Biocatalytic potential of Pseudolycoriella CAZymes (Sciaroidea, Diptera) in degrading plant and fungal cell wall polysaccharides.

Soil biota has a crucial impact on soil ecology, global climate changes, and effective crop management and studying the diverse ecological roles of dipteran larvae deepens the understanding of soil food webs. A multi-omics study of Pseudolycoriella hygida comb. nov. (Diptera: Sciaroidea: Sciaridae) aimed to characterize carbohydrate-active enzymes (CAZymes) for litter degradation in this species. Manual curation of 17,881 predicted proteins in the Psl. hygida genome identified 137 secreted CAZymes, of which 33 are present in the saliva proteome, and broadly confirmed by saliva CAZyme catalytic profiling against plant cell wall polysaccharides and pNP-glycosyl substrates. Comparisons with two other sciarid species and the outgroup Lucilia cuprina (Diptera: Calliphoridae) identified 42 CAZyme families defining a sciarid CAZyme profile. The litter-degrading potential of sciarids corroborates their significant role as decomposers, yields insights to the evolution of insect feeding habits, and highlights the importance of insects as a source of biotechnologically relevant enzymes.

Selective xyloglucan oligosaccharide hydrolysis by a GH31 α-xylosidase from Escherichia coli.

Xyloglucan is ubiquitous in the cell walls of land plants and is also an essential storage polymer in seeds of many species. We studied the hydrolysis of the non-reducing end xylosyl residue of xyloglucan oligosaccharides (XGOs) by the Escherichia coli α-xylosidase (YicI). Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS) and ion fragmentation analysis together with high performance anion exchange chromatography with pulsed amperometric detection revealed that YicI preferentially removes the xylosyl residue from the glycosyl residue of non-galactosylated oligosaccharides. The YicI shows decreasing activity against the galactosylated oligosaccharides XXXG>XXLG≥XLXG. Studies of the XGOs interaction with active site residues by molecular dynamics simulations suggested that hydrogen bond interactions between the D49 and galactosylated oligosaccharides play an important role in enzyme-XGO interactions. This was confirmed by site-directed mutagenesis, where the D49A mutant affected catalytic efficiency against galactosylated XGOs. Our findings advance xyloglucan disassembly models and highlight the importance of YicI for biotechnology applications.

Covalent Immobilization of Chondrostereum purpureum Endopolygalacturonase on Ferromagnetic Nanoparticles: Catalytic Properties and Biotechnological Application.

Pectinases are widely used in a variety of industrial processes. However, their application is limited by low catalytic processivity, reduced stability, high cost, and poor re-use compatibility. These drawbacks may be overcome by enzyme immobilization with ferromagnetic nanoparticles, which are easily recovered by a magnetic field. In this work, an endopolygalacturonase from Chondrostereum purpureum (EndoPGCp) expressed in Pichia pastoris was immobilized on glutaraldehyde-activated chitosan ferromagnetic nanoparticles (EndoPGCp-MNP) and used to supplement a commercial enzyme cocktail. No significant differences in biochemical and kinetic properties were observed between EndoPGCp-MNP and EndoPGCp, although the EndoPGCp-MNP showed slightly increased thermostability. Cocktail supplementation with EndoPGCp-MNP increased reducing sugar release from orange wastes by 1.8-fold and showed a synergistic effect as compared to the free enzyme. Furthermore, EndoPGCp-MNP retained 65% of the initial activity after 7 cycles of re-use. These properties suggest that EndoPGCp-MNP may find applications in the processing of pectin-rich agroindustrial residues.

Enhanced hydrolytic efficiency of an engineered CBM11-glucanase enzyme chimera against barley ß-d-glucan extracts.

The ß-d-glucans are abundant cell wall polysaccharides in many cereals and contain both (1,3)- and (1,4)-bonds. The ß-1,3-1,4-glucanases (EC 3.2.1.73) hydrolyze ß-(1,4)-d-glucosidic linkages in glucans, and have applications in both animal and human food industries. A chimera between the family 11 carbohydrate-binding module from Ruminoclostridium (Clostridium)thermocellumcelH (RtCBM11), with the ß-1,3-1,4-glucanase from Bacillus subtilis (BglS) was constructed by end-to-end fusion (RtCBM11-BglS) to evaluate the effects on the catalytic function and its application in barley ß-glucan degradation for the brewing industry. The parental and chimeric BglS presented the same optimum pH (6.0) and temperature (50 °C) for maximum activity. The RtCBM11-BglS showed increased thermal stability and 30% higher hydrolytic efficiency against purified barley ß-glucan, and the rate of hydrolysis of ß-1,3-1,4-glucan in crude barley extracts was significantly increased. The enhanced catalytic performance of the RtCBM11-BglS may be useful for the treatment of crude barley extracts in the brewing industry.

Biochemical and kinetic characterization of the recombinant GH28 Stereum purpureum endopolygalacturonase and its biotechnological application.

Endopolygalacturonase (EndoPG) from Stereum purpureum was expressed as a soluble protein in Pichia pastoris GS115, where after 3 days methanol induction the enzyme activity in the culture supernatant was 40 U mL-1. After purification by IMAC, SDS-PAGE analysis showed that the molecular weight of EndoPG was approximately 60.0 kDa. The carbohydrate content of the recombinant enzyme was estimated to be 67.0% (w/w). The optimum temperature and pH of catalysis were 60-70 °C and pH of 4.5, respectively. The enzyme was highly stable over the pH range 6.0-8.0 and retained approximately 60% of its initial activity after incubation at 70 °C for 30 min. The enzyme showed a specific activity of 5040.0 ±â€¯217 U mg-1 and hydrolyzed citrus pectin with Vmax and a KM of 4947.10 ±â€¯393.63 U mg-1 and 2.45 ±â€¯0.23 mg mL-1, respectively, and showed a catalytic efficiency of 2052.90 ±â€¯193.54 mL mg-1 s-1. EndoPG alone reduced the viscosity of papaya juice by 20% after 30 min, and increased its transmittance about 50% with a concomitant reduction of the color by about 55% after 5 h of enzymatic treatment. For apple juice, the relative reduction of viscosity was 30% after 5 h, and the reduction of the color was 30% with a 12% increase in transmittance. Supplementation of a commercial enzymatic cocktail for lignocellulose saccharification with EndoPG increased total reducing sugar release by 8.6 ±â€¯2.1% against sugar cane bagasse, indicating improved access of the cellulolytic enzymes to the biomass polysaccharides.

Immobilization of a ß-glucosidase and an endoglucanase in ferromagnetic nanoparticles: A study of synergistic effects.

Nanoparticles can act as support materials for enzymatic immobilization, introducing a balance of characteristics that modulate the efficiency of biocatalysts, such as specific surface area, resistance to mass transfer and effective enzymatic loading. Magnetic nanoparticles can be easily separated using an external magnetic field, and in this work two recombinant enzymes, the ß-glucosidase from Humicola insolens (Bglhi) and the endoglucanase from Scytalidium thermophilum (Egst) were immobilized on synthetized Fe3O4 nanoparticles derivatized with chitosan/glutaraldehyde/N-(5-amino-1-carboxy-pentyl) iminodiacetic acid and functionalized with NiCl2. The immobilization yields were about 20% for Bglhi and Egst with efficiencies of 132% and 115%, respectively. The two enzymes were also co-immobilized with yield was about 49%. The optimal temperatures of the immobilized enzymes were 70 °C and 55 °C for Egst and Bglhi, respectively. Egst hydrolyzed CMC in the presence of 4 mM MnCl2 with Vmax = 625.0 ±â€¯6.7 U mg-1 and KM = 6.4 ±â€¯0.5 mg mL-1 resulting in a catalytic efficiency (kcat/KM) of 107.4 ±â€¯5.4 mg-1 s-1 mL. Bglhi hydrolyzed pNP-Glc with Vmax = 52.7 ±â€¯2.7 U mg-1 and KM = 0.23 ±â€¯0.01 mM resulting in a catalytic efficiency (kcat/KM) of 214.3 ±â€¯10.2 s-1 mM-1. The individually immobilized enzymes when combined showed a synergistic effect on the substrates tested and a very similar action when compared to the co-immobilized enzymes, suggesting excellent potential for application in biotechnological processes.

Glucose tolerant and glucose stimulated ß-glucosidases - A review.

The ß-glucosidases (ß-D-glucoside glucohydrolase, EC 3.2.1.21) hydrolyze glycosidic bonds of alkyl-, amino-, or aryl-ß-D-glucosides, cyanogenic glucosides, disaccharides and short oligosaccharides and can also catalyze the synthesis of glycosyl-bonds between different molecules via transglycosylation. Due to their ubiquitous phylogenetic distribution, substrate diversity and ability to both hydrolyze and synthesize glycosidic bonds, the catalysis and regulation of ß-glucosidases have been extensively studied. Many ß-glucosidases are inhibited by the reaction product glucose, and reduced catalytic activity may limit the biotechnological and industrial applications of these enzymes and this has stimulated the search for ß-glucosidases that maintain their activity at high glucose concentrations. Studies of many glucose tolerant enzymes have been reported and due to the ongoing interest in these enzymes, here it has been reviewed this accumulated body of knowledge which provides valuable insights as to the kinetics, structure, regulation and evolution of glucose tolerant and glucose stimulated ß-glucosidases.

A halotolerant bifunctional ß-xylosidase/α-l-arabinofuranosidase from Colletotrichum graminicola: Purification and biochemical characterization.

A ß-xylosidase from Colletotrichum graminicola (Bxcg) was purified. The enzyme showed high halotolerance, retaining about 63% of the control activity in the presence of 2.5molL-1 NaCl. The presence of NaCl has not affected the optimum reaction temperature (65°C), but the optimum pH was slightly altered (from 4.5 to 5.0) at high salt concentrations. Bxcg was fully stable at 50°C for 24h and over a wide pH range even in the presence of NaCl. In the absence of salt Bxcg hydrolyzed p-nitrophenyl-ß-d-xylopyranoside with maximum velocity of 348.8±11.5Umg-1 and high catalytic efficiency (1432.7±47.3Lmmol-1s-1). Bxcg revealed to be a bifunctional enzyme with both ß-xylosidase and α-l-arabinofuranosidase activities, and hydrolyzed xylooligosaccharides containing up to six pentose residues. The enzyme showed high synergistic effect (3.1-fold) with an endo-xylanase for the hydrolysis of beechwood xylan, either in the absence or presence of 0.5molL-1 NaCl, and was tolerant to different organic solvents and surfactants. This is the first report of a halotolerant bifunctional ß-xylosidase/α-l-arabinofuranosidase from C. graminicola, and the enzyme showed attractive properties for application in lignocellulose hydrolysis, particularly under high salinity and/or in the presence of residues of pretreatment steps.

Overexpression of a Cellobiose-Glucose-Halotolerant Endoglucanase from Scytalidium thermophilum.

Enzyme reaction products and by-products from pretreatment steps can inhibit endoglucanases and are major factors limiting the efficiency of enzymatic lignocellulosic biomass hydrolysis. The gene encoding the endoglucanase from Scytalidium thermophilum (egst) was cloned and expressed as a soluble protein in Pichia pastoris GS115. The recombinant enzyme (Egst) was monomeric (66 kDa) and showed an estimated carbohydrate content of 53.3% (w/w). The optimum temperature and pH of catalysis were 60-70 °C and pH of 5.5, respectively. The enzyme was highly stable at pH 3.0-8.0 with a half-life in water of 100 min at 65 °C. The Egst presented good halotolerance, retaining 84.1 and 71.4% of the control activity in the presence of 0.5 and 2.0 mol L-1 NaCl, respectively. Hydrolysis of medium viscosity carboxymethylcellulose (CMC) by Egst was stimulated 1.77-, 1.84-, 1.64-, and 1.8-fold by dithiothreitol, ß-mercaptoethanol, cysteine, and manganese at 10, 10, 10, and 5 mmol L-1 concentration, respectively. The enzyme hydrolyzed CMC with maximal velocity and an apparent affinity constant of 432.10 ± 16.76 and 10.5 ± 2.53 mg mL-1, respectively. Furthermore, the Egst was tolerant to reaction products and able to act on pretreated fractions sugarcane bagasse demonstrating excellent properties for application in the hydrolysis of lignocellulosic biomass.

Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced ß-glucanase activity monitored by electron paramagnetic resonance.

BACKGROUND: The Bacillus subtilis endo-ß-1,4-glucanase (BsCel5A) hydrolyzes ß-1,3-1,4-linked glucan, and the enzyme includes a family 3 carbohydrate-binding module (CBM3) that binds ß-1,4-linked glucan. METHODS: Here we investigate the BsCel5A ß-1,3-1,4 glucanase activity after exchanging the CBM3 domain for the family 11 CBM from Ruminiclostridium thermocellum celH (RtCBM11) having ß-1,3-1,4 glucan affinity. RESULTS: The BsCel5A-RtCBM11 presents a 50.4% increase in Vmax, a 10% reduction in K0.5, and a 2.1-fold increase in catalytic efficiency. Enzyme mobility and binding to barley ß-1,3-1,4 glucan and pre-treated sugarcane bagasse were investigated using Electron Paramagnetic Resonance (EPR) with Site-Directed Spin Labeling (SDSL) of the binding site regions of the CBM3 and RtCBM11 domains in the BsCel5A-CBM3 and BsCel5A-RtCBM11, respectively. Although higher mobility than the RtCBM11 was shown, no interaction of the spin-labeled CBM3 with ß-1,3-1,4 glucan was observed. In contrast, a Ka value of 0.22 mg/mL was estimated from titration of the BsCel5A-RtCBM11 with ß-1,3-1,4 glucan. Enzyme binding as inferred from altered EPR spectra of the BsCel5A-RtCBM11 was observed only after xylan or lignin extraction from sugarcane bagasse. Binding to xylan- or lignin-free lignocellulose was correlated with a 4.5- to 5-fold increase in total reducing sugar release as compared to the milled intact sugarcane bagasse, suggesting that xylan impedes enzyme access to the ß-1,3-1,4 glucan. CONCLUSIONS: These results show that the non-specific binding of the BsCel5A-RtCBM11 to the lignin component of the cell wall is minimal, and represent the first reported use of EPR to directly study the interaction of glycoside hydrolyse enzymes with natural insoluble substrates.

Engineering the GH1 ß-glucosidase from Humicola insolens: Insights on the stimulation of activity by glucose and xylose.

The activity of the GH1 ß-glucosidase from Humicola insolens (Bglhi) against p-nitrophenyl-ß-D-glucopyranoside (pNP-Glc) and cellobiose is enhanced 2-fold by glucose and/or xylose. Kinetic and transglycosylation data showed that hydrolysis is preferred in the absence of monosaccharides. Stimulation involves allosteric interactions, increased transglycosylation and competition of the substrate and monosaccharides for the -1 glycone and the +1/+2 aglycone binding sites. Protein directed evolution has been used to generate 6 mutants of Bglhi with altered stimulation patterns. All mutants contain one of three substitutions (N235S, D237V or H307Y) clustered around the +1/+2 aglycone binding sites. Two mutants with the H307Y substitution preferentially followed the transglycosylation route in the absence of xylose or glucose. The strong stimulation of their pNP-glucosidase and cellobiase activities was accompanied by increased transglycosylation and higher monosaccharide tolerance. The D237V mutation favoured hydrolysis over transglycosylation and the pNP-glucosidase activity, but not the cellobiase activity, was stimulated by xylose. The substitution N235S abolished the preference for hydrolysis or transglycosylation; the cellobiase, but not the pNP-glucosidase activity of the mutants was strongly inhibited by xylose. Both the D237V and N235S mutations lowered tolerance to the monosaccharides. These results provide evidence that the fine modulation of the activity of Bglhi and mutants by glucose and/or xylose is regulated by the relative affinities of the glycone and aglycone binding sites for the substrate and the free monosaccharides.

A High Redox Potential Laccase from Pycnoporus sanguineus RP15: Potential Application for Dye Decolorization.

Laccase production by Pycnoporus sanguineus RP15 grown in wheat bran and corncob under solid-state fermentation was optimized by response surface methodology using a Central Composite Rotational Design. A laccase (Lacps1) was purified and characterized and the potential of the pure Lacps1 and the crude culture extract for synthetic dye decolorization was evaluated. At optimal conditions (eight days, 26 °C, 18% (w/w) milled corncob, 0.8% (w/w) NH4Cl and 50 mmol·L(-1) CuSO4, initial moisture 4.1 mL·g(-1)), the laccase activity reached 138.6 ± 13.2 U·g(-1). Lacps1 was a monomeric glycoprotein (67 kDa, 24% carbohydrate). Optimum pH and temperature for the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) were 4.4 and 74.4 °C, respectively. Lacps1 was stable at pH 3.0-8.0, and after two hours at 55-60 °C, presenting high redox potential (0.747 V vs. NHE). ABTS was oxidized with an apparent affinity constant of 147.0 ± 6.4 µmol·L(-1), maximum velocity of 413.4 ± 21.2 U·mg(-1) and catalytic efficiency of 3140.1 ± 149.6 L·mmol(-1)·s(-1). The maximum decolorization percentages of bromophenol blue (BPB), remazol brilliant blue R and reactive blue 4 (RB4), at 25 or 40 °C without redox mediators, reached 90%, 80% and 60%, respectively, using either pure Lacps1 or the crude extract. This is the first study of the decolorization of BPB and RB4 by a P. sanguineus laccase. The data suggested good potential for treatment of industrial dye-containing effluents.