Endo-β-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family

The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).     

Heterologous expression of glycoside hydrolase family 2 and 42 β-galactosidases of lactic acid bacteria in Lactococcus lactis

This study characterized a glycoside hydrolase family 42 (GH42) β-galactosidase of Lactobacillus acidophilus (LacA) and compared lactose hydrolysis, hydrolysis of oNPG, pNPG and pNPG-analogues and galactooligosaccharides (GOSs) formation to GH2 β-galactosidases of Streptococcus thermophilus (LacZ type), Lactobacillus plantarum and Leuconostoc mesenteroides subsp. cremoris (both LacLM type). Beta-galactosidases were heterologously expressed in Lactococcus lactis using a p170 derived promoter; experiments were performed with L. lactis crude cell extract (CCE). The novel GH42 β-galactosidase of Lb. acidophilus had lower activity on lactose, oNPG and pNPG but higher relative activity on pNP analogues compared to GH2 β-galactosidases, and did not transgalactosylate at high lactose concentrations. Temperature and pH optima for lactose hydrolysis varied between GH2 β-galactosidases. oNPG and pNPG were the preferred substrates for hydrolysis; in comparison, activity on pNPG-analogues was less than 1.5%. GH2 β-galactosidases formed structurally similar GOS with varying preferences.     

In silico analysis of expression pattern of a Wnt/β-catenin responsive gene ANLN in gastric cancer

Actin-binding protein anillin (ANLN) is primarily involved in the cytokinesis and known to be dysregulated in many cancers including gastric cancer (GC). However, the regulation and clinical significance of ANLN in GC are far less clear. In the present study, we aimed to investigate the clinical significance and possible regulators of ANLN in GC. We have identified the Wnt/β-catenin associated regulation of ANLN by analyzing the in vitro perturbed β-catenin mRNA expression profiles. Investigating the gastric tumors from publicly available genome-wide mRNA expression profiles, we have identified the over expression of ANLN in gastric tumors. Association between ANLN expression and clinical characteristics of GC showed elevated expression in intestinal type GC. Performing a single sample prediction method across GC mRNA expression profiles, we have identified the over expression of ANLN in proliferative type gastric tumors compared to the invasive and metabolic type gastric tumors. In silico pathway prediction analysis revealed the association between Wnt/β-catenin signaling and ANLN expression in gastric tumors. Our results highlight that expression of a Wnt/β-catenin responsive gene ANLN in GC is a molecular predictor of intestinal and proliferative type gastric tumors.     

A conserved π-helix plays a key role in thermoadaptation of catalysis in the glycoside hydrolase family 4

Here, we characterize the role of a π-helix in the molecular mechanisms underlying thermoadaptation in the glycoside hydrolase family 4 (GH4). The interspersed π-helix present in a subgroup is evolutionarily related to a conserved α-helix in other orthologs by a single residue insertion/deletion event. The insertional residue, Phe407, in a hyperthermophilic α-glucuronidase, makes specific interactions across the inter-subunit interface. In order to establish the sequence-structure-stability implications of the π-helix, the wild-type and the deletion variant (Δ407) were characterized. The variant showed a significant lowering of melting temperature and optimum temperature for the highest activity. Crystal structures of the proteins show a transformation of the π-helix to a continuous α-helix in the variant, identical to that in orthologs lacking this insertion. Thermodynamic parameters were determined from stability curves representing the temperature dependence of unfolding free energy. Though the proteins display maximum stabilities at similar temperatures, a higher melting temperature in the wild-type is achieved by a combination of higher enthalpy and lower heat capacity of unfolding. Comparisons of the structural changes, and the activity and thermodynamic profiles allow us to infer that specific non-covalent interactions, and the existence of residual structure in the unfolded state, are crucial determinants of its thermostability. These features permit the enzyme to balance the preservation of structure at a higher temperature with the thermodynamic stability required for optimum catalysis.     

Cloning, expression, and characterization of a glycoside hydrolase family 50 β-agarase from a marine Agarivorans isolate

The gene for a thermostable β-agarase from Agarivorans sp. JA-1 was cloned and sequenced. It comprised an open reading frame of 2,988 base pairs, which encode a protein of 109,450 daltons consisting of 995 amino acid residues. A comparison of the entire sequence showed that the enzyme has 98.8% sequence similarities to β-agarase from Vibrio sp. JT1070, indicating that it belongs to the family glycoside hydrolase (GH)-50. The gene corresponding to a mature protein of 976 amino acids was inserted and expressed in Escherichia coli. The recombinant β-agarase was purified to homogeneity. It had maximal activity at 40°C and pH 8.0 in the presence of 1 mM NaCl and 1 mM CaCl₂. The enzyme hydrolyzed agarose as well as neoagarohexaose and neoagarotetraose to yield neoagarobiose as the main product. Thus, the enzyme would be useful for the industrial production of neoagarobiose.     

Characterization of a new acid stable exo-β-1,3-glucanase of Rhizoctonia solani and its action on microbial polysaccharides

A new acid stable exo-β-1,3-glucanase of Rhizoctonia solani purified from a commercial source ‘Kitarase-M’, by a combination of ammonium sulfate precipitation, ion-exchange and gel filtration methods, had specific activity of 0.26 U/mg protein, K_m and V_max values of 0.78 mg/ml and 0.27 mM/min/mg protein, respectively. It had molecular weight of 62 kDa with optimum activity at 40 °C temperature and pH 5.0, with high stability at pH of 3–7. Unique amino acid sequence was found at N-terminal end. The substrate specificity studies confirmed that it is an exo-β-1,3-glucanase. It could hydrolyze curdlan powder to release glucose.     

Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) β-glucosidase from Aspergillus niger ASKU28

Background

The commercially important glycoside hydrolase family 3 (GH3) β-glucosidases from Aspergillus niger are anomeric-configuration-retaining enzymes that operate through the canonical double-displacement glycosidase mechanism. Whereas the catalytic nucleophile is readily identified across all GH3 members by sequence alignments, the acid/base catalyst in this family is phylogenetically variable and less readily divined.

Methods

In this report, we employed three-dimensional structure homology modeling and detailed kinetic analysis of site-directed mutants to identify the catalytic acid/base of a GH3 β-glucosidase from A. niger ASKU28.

Results

In comparison to the wild-type enzyme and other mutants, the E490A variant exhibited greatly reduced k_cat and k_cat/K_m values toward the natural substrate cellobiose (67,000- and 61,000-fold, respectively). Correspondingly smaller kinetic effects were observed for artificial chromogenic substrates p-nitrophenyl β-d-glucoside and 2,4-dinitrophenyl β-d-glucoside, the aglycone leaving groups of which are less dependent on acid catalysis, although changes in the rate-determining catalytic step were revealed for both. pH-rate profile analyses also implicated E490 as the general acid/base catalyst. Addition of azide as an exogenous nucleophile partially rescued the activity of the E490A variant with the aryl β-glucosides and yielded β-glucosyl azide as a product.

Conclusions and general significance

These results strongly support the assignment of E490 as the acid/base catalyst in a β-glucosidase from A. niger ASKU28, and provide crucial experimental support for the bioinformatic identification of the homologous residue in a range of related GH3 subfamily members.     

Characterization and expression of chitinase and 1,3-β-glucanase genes in cotton

We have isolated cDNA clones representing mRNAs encoding chitinase and 1,3--glucanase in cotton (Gossypium hirsutum L.) leaves. The chitinase clones were sequenced and found to encode a 28,806 Da protein with 71% amino acid sequence similarity to the SK2 chitinase from potato (Solanum tuberosum). The 1,3--glucanase clones encoded a 37,645 Da protein with 57.6% identity to a 1,3--glucanase from soybean (Glycine max). Northern blot analyses showed that chitinase mRNA is induced in plants treated with ethaphon or salicylic acid, whereas the levels of 1,3--glucanase mRNA are relatively unaffected. Southern blots of cotton genomic DNA and genomic clones indicated chitinase is encoded by a small gene family of which two members, Chi 2;1 and Chi 2;2, were characterized. These genes share 97% sequence identity in their transcribed regions. The genes were found to have three exons which are 309, 154 and 550 bp long, and two introns 99 and 154 bp in length. The 5-flanking regions of Chi 2;1 and Chi 2;2 exhibit a large degree of similarity and may contain sequences important for gene response to chemical agents and fungal attack.     

Structural and enzymatic characterization of a glycoside hydrolase family 31 α-xylosidase from Cellvibrio japonicus involved in xyloglucan saccharification

The desire for improved methods of biomass conversion into fuels and feedstocks has re-awakened interest in the enzymology of plant cell wall degradation. The complex polysaccharide xyloglucan is abundant in plant matter, where it may account for up to 20% of the total primary cell wall carbohydrates. Despite this, few studies have focused on xyloglucan saccharification, which requires a consortium of enzymes including endo-xyloglucanases, α-xylosidases, β-galactosidases and α-L-fucosidases, among others. In the present paper, we show the characterization of Xyl31A, a key α-xylosidase in xyloglucan utilization by the model Gram-negative soil saprophyte Cellvibrio japonicus. CjXyl31A exhibits high regiospecificity for the hydrolysis of XGOs (xylogluco-oligosaccharides), with a particular preference for longer substrates. Crystallographic structures of both the apo enzyme and the trapped covalent 5-fluoro-β-xylosyl-enzyme intermediate, together with docking studies with the XXXG heptasaccharide, revealed, for the first time in GH31 (glycoside hydrolase family 31), the importance of a PA14 domain insert in the recognition of longer oligosaccharides by extension of the active-site pocket. The observation that CjXyl31A was localized to the outer membrane provided support for a biological model of xyloglucan utilization by C. japonicus, in which XGOs generated by the action of a secreted endo-xyloglucanase are ultimately degraded in close proximity to the cell surface. Moreover, the present study diversifies the toolbox of glycosidases for the specific modification and saccharification of cell wall polymers for biotechnological applications.     

Cloning and expression of a thermostable exo-α-1,4-glucosidase gene from Bacillus stearothermophilus ATCC12016 in Escherichia coli

The gene coding for a thermostable exo--1,4-glucosidase (-glucoside glucohydrolase: EC 3.2.1.20) of Bacillus stearothermophilus ATCC 12016 was cloned within a 2.8-kb AvaI fragment of DNA using the plasmid pUC19 as a vector and Escherichia coli JM109 as a host. E. coli with the hybrid plasmid accumulated exo--1,4-glucosidase mainly in the cytoplasm. The level of enzyme production was about sevenfold higher than that observed for B. stearothermophilus. The cloned enzyme coincided absolutely with the B. stearothermophilus enzyme in its relative molecular mass (62 000), isoelectric point (5.0), amino-terminal sequence of 15 residues (Met-Lys-Lys-Thr-Trp-Trp-Lys-Glu-Gly-Val-Ala-Tyr-Gln-Ile-Tyr-), the temperature dependency of its activity and stability, and its antigenic determinants.Correspondence to: Y. Suzuki     

A (1→6)-β-glucan from a somatic hybrid of Pleurotus florida and Volvariella volvacea: isolation, characterization, and study of immunoenhancing properties

A water-soluble immunoenhancing polysaccharide was isolated from the aqueous extract of fruit bodies of somatic hybrid (Pflo Vv5 FB), obtained through protoplast fusion between Pleurotus florida and Volvariella volvacea strains. On the basis of acid hydrolysis, the polysaccharide was found to contain glucose only. Methylation analysis, periodate oxidation along with ¹H, DEPT-135, and ¹³C NMR spectroscopy, including two-dimensional TOCSY, DQF-COSY, NOESY, ROESY, 1H,13C-HMQC, and HMBC experiments showed that the polysaccharide was a (1→6)-β-d-glucan, which was not a constituent of any of the parent mushrooms previously reported.This glucan stimulated the macrophages, splenocytes, and thymocytes.     

Cloning and expression analysis of an endo-1,3-β-d-glucosidase from Phytophthora cinnamomi

Molecular Biology Reports - Phytophthora is considered one of the most destructive genus for many agricultural plant species worldwide, with a strong environmental and economic impact. Phytophthora...     

Cloning and expression of a β -1,3-glucanase gene from Bacillus circulans KCTC3004 in Escherichia coli

Summary A new gene encoding the -1,3-glucanase(laminarinase) of Bacillus circulans KCTC3004 was cloned into Escherichia coli using pUC19 as a vector. The gene localized in the 5.3 kb PstI DNA fragment was expressed independently of its orientation in the cloning vector showing enzyme activity about 33 times greater than that produced by the original B. circulans. The optimum pH and temperature of the cloned enzyme were pH 5.4 and 50°C, respectively. The molecular weight of the enzyme was about 38,000 and the processing of the enzyme molecule within the E. coli cell was not observed. The enzyme hydrolyzed laminarin to produce laminaritriose, laminaribiose, and glucose as main products, but it was inactive for lichenan, CMC, or xylan.     

Genome-wide classification and expression analysis of nucleobase–ascorbate transporter (NAT) gene family in tomato

Ascorbic acid or vitamin C is a wide spectrum antioxidant and plays a crucial role in a many metal-containing enzymes essential for humans, which are unable to synthesize the vitamin C and must obtain it from dietary sources. Ascorbic acid is transported by sodium-coupled ascorbic acid transporters or SVCTs in humans. However, little information is available about the nucleobase–ascorbate transporters (NATs) in tomato (Solanum lycopersicum). In the current study, we identified 12 NAT genes by screening SGN genome databases in tomato. A complete overview of this gene family in tomato is presented, including gene structures, chromosome distribution and localization, phylogenies, motif analysis and expression profiles. The SlNAT genes contained 14 exons, mostly, and dispersed on all the chromosomes except chromosome 8 and 9. All the SlNATs were located to plasma membrane, chloroplast thylakoid membrane, Golgi body, and endoplasmic reticulum (membrane). The phylogenetic tree showed that the plant NATs were divided into 4 clades, well-supported by the distribution of conserved motifs, and the SlNAT proteins shared higher similarity and clustered more closely with AtNAT proteins. Furthermore, the expression profiles of SlNAT genes in various organs showed 9 out of 12 SlNAT genes were constituently expression with differential expression levels under normal growth conditions. Our systematic analysis will provide a useful platform for molecular clone and functional identification of NAT genes in tomato and probably other Solanaceae plants.     

Gene cloning and expression of a new acidic family 7 endo-β-1,3-1,4-glucanase from the acidophilic fungus Bispora sp. MEY-1

Most reported microbial β-1,3-1,4-glucanases belong to the glycoside hydrolase family 16. Here, we report a new acidic family 7 endo-β-1,3-1,4-glucanase (Bgl7A) from the acidophilic fungus Bispora sp. MEY-1. The cDNA of Bgl7A was isolated and over-expressed in Pichia pastoris, with a yield of about 1,000 U ml^–1 in a 3.7-l fermentor. The purified recombinant Bgl7A had three activity peaks at pH 1.5, 3.5, and 5.0 (maximum), respectively, and a temperature optimum at 60°C. The enzyme was stable at pH 1.0–8.0 and highly resistant to both pepsin and trypsin. Belonging to the group of non-specific endoglucanase, Bgl7A can hydrolyze not only β-glucan and cellulose but also laminarin and oat spelt xylan. The specific activity of Bgl7A against barley β-glucan and lichenan (4,040 and 2,740 U mg^–1) was higher than toward carboxymethyl cellulose sodium (395 U mg^–1), which was different from other family 7 endo-β-glucanases.     

Cloning and characterization of a novel cellobiase gene, cba3, encoding the first known β-glucosidase of glycoside hydrolase family 1 of Cellulomonas biazotea

A novel cellobiase gene, designated cba3, was cloned from Cellulomonas biazotea. Although cellobiase genes of C. biazotea were previously cloned, published and/or patented, they encoded β-glucosidases all belonging to glycoside hydrolase family 3 (GH3); the new Cba3 cellobiase was identified to be a glycoside hydrolase family 1 (GH1) member, which represents the first discovered GH1 β-glucosidase of C. biazotea. Escherichia coli transformants expressing recombinant Cba3 were shown to grow readily in minimal media using cellobiose as the sole carbon source, supporting the conclusion that Cba3 is a genuine cellobiase. The full-length cba3 gene was revealed by sequencing to be 1344 bp long. Cba3 deletants lacking either the N-terminal 10 amino acids or the C-terminal 10 residues were found to be biologically inactive, supporting the importance of both ends in catalysis. Like other GH1 β-glucosidases, Cba3 was shown to contain the highly conserved NEP and ENG motifs, which are crucial for enzymatic activity. Despite lacking a classical N-terminal signal peptide, Cba3 was demonstrated to be a secretory protein. The findings that Cba3 is a cellobiase, and that it was expressed well as an extracellular protein in E. coli, support the potential of Cba3 for use with other cellulases in the hydrolysis of cellulosic biomass.