Structural and Functional Characterization of a Gene Cluster Responsible for Deglycosylation of C-glucosyl Flavonoids and Xanthonoids by Deinococcus aerius.

Plant C-glycosylated aromatic polyketides are important for plant and animal health. These are specialized metabolites that perform functions both within the plant, and in interaction with soil or intestinal microbes. Despite the importance of these plant compounds, there is still limited knowledge of how they are metabolized. The Gram-positive aerobic soil bacterium Deinococcus aerius strain TR0125 and other Deinococcus species thrive in a wide range of harsh environments. In this work, we identified a C-glycoside deglycosylation gene cluster in the genome of D. aerius. The cluster includes three genes coding for a GMC-type oxidoreductase (DaCGO1) that oxidizes the glucosyl C3 position in aromatic C-glucosyl compounds, which in turn provides the substrate for the C-glycoside deglycosidase (DaCGD; composed of α+ß subunits) that cleaves the glucosyl-aglycone C-C bond. Our results from size-exclusion chromatography, single particle cryo-electron microscopy and X-ray crystallography show that DaCGD is an α2ß2 heterotetramer, which represents a novel oligomeric state among bacterial CGDs. Importantly, the high-resolution X-ray structure of DaCGD provides valuable insights into the activation of the catalytic hydroxide ion by Lys261. DaCGO1 is specific for the 6-C-glucosyl flavones isovitexin, isoorientin and the 2-C-glucosyl xanthonoid mangiferin, and the subsequent C-C-bond cleavage by DaCGD generated apigenin, luteolin and norathyriol, respectively. Of the substrates tested, isovitexin was the preferred substrate (DaCGO1, Km 0.047 mM, kcat 51 min-1; DaCGO1/DaCGD, Km 0.083 mM, kcat 0.42 min-1).

Crystal structure of the feruloyl esterase from Lentilactobacillus buchneri reveals a novel homodimeric state.

Ferulic acid is a common constituent of the plant cell-wall matrix where it decorates and can crosslink mainly arabinoxylans to provide structural reinforcement. Microbial feruloyl esterases (FAEs) specialize in catalyzing hydrolysis of the ester bonds between phenolic acids and sugar residues in plant cell-wall polysaccharides such as arabinoxylan to release cinnamoyl compounds. Feruloyl esterases from lactic acid bacteria (LAB) have been highlighted as interesting enzymes for their potential applications in the food and pharmaceutical industries; however, there are few studies on the activity and structure of FAEs of LAB origin. Here, we report the crystal structure and biochemical characterization of a feruloyl esterase (LbFAE) from Lentilactobacillus buchneri, a LAB strain that has been used as a silage additive. The LbFAE structure was determined in the absence and presence of product (FA) and reveals a new type of homodimer association not previously observed for fungal or bacterial FAEs. The two subunits associate to restrict access to the active site such that only single FA chains attached to arabinoxylan can be accommodated, an arrangement that excludes access to FA cross-links between arabinoxylan chains. This narrow specificity is further corroborated by the observation that no FA dimers are produced, only FA, when feruloylated arabinoxylan is used as substrate. Docking of arabinofuranosyl-ferulate in the LbFAE structure highlights the restricted active site and lends further support to our hypothesis that LbFAE is specific for single FA side chains in arabinoxylan.

Crystal structure of a homotrimeric verrucomicrobial exo-ß-1,4-mannosidase active in the hindgut of the wood-feeding termite Reticulitermes flavipes.

The termite Reticulitermes flavipes causes extensive damage due to the high efficiency and broad specificity of the ligno- and hemicellulolytic enzyme systems produced by its symbionts. Thus, the R. flavipes gut microbiome is expected to constitute an excellent source of enzymes that can be used for the degradation and valorization of plant biomass. The symbiont Opitutaceae bacterium strain TAV5 belongs to the phylum Verrucomicrobia and thrives in the hindgut of R. flavipes. The sequence of the gene with the locus tag opit5_10225 in the Opitutaceae bacterium strain TAV5 genome has been classified as a member of glycoside hydrolase family 5 (GH5), and provisionally annotated as an endo-ß-mannanase. We characterized biochemically and structurally the opit5_10225 gene product, and show that the enzyme, Op5Man5, is an exo-ß-1,4-mannosidase [EC 3.2.1.25] that is highly specific for ß-1,4-mannosidic bonds in mannooligosaccharides and ivory nut mannan. The structure of Op5Man5 was phased using electron cryo-microscopy and further determined and refined at 2.2 Šresolution using X-ray crystallography. Op5Man5 features a 200-kDa large homotrimer composed of three modular monomers. Despite insignificant sequence similarity, the structure of the monomer, and homotrimeric assembly are similar to that of the GH42-family ß-galactosidases and the GH164-family exo-ß-1,4-mannosidase Bs164 from Bacteroides salyersiae. To the best of our knowledge Op5Man5 is the first structure of a glycoside hydrolase from a bacterial symbiont isolated from the R. flavipes digestive tract, as well as the first example of a GH5 glycoside hydrolase with a GH42 ß-galactosidase-type homotrimeric structure.

A homodimeric bacterial exo-ß-1,3-glucanase derived from moose rumen microbiome shows a structural framework similar to yeast exo-ß-1,3-glucanases.

The impact of various ß-glucans on the gut microbiome and immune system of vertebrates is becoming increasingly recognized. Besides the fundamental interest in understanding how ß-glucans support human and animal health, enzymes that metabolize ß-glucans are of interest for hemicellulose bioprocessing. Our earlier metagenomic analysis of the moose rumen microbiome identified a gene coding for a bacterial enzyme with a possible role in ß-glucan metabolization. Here, we report that the enzyme, mrbExg5, has exo-ß-1,3-glucanase activity on ß-1,3-linked glucooligosaccharides and laminarin, but not on ß-1,6- or ß-1,4-glycosidic bonds. Longer oligosaccharides are good substrates, while shorter substrates are readily transglycosylated into longer products. The enzyme belongs to glycoside hydrolase subfamily GH5_44, which is a close phylogenetic neighbor of the subfamily GH5_9 exo-ß-1,3-glucanases of the yeasts Saccharomyces cerevisiae and Candida albicans. The crystal structure shows that unlike the eukaryotic relatives, mrbExg5 is a functional homodimer with a binding region characterized by: (i) subsite +1 can accommodate a branched sugar on the ß-1,3-glucan backbone; (ii) subsite +2 is restricted to exclude backbone substituents; and (iii) a fourth subsite (+3) formed by a unique loop. mrbExg5 is the first GH5_44 enzyme to be structurally characterized, and the first bacterial GH5 with exo-ß-1,3-glucanase activity.

A Transmembrane Crenarchaeal Mannosyltransferase Is Involved in N-Glycan Biosynthesis and Displays an Unexpected Minimal Cellulose-Synthase-like Fold.

Protein glycosylation constitutes a critical post-translational modification that supports a vast number of biological functions in living organisms across all domains of life. A seemingly boundless number of enzymes, glycosyltransferases, are involved in the biosynthesis of these protein-linked glycans. Few glycan-biosynthetic glycosyltransferases have been characterized in vitro, mainly due to the majority being integral membrane proteins and the paucity of relevant acceptor substrates. The crenarchaeote Pyrobaculum calidifontis belongs to the TACK superphylum of archaea (Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota) that has been proposed as an eukaryotic ancestor. In archaea, N-glycans are mainly found on cell envelope surface-layer proteins, archaeal flagellins and pili. Archaeal N-glycans are distinct from those of eukaryotes, but one noteworthy exception is the high-mannose N-glycan produced by P. calidifontis, which is similar in sugar composition to the eukaryotic counterpart. Here, we present the characterization and crystal structure of the first member of a crenarchaeal membrane glycosyltransferase, PcManGT. We show that the enzyme is a GDP-, dolichylphosphate-, and manganese-dependent mannosyltransferase. The membrane domain of PcManGT includes three transmembrane helices that topologically coincide with "half" of the six-transmembrane helix cellulose-binding tunnel in Rhodobacter spheroides cellulose synthase BcsA. Conceivably, this "half tunnel" would be suitable for binding the dolichylphosphate-linked acceptor substrate. The PcManGT gene (Pcal_0472) is located in a large gene cluster comprising 14 genes of which 6 genes code for glycosyltransferases, and we hypothesize that this cluster may constitute a crenarchaeal N-glycosylation (PNG) gene cluster.

Can laccases catalyze bond cleavage in lignin?

Modification of lignin is recognized as an important aspect of the successful refining of lignocellulosic biomass, and enzyme-assisted processing and upcycling of lignin is receiving significant attention in the literature. Laccases (EC 1.10.3.2) are taking the centerstage of this attention, since these enzymes may help degrading lignin, using oxygen as the oxidant. Laccases can catalyze polymerization of lignin, but the question is whether and how laccases can directly catalyze modification of lignin via catalytic bond cleavage. Via a thorough review of the available literature and detailed illustrations of the putative laccase catalyzed reactions, including the possible reactions of the reactive radical intermediates taking place after the initial oxidation of the phenol-hydroxyl groups, we show that i) Laccase activity is able to catalyze bond cleavage in low molecular weight phenolic lignin model compounds; ii) For laccases to catalyze inter-unit bond cleavage in lignin substrates, the presence of a mediator system is required. Clearly, the higher the redox potential of the laccase enzyme, the broader the range of substrates, including o- and p-diphenols, aminophenols, methoxy-substituted phenols, benzenethiols, polyphenols, and polyamines, which may be oxidized. In addition, the currently available analytical methods that can be used to detect enzyme catalyzed changes in lignin are summarized, and an improved nomenclature for unequivocal interpretation of the action of laccases on lignin is proposed.

Important nutritional constituents, flavour components, antioxidant and antibacterial properties of Pleurotus sajor-caju.

Oyster mushroom (Pleurotus sajor-caju) cultivated in the laboratory was studied for nutritional constituents, flavor components, antioxidant and antibacterial properties. Nutritional constituents estimated per 100 g dry weight (d.w.) include protein (29.3 g), carbohydrate (62.97 g), crude fat (0.91 g), ash (6.82 g) and crude fiber (12.3 g). Energy value of this mushroom was about 297.5 kcal/100 g d.w. Major mineral components estimated include Ca, Fe, and Mg with highest level of 505.0, 109.5 and 108.7 mg/100 g respectively. Methanolic extract containing significant amounts of phenols and flavonoids showed free radical scavenging potential and antibacterial activities against various spp. of Gram positive and Gram negative bacteria. Compounds responsible for antibacterial activities analyzed by GC-MS include ß- Sistosterol, Cholestanol, 1,5-Dibenzoylnaphthalene and 1,2-Benzenedicarboxylic acid. Flavor components extracted by hot extraction method were found to be higher in number and concentration than the cold extraction method. The characteristic flavor component of mushroom i.e. 1-Octen-3-ol was better extracted by hot than the cold.

Decolorization of dyehouse effluent and biodegradation of Congo red by Bacillus thuringiensis RUN1.

A dye-decolorizing bacterium was isolated from a soil sample and identified as Bacillus thuringiensis using 16S rRNA sequencing. The bacterium was able to decolorize three different textile dyes, namely, Reactive blue 13, Reactive red 58, and Reactive yellow 42, and a real dyehouse effluent up to 80-95% within 6 h. Some non-textile industrially important dyes were also decolorized to different extents. Fourier transform infrared spectroscopy and gas chromatography-mass spectrometer analysis of the ethyl acetate extract of Congo red dye and its metabolites showed that the bacterium could degrade it by the asymmetric cleavage of the azo bonds to yield sodium (4- amino-3-diazenylnaphthalene-1-sulfonate) and phenylbenzene. Sodium (4-amino-3-diazenylnaphthalene-1-sulfonate) was further oxidized by the ortho-cleavage pathway to yield 2- (1-amino-2-diazenyl-2-formylvinyl) benzoic acid. There was induction of the activities of laccase and azoreductase during the decolorization of Congo red, which suggests their probable role in the biodegradation. B. thuringiensis was found to be versatile and could be used for industrial effluent biodegradation.

Biodecolorization of azo dye Remazol orange by Pseudomonas aeruginosa BCH and toxicity (oxidative stress) reduction in Allium cepa root cells.

In this report a textile azo dye Remazol orange was degraded and detoxified by bacterium Pseudomonas aeruginosa BCH in plain distilled water. This bacterial decolorization performance was found to be pH and temperature dependent with maximum decolorization observed at pH 8 and temperature 30 °C. Bacterium tolerated higher dye concentrations up to 400 mg l(-1). Effect of initial cell mass showed that higher cell mass concentration can accelerate decolorization process with maximum of 92 % decolorization observed at 2.5 g l(-1) cell mass within 6.5 h. Effect of various metal ions showed Mn has inducing effect whereas Zn strongly inhibited the decolorization process at 5 mM concentration. Analysis of biodegradation products carried out with UV-vis spectroscopy, HPTLC and FTIR confirmed the decolorization and degradation of Remazol orange. Possible route for the degradation of dye was proposed based on GC-MS analysis. During toxicological scrutiny in Allium cepa root cells, induction in the activities of superoxide dismutase (SOD), guaiacol peroxidase (GPX) and inhibition of catalase (CAT) along with raised levels of lipid peroxidation and protein oxidation in dye treated samples were detected which conclusively indicated the generation of oxidative stress. Less toxic nature of the dye degraded products was observed after bacterial treatment.

Ecofriendly degradation, decolorization and detoxification of textile effluent by a developed bacterial consortium.

Present study illustrates the effectual decolorization and degradation of the textile effluent using a developed bacterial consortium SDS, consisted of bacterial species Providencia sp. SDS and Pseudomonas aeuroginosa strain BCH, originally isolated from dye contaminated soil. The intensive metabolic activity of the consortium SDS led to complete decolorization of textile effluent within 20 h at pH 7 and temperature 30°C. Significant induction in the activities of veratryl alcohol oxidase, laccase, azoreductase and DCIP reductase were observed during decolorization, which indicates their involvement in decolorization and degradation process. The decolorization and biodegradation was monitored using UV-vis spectroscopy, IR spectroscopy, HPLC and HPTLC analysis. Toxicological analysis of effluent before and after treatment was performed using classical Allium cepa test. Investigations of various toxicological parameters viz, oxidative stress response, cytotoxicity, genotoxicity and phytotoxicity, collectively concludes that, the toxicity of effluent reduces significantly after treatment with consortium SDS.

Biochemical characteristics of a textile dye degrading extracellular laccase from a Bacillus sp. ADR.

Bacillus sp. ADR secretes an extracellular laccase in nutrient broth, and this enzyme was purified up to 56-fold using acetone precipitation and DEAE-cellulose anion exchange chromatography. The molecular weight of purified laccase was estimated to be 66 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified laccase oxidized 2,6-dimethoxy phenol, o-tolidine, hydroquinone, L-DOPA and guaiacol. The optimum pH for oxidation of o-tolidine, 2,6-dimethoxy phenol and guaiacol were 3.0, 4.0 and 5.0, respectively. The purified laccase contained 2.7 mol/mol of copper. The laccase was stable up to 40 °C and within the pH range of 7.0-9.0. Well-known inhibitors of multicopper oxidases such as, sodium azide, L-cysteine and dithiothreitol showed significant inhibition of laccase activity. The purified enzyme decolorized structurally different azo dyes with variable decolorization rates and efficiencies of 68-90%. This study is useful for understanding the precise use of Bacillus sp. ADR in the decolorization of textile dyes containing industrial wastewater.

Textile dye degradation by bacterial consortium and subsequent toxicological analysis of dye and dye metabolites using cytotoxicity, genotoxicity and oxidative stress studies.

The present study aims to evaluate Red HE3B degrading potential of developed microbial consortium SDS using two bacterial cultures viz. Providencia sp. SDS (PS) and Pseudomonas aeuroginosa strain BCH (PA) originally isolated from dye contaminated soil. Consortium was found to be much faster for decolorization and degradation of Red HE3B compared to the individual bacterial strain. The intensive metabolic activity of these strains led to 100% decolorization of Red HE3B (50 mg l(-1)) with in 1h. Significant induction of various dye decolorizing enzymes viz. veratryl alcohol oxidase, laccase, azoreductase and DCIP reductase compared to control, point out towards their involvement in overall decolorization and degradation process. Analytical studies like HPLC, FTIR and GC-MS were used to scrutinize the biodegradation process. Toxicological studies before and after microbial treatment was studied with respect to cytotoxicity, genotoxicity, oxidative stress, antioxidant enzyme status, protein oxidation and lipid peroxidation analysis using root cells of Allium cepa. Toxicity analysis with A. cepa signifies that dye Red HE3B exerts oxidative stress and subsequently toxic effect on the root cells where as biodegradation metabolites of the dye are relatively less toxic in nature. Phytotoxicity studies also indicated that microbial treatment favors detoxification of Red HE3B.

Influence of organic and inorganic compounds on oxidoreductive decolorization of sulfonated azo dye C.I. Reactive Orange 16.

An isolated bacterial strain is placed in the branch of the Bacillus genus on the basis of 16S rRNA sequence and biochemical characteristics. It decolorized an individual and mixture of dyes, including reactive, disperse and direct. Bacillus sp. ADR showed 88% decolorization of sulfonated azo dye C.I. Reactive Orange 16 (100 mg L(-1)) with 2.62 mg of dye decolorized g(-1) dry cells h(-1) as specific decolorization rate along with 50% reduction in COD under static condition. The optimum pH and temperature for the decolorization was 7-8 and 30-40 degrees C, respectively. It was found to tolerate the sulfonated azo dye concentration up to 1.0 g L(-1). Significant induction in the activity of an extracellular phenol oxidase and NADH-DCIP reductase enzymes during decolorization of C.I. Reactive Orange 16 suggest their involvement in the decolorization. The metal salt (CaCl2), stabilizers (3,4-dimethoxy benzyl alcohol and o-tolidine) and electron donors (sodium acetate, sodium formate, sodium succinate, sodium citrate and sodium pyruvate) enhanced the C.I. Reactive Orange 16 decolorization rate of Bacillus sp. ADR. The 6-nitroso naphthol and dihydroperoxy benzene were final products obtained after decolorization of C.I. Reactive Orange 16 as characterized using FTIR and GC-MS.

Coordinate action of exiguobacterial oxidoreductive enzymes in biodegradation of reactive yellow 84A dye.

A novel bacterial species identified as Exiguobacterium sp. RD3 degraded the diazo dye reactive yellow 84A (50 mg l(-1)) within 48 h at static condition, at 30 degrees C and pH 7. Lower salinity conditions were found to be favorable for growth and decolorization. Enzymatic activities of an H(2)O(2) independent oxidase along with laccase and an azoreductase suggest their prominent role during the decolorization of reactive yellow 84A. Presence of an H(2)O(2) independent oxidase in Exiguobacterium sp. RD3 was confirmed and hydrogen peroxide produced was detected by a coupled iodometric assay. Azoreductase activity was prominent in presence of cofactors NADH and NADP in mineral salt medium. Considerable depletion of COD of the dye solution during degradation of dye was indicative of conversion of complex dye into simple oxidizable products. Products of degradation were analyzed by HPLC, FTIR and GCMS. A possible product of the degradation was identified by GCMS. Degradation of dye resulted with significant reduction of phytotoxicity, confirming the environmentally safe nature of the degradation metabolites.