active enzymes involved in carbohydrate metabolism are defined as carbohydrate-active enzymes (CAZymes), and the most abundant family in the CAZy database is the glycoside hydrolases. The enzymes of this family play a fundamental role in the decomposition of plant biomass.ResultsIn this study, the CAZymes of T. harzianum were identified and classified using bioinformatic approaches after which the expression profiles of all annotated CAZymes were assessed via RNA-Seq, and a phylogenetic analysis was performed. A total of 430 CAZymes (3.7% of the total proteins for this organism) were annotated in T. harzianum, including 259 glycoside hydrolases (GHs), 101 glycosyl transferases (GTs), 6 polysaccharide lyases (PLs), 22 carbohydrate esterases (CEs), 42 auxiliary activities (AAs) and 46 carbohydrate-binding modules (CBMs). Among the identified T. harzianum CAZymes, 47% were predicted to harbor a signal peptide sequence and were therefore classified as secreted proteins. The GH families were the CAZyme class with the greatest number of expressed genes, including GH18 (23 genes), GH3 (17 genes), GH16 (16 genes), GH2 (13 genes) and GH5 (12 genes). A phylogenetic analysis of the proteins in the AA9/GH61, CE5 and GH55 families showed high functional variation among the proteins.ConclusionsIdentifying the main proteins used by T. harzianum for biomass degradation can ensure new advances in the biofuel production field. Herein, we annotated and characterized the expression levels of all of the CAZymes from T. harzianum, which may contribute to future studies focusing on the functional and structural characterization of the identified proteins.

Ten years of CAZypedia: a living encyclopedia of carbohydrate-active enzymes

CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate-active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification and degradation of complex carbohydrates. CAZypedia is closely connected with the actively curated CAZy database, which provides a sequence-based foundation for the biochemical, mechanistic and structural characterization of these diverse proteins. Now celebrating its 10th anniversary online, CAZypedia is a successful example of dynamic, community-driven and expert-based biocuration. CAZypedia is an open-access resource available at URL http://www.cazypedia.org.

Novel archaeal thermostable cellulases from an oil reservoir metagenome

Microbial assemblages were sampled from an offshore deep sub-surface petroleum reservoir 2.5 km below the ocean floor off the coast of Norway, providing conditions of high temperature and pressure, to identify new thermostable enzymes. In this study, we used DNA sequences obtained directly from the sample metagenome and from a derived fosmid library to survey the functional diversity of this extreme habitat. The metagenomic fosmid library containing 11,520 clones was screened using function- and sequence-based methods to identify recombinant clones expressing carbohydrate-degrading enzymes. Open reading frames (ORFs) encoding carbohydrate-degrading enzymes were predicted by BLAST against the CAZy database, and many fosmid clones expressing carbohydrate-degrading activities were discovered by functional screening using Escherichia coli as a heterologous host. Each complete ORF predicted to encode a cellulase identified from sequence- or function-based screening was subcloned in an expression vector. Five subclones was found to have significant activity using a fluorescent cellulose model substrate, and three of these were observed to be highly thermostable. Based on phylogenetic analyses, the thermostable cellulases were derived from thermophilic Archaea and are distinct from known cellulases. Cellulase F1, obtained from function-based screening, contains two distinct cellulase modules, perhaps resulting from fusion of two archaeal cellulases and with a novel protein structure that may result in enhanced activity and thermostability. This enzyme was found to exhibit exocellulase function and to have a remarkably high activity compared to commercially available enzymes. Results from this study highlight the complementarity of hybrid approaches for enzyme discovery, combining sequence- and function-based screening.

Metagenomic mining pectinolytic microbes and enzymes from an apple pomace-adapted compost microbial community

BackgroundDegradation of pectin in lignocellulosic materials is one of the key steps for biofuel production. Biological hydrolysis of pectin, i.e., degradation by pectinolytic microbes and enzymes, is an attractive paradigm because of its obvious advantages, such as environmentally friendly procedures, low in energy demand for lignin removal, and the possibility to be integrated in consolidated process. In this study, a metagenomics sequence-guided strategy coupled with enrichment culture technique was used to facilitate targeted discovery of pectinolytic microbes and enzymes. An apple pomace-adapted compost (APAC) habitat was constructed to boost the enrichment of pectinolytic microorganisms.ResultsAnalyses of 16S rDNA high-throughput sequencing revealed that microbial communities changed dramatically during composting with some bacterial populations being greatly enriched. Metagenomics data showed that apple pomace-adapted compost microbial community (APACMC) was dominated by Proteobacteria and Bacteroidetes. Functional analysis and carbohydrate-active enzyme profiles confirmed that APACMC had been successfully enriched for the targeted functions. Among the 1756 putative genes encoding pectinolytic enzymes, 129 were predicted as novel (with an identity <30% to any CAZy database entry) and only 1.92% were more than 75% identical with proteins in NCBI environmental database, demonstrating that they have not been observed in previous metagenome projects. Phylogenetic analysis showed that APACMC harbored a broad range of pectinolytic bacteria and many of them were previously unrecognized.ConclusionsThe immensely diverse pectinolytic microbes and enzymes found in our study will expand the arsenal of proficient degraders and enzymes for lignocellulosic biofuel production. Our study provides a powerful approach for targeted mining microbes and enzymes in numerous industries.

The starch-binding domain family CBM41-An in silico analysis of evolutionary relationships.

Within the CAZy database, there are 81 carbohydrate-binding module (CBM) families. A CBM represents a non-catalytic domain in a modular arrangement of glycoside hydrolases (GHs). The present in silico study has been focused on starch-binding domains from the family CBM41 that are usually part of pullulanases from the α-amylase family GH13. Currently there are more than 1,600 sequences classified in the family CBM41, almost exclusively from Bacteria, and so a study was undertaken in an effort to divide the members into relevant groups (subfamilies) and also to contribute to the evolutionary picture of family CBM41. The CBM41 members adopt a β-sandwich fold (∼100 residues) with one carbohydrate-binding site formed by the side-chains of three aromatic residues that interact with carbohydrate. The family CBM41 can be divided into two basic subdivisions, distinguished from each other by a characteristic sequence pattern or motif of the three essential aromatics as follows: (i) "W-W-∼10aa-W" (the so-called Streptococcus/Klebsiella-type); and (ii) "W-W-∼30aa-W" (Thermotoga-type). Based on our bioinformatics analysis it is clear that the first and second positions of the motif can be occupied by aromatic residues (Phe, Tyr, His) other than tryptophan, resulting in the existence of six different carbohydrate-binding CBM41 groups, that reflect mostly differences in taxonomy, but which should retain the ability to bind an α-glucan. In addition, three more groups have been proposed that, although lacking the crucial aromatic motif, could possibly employ other residues from remaining parts of their sequence for binding carbohydrate. Proteins 2017; 85:1480-1492. © 2017 Wiley Periodicals, Inc.

Homology to peptide pattern for annotation of carbohydrate-active enzymes and prediction of function

BackgroundCarbohydrate-active enzymes are found in all organisms and participate in key biological processes. These enzymes are classified in 274 families in the CAZy database but the sequence diversity within each family makes it a major task to identify new family members and to provide basis for prediction of enzyme function. A fast and reliable method for de novo annotation of genes encoding carbohydrate-active enzymes is to identify conserved peptides in the curated enzyme families followed by matching of the conserved peptides to the sequence of interest as demonstrated for the glycosyl hydrolase and the lytic polysaccharide monooxygenase families. This approach not only assigns the enzymes to families but also provides functional prediction of the enzymes with high accuracy.ResultsWe identified conserved peptides for all enzyme families in the CAZy database with Peptide Pattern Recognition. The conserved peptides were matched to protein sequence for de novo annotation and functional prediction of carbohydrate-active enzymes with the Hotpep method. Annotation of protein sequences from 12 bacterial and 16 fungal genomes to families with Hotpep had an accuracy of 0.84 (measured as F1-score) compared to semiautomatic annotation by the CAZy database whereas the dbCAN HMM-based method had an accuracy of 0.77 with optimized parameters. Furthermore, Hotpep provided a functional prediction with 86% accuracy for the annotated genes. Hotpep is available as a stand-alone application for MS Windows.ConclusionsHotpep is a state-of-the-art method for automatic annotation and functional prediction of carbohydrate-active enzymes.

New Ulvan-Degrading Polysaccharide Lyase Family: Structure and Catalytic Mechanism Suggests Convergent Evolution of Active Site Architecture.

Ulvan is a complex sulfated polysaccharide biosynthesized by green seaweed and contains predominantly rhamnose, xylose, and uronic acid sugars. Ulvan-degrading enzymes have only recently been identified and added to the CAZy ( www.cazy.org ) database as family PL24, but neither their structure nor catalytic mechanism(s) are yet known. Several homologous, new ulvan lyases, have been discovered in Pseudoalteromonas sp. strain PLSV, Alteromonas LOR, and Nonlabens ulvanivorans, defining a new family PL25, with the lyase encoded by the gene PLSV_3936 being one of them. This enzyme cleaves the glycosidic bond between 3-sulfated rhamnose (R3S) and glucuronic acid (GlcA) or iduronic acid (IdoA) via a β-elimination mechanism. We report the crystal structure of PLSV_3936 and its complex with a tetrasaccharide substrate. PLSV_3936 folds into a seven-bladed β-propeller, with each blade consisting of four antiparallel β-strands. Sequence conservation analysis identified a highly conserved region lining at one end of a deep crevice on the protein surface. The putative active site was identified by mutagenesis and activity measurements. Crystal structure of the enzyme with a bound tetrasaccharide substrate confirmed the identity of base and acid residues and allowed determination of the catalytic mechanism and also the identification of residues neutralizing the uronic acid carboxylic group. The PLSV_3936 structure provides an example of a convergent evolution among polysaccharide lyases toward a common active site architecture embedded in distinct folds.

Polysaccharide Degradation Capability of Actinomycetales Soil Isolates from a Semiarid Grassland of the Colorado Plateau.

Among the bacteria, members of the order Actinomycetales are considered quintessential degraders of complex polysaccharides in soils. However, studies examining complex polysaccharide degradation by Actinomycetales (other than Streptomyces spp.) in soils are limited. Here, we examine the lignocellulolytic and chitinolytic potential of 112 Actinomycetales strains, encompassing 13 families, isolated from a semiarid grassland of the Colorado Plateau in Utah. Members of the Streptomycetaceae, Pseudonocardiaceae, Micromonosporaceae, and Promicromonosporaceae families exhibited robust activity against carboxymethyl cellulose, xylan, chitin, and pectin substrates (except for low/no pectinase activity by the Micromonosporaceae). When incubated in a hydrated mixture of blended Stipa and Hilaria grass biomass over a 5-week period, Streptomyces and Saccharothrix (a member of the Pseudonocardiaceae) isolates produced high levels of extracellular enzyme activity, such as endo- and exocellulase, glucosidase, endo- and exoxylosidase, and arabinofuranosidase. These characteristics make them well suited to degrade the cellulose and hemicellulose components of grass cell walls. On the basis of the polysaccharide degradation profiles of the isolates, relative abundance of Actinomycetales sequences in 16S rRNA gene surveys of Colorado Plateau soils, and analysis of genes coding for polysaccharide-degrading enzymes among 237 Actinomycetales genomes in the CAZy database and 5 genomes from our isolates, we posit that Streptomyces spp. and select members of the Pseudonocardiaceae and Micromonosporaceae likely play an important role in the degradation of hemicellulose, cellulose, and chitin substances in dryland soils.IMPORTANCE Shifts in the relative abundance of Actinomycetales taxa have been observed in soil microbial community surveys during large, manipulated climate change field studies. However, our limited understanding of the ecophysiology of diverse Actinomycetales taxa in soil systems undermines attempts to determine the underlying causes of the population shifts or their impact on carbon cycling in soil. This study combines a systematic analysis of the polysaccharide degradation potential of a diverse collection of Actinomycetales isolates from surface soils of a semiarid grassland with analysis of genomes from five of these isolates and publicly available Actinomycetales genomes for genes encoding polysaccharide-active enzymes. The results address an important gap in knowledge of Actinomycetales ecophysiology-identification of key taxa capable of facilitating lignocellulose degradation in dryland soils. Information from this study will benefit future metagenomic studies related to carbon cycling in dryland soils by providing a baseline linkage of Actinomycetales phylogeny with lignocellulolytic functional potential.

The evolutionary origin and possible functional roles of FNIII domains in two Microbacterium aurum B8.A granular starch degrading enzymes, and in other carbohydrate acting enzymes

AbstractFibronectin type III (FNIII) domains were first identified in the eukaryotic plasma protein fibronectin, where they act as structural spacers or enable protein-protein interactions. Recently we characterized two large and multi-domain amylases in Microbacterium aurum B8.A that both carry multiple FNIII and carbohydrate binding modules (CBMs). The role of (multiple) FNIII domains in such carbohydrate acting enzymes is currently unclear. Four hypothetical functions are considered here: a substrate surface disruption domain, a carbohydrate binding module, as a stable linker, or enabling protein-protein interactions. We performed a phylogenetic analysis of all FNIII domains identified in proteins listed in the CAZy database. These data clearly show that the FNIII domains in eukaryotic and archaeal CAZy proteins are of bacterial origin and also provides examples of interkingdom gene transfer from Bacteria to Archaea and Eucarya. FNIII domains occur in a wide variety of CAZy enzymes acting on many different substrates, suggesting that they have a non-specific role in these proteins. While CBM domains are mostly found at protein termini, FNIII domains are commonly located between other protein domains. FNIII domains in carbohydrate acting enzymes thus may function mainly as stable linkers to allow optimal positioning and/or flexibility of the catalytic domain and other domains, such as CBM.

Performance of Hidden Markov Models in Recovering the Standard Classification of Glycoside Hydrolases.

Glycoside hydrolases (GHs) are carbohydrate-active enzymes that assist the hydrolysis of glycoside bonds of complex sugars into carbohydrates. The current standard GH family classification is available in the CAZy database, which is based on the similarities of amino acid sequences and curated semi-automatically. However, with the exponential increase in data availability from genome sequences, automated classification methods are required for the fast annotation of coding sequences. Currently, the dbCAN database offers automatic annotations of signature domains from CAZy-defined classifications using a statistical approach, the hidden Markov models (HMMs). However, dbCAN does not contain the entire set of CAZy GH families. Moreover, no evaluation has been conducted so far of the viability of using HMM profiles as a means of automatically assigning GH amino acid sequences to the standard CAZy GH family classification itself. In this work, we performed a meta-analysis in which amino acid sequences from CAZy-defined GH families were used to build HMM family-specific profiles. We then queried a set with ~300 000 GH sequences against our database of HMM profiles estimated from CAZy families. We conducted the same evaluation against the available dbCAN HMM profiles. Our analyses recovered 65% of matches with the standard CAZy classification, whereas dbCAN HMMs resulted in 61% of matches. We also provided an analysis of the types of errors commonly found when HMMs are used to recover CAZy-based classifications. Although the performance of HMM was good, further developments are necessary for a fully automated classification of GH, allowing the standardization of GH classification among protein databases.

An Effective Bacterial Fucosidase for Glycoprotein Remodeling.

Fucose is an important component of many oligo- and polysaccharide structures as well as glycoproteins and glycolipids, which are often associated with a variety of physiological processes ranging from fertilization, embryogenesis, signal transduction, and disease progression, such as rheumatoid arthritis, inflammation, and cancer. The enzyme α-l-fucosidase is involved in the cleavage of the fucosidic bond in glycans and glycoconjugates, particularly the Fuc-α-1,2-Gal, Fuc-α-1,3/4-GlcNAc, and Fuc-α-1,6-GlcNAc linkages. Here, we report a highly efficient fucosidase, designated as BfFucH identified from a library of bacterial glycosidases expressed in E. coli from the CAZy database, which is capable of hydrolyzing the aforementioned fucosidic linkages, especially the α-1,6-linkage from the N-linked Fuc-α-1,6-GlcNAc residue on glycoproteins. Using BfFucH coupled with endoglycosidases and the emerging glycosynthases allows glycoengineering of IgG antibodies to provide homogeneous glycoforms with well-defined glycan structures and optimal effector functions.

Identification of the Core Set of Carbon-Associated Genes in a Bioenergy Grassland Soil.

Despite the central role of soil microbial communities in global carbon (C) cycling, little is known about soil microbial community structure and even less about their metabolic pathways. Efforts to characterize soil communities often focus on identifying differences in gene content across environmental gradients, but an alternative question is what genes are similar in soils. These genes may indicate critical species or potential functions that are required in all soils. Here we identified the “core” set of C cycling sequences widely present in multiple soil metagenomes from a fertilized prairie (FP). Of 226,887 sequences associated with known enzymes involved in the synthesis, metabolism, and transport of carbohydrates, 843 were identified to be consistently prevalent across four replicate soil metagenomes. This core metagenome was functionally and taxonomically diverse, representing five enzyme classes and 99 enzyme families within the CAZy database. Though it only comprised 0.4% of all CAZy-associated genes identified in FP metagenomes, the core was found to be comprised of functions similar to those within cumulative soils. The FP CAZy-associated core sequences were present in multiple publicly available soil metagenomes and most similar to soils sharing geographic proximity. In soil ecosystems, where high diversity remains a key challenge for metagenomic investigations, these core genes represent a subset of critical functions necessary for carbohydrate metabolism, which can be targeted to evaluate important C fluxes in these and other similar soils.

Characterization of a new α-l-fucosidase isolated from Fusarium proliferatum LE1 that is regioselective to α-(1 → 4)-l-fucosidic linkage in the hydrolysis of α-l-fucobiosides

Here, we report the biochemical characterization of a novel α-l-fucosidase with broad substrate specificity (FpFucA) isolated from the mycelial fungus Fusarium proliferatum LE1. Highly purified α-l-fucosidase was obtained from several chromatographic steps after growth in the presence of l-fucose. The purified α-l-fucosidase appeared to be a monomeric protein of 67 ± 1 kDa that was able to hydrolyze the synthetic substrate p-nitrophenyl α-l-fucopyranoside (pNPFuc), with Km = 1.1 ± 0.1 mM and kcat = 39.8 ± 1.8 s−1. l-fucose, 1-deoxyfuconojirimycin and tris(hydroxymethyl)aminomethane inhibited pNPFuc hydrolysis, with inhibition constants of 0.2 ± 0.05 mM, 7.1 ± 0.05 nM, and 12.2 ± 0.1 mM, respectively. We assumed that the enzyme belongs to subfamily A of the GH29 family (CAZy database) based on its ability to hydrolyze practically all fucose-containing oligosaccharides used in the study and the phylogenetic analysis. We found that this enzyme was a unique α-l-fucosidase that preferentially hydrolyzes the α-(1 → 4)-L-fucosidic linkage present in α-L-fucobiosides with different types of linkages. As a retaining glycosidase, FpFucA is capable of catalyzing the transglycosylation reaction with alcohols (methanol, ethanol, and 1-propanol) and pNP-containing monosaccharides as acceptors. These features make the enzyme an important tool that can be used in the various modifications of valuable fucose-containing compounds.