Soluble Β-glucosidase Research Articles

Polyester fabric modification by chemical treatment to enhancing the β-glucosidase immobilization

This work reports the first approach to immobilizing the β-glucosidase enzyme on a modified polyester fabric support matrix. Herein, polyester fabric was successfully fabricated with hydrazide groups incorporated with graphene oxide, followed by glyoxal as the crosslinker. Various techniques, including Fourier transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, differential scanning calorimeter, and zeta potential analysis, were used to investigate their microstructural, dispersive, thermal, and physicochemical properties. β-glucosidase immobilization exhibited maximum activity at pH 6.0 with an immobilization yield (89.5%), immobilization efficiency (92%), and enzyme activity yields (82.3%). After fifteen reaction cycles, the remaining enzyme activity was 59%. Stored at 4 °C, immobilized β-glucosidase retained 74% of its activity, compared to a retain of 43% for soluble β-glucosidase, during the 6-weeks period. Soluble and immobilized enzyme exhibited similar optimal catalytic temperature at 60 °C, while the optimal catalytic pH was 5 and 6, respectively. Both soluble and immobilized β-glucosidase presented Michaelis–Menten kinetics with Vmax values of 1.82 and 2.94 U/mg, and Km values of 2.94 and 5.15 mM, respectively. This research provided a potential directed immobilization method for β-glucosidase, and robust biocatalyst for industrial applications.

Xyloglucan exoglycosidases in the monocot model Brachypodium distachyon and the conservation of xyloglucan disassembly in angiosperms.

Brachypodium distachyon has a full set of exoglycosidases active on xyloglucan, including α-xylosidase, β-galactosidase, soluble and membrane-bound β-glucosidases and two α-fucosidases. However, unlike in Arabidopsis, both fucosidases are likely cytosolic. Xyloglucan is present in primary walls of all angiosperms. While in most groups it regulates cell wall extension, in Poaceae its role is still unclear. Five exoglycosidases participate in xyloglucan hydrolysis in Arabidopsis: α-xylosidase, β-galactosidase, α-fucosidase, soluble β-glucosidase and GPI-anchored β-glucosidase. Mutants in the corresponding genes show alterations in xyloglucan composition. In this work putative orthologs in the model grass Brachypodium distachyon were tested for their ability to complement Arabidopsis mutants. Xylosidase and galactosidase mutants were complemented, respectively, by BdXYL1 (Bd2g02070) and BdBGAL1 (Bd2g56607). BdBGAL1, unlike other xyloglucan β-galactosidases, is able to remove both galactoses from XLLG oligosaccharides. In addition, soluble β-glucosidase BdBGLC1 (Bd1g08550) complemented a glucosidase mutant. Closely related BdBGLC2 (Bd2g51280), which has a putative GPI-anchor sequence, was found associated with the plasma membrane and only a truncated version without GPI-anchor complemented the mutant, proving that Brachypodium also has soluble and membrane-bound xyloglucan glucosidases. Both BdXFUC1 (Bd3g25226) and BdXFUC2 (Bd1g28366) can hydrolyze fucose from xyloglucan oligosaccharides but were unable to complement a fucosidase mutant. Fluorescent protein fusions of BdXFUC1 localized to the cytosol and both proteins lack a signal peptide. Signal peptides appear to have evolved only in some eudicot lineages of this family, like the one leading to Arabidopsis. These results could be explained if cytosolic xyloglucan α-fucosidases are the ancestral state in angiosperms, with fucosylated oligosaccharides transported across the plasma membrane.

Immobilization and high stability of an extracellular β-glucosidase from Aspergillus japonicus by ionic interactions

Abstract A β-glucosidase from Aspergillus japonicus was produced on submerged fermentation using sugar cane bagasse in nature as carbon source, and incubated at 30 °C, for 72 h. After that, the dialyzed crude extract containing the active β-glucosidase was purified from three successive steps using DEAE-fractogel, MANAE-agarose and octyl-sepharose chromatographic columns. The enzyme migrated as a single band on SDS-PAGE and its molecular weight was estimated to be 114 kDa. The amino acid sequence determined by mass spectrometry demonstrated a similar structure for the β-glucosidase from Aspergillus niger and A. kawachii. The purified enzyme was immobilized by entrapment with sodium alginate beads; covalent attachment using activated CNBr-agarose and ionic interaction on MANAE-agarose and DEAE-cellulose. Soluble β-glucosidase presented a half-life of 20 min, at 60 °C, while the MANAE-agarose and DEAE-cellulose derivatives presented a half-life of 25 and 48 h, respectively. The optima pH for soluble β-glucosidase, MANAE- and DEAE derivatives was 4.5. The optimal temperature for DEAE derivative and soluble enzyme was 60 °C and for MANAE-agarose derivative was 65 °C. The Km values for soluble enzyme, MANAE-agarose and DEAE-cellulose derivatives using β-pNPG as substrate were 1.4, 2.6 and 2.0 mg/mL, respectively. Using cellobiose as substrate the Km values were 2.8, 3.4 and 3.7 mg/mL, respectively. The Vmax values using β-pNPG were 24, 25.8 and 13.5 U/mg proteins for the soluble enzyme, MANAE-agarose and DEAE-cellulose derivatives, respectively. Using cellobiose as substrate the Vmax values were 146, 152 and 100.5 U/mg protein. The MANAE and DEAE derivatives presented a good operational stability being evidenced 50 and 60% residual activity after 5 cycles of reaction with the substrate pNPβ- d -glucopyranoside.

Co-expression of the small heat shock protein, Lo18, with β-glucosidase in Escherichia coli improves solubilization and reveals various associations with overproduced heterologous protein, GroEL/ES

We developed a new system to improve the overproduction of soluble proteins in E. coli based on a plasmid encoding the small heat-shock protein, Lo18, derived from the lactic acid bacterium Oenococcus oeni. The efficiency of this system was compared with that of another system based on production of the E. coli universal chaperone GroEL/ES. A compatible plasmid encoding β-glucosidase was constructed for the overproduction and aggregation of this enzyme. Co-expression with Lo18 resulted in an increase in soluble β-glucosidase levels similar to that obtained in the GroEL/ES co-expression system. Lo18 was found preferentially in the insoluble fraction, associated with aggregated enzyme. By contrast, GroEL/ES was more abundant in the soluble fraction.

β-Glucosidase immobilized and stabilized on agarose matrix functionalized with distinct reactive groups

The bioconversion of lignocellulosic biomass to fermentable sugars for production of ethanol requires a multienzyme system named cellulase. This system contains enzymes that act synergistically in the hydrolysis of cellulose: endoglucanase, cellobiohydrolase and β-glucosidase. The first two enzymes act directly on cellulose, yielding mainly glucose and cellobiose, which is hydrolyzed into glucose by β-glucosidase. An industrial process would be more economical by using immobilized systems that allow the reuse of the enzyme and improve the enzyme stability against different inactivation agents. Particularly, the hydrolysis of cellobiose would be performed using immobilized enzyme because cellobiose molecules are soluble in the reaction medium. In this work, β-glucosidase was immobilized on agarose matrix derivatized with different reactive groups, e.g. polyethylenimine (PEI), glyoxyl (linear aliphatic aldehydes) and amine-epoxy, trying to optimize the stability and activity of the immobilized enzyme. Using reversible attachment (immobilization by anion exchange), the derivatives were active, but with poor thermal stability, e.g. PEI agarose derivative was approximately 7 times more stable than the soluble β-glucosidase. However, these derivatives have important characteristics for an industrial process: reuse of the enzyme and/or the application of continuous systems. Among the activated supports with irreversible attachment (covalent immobilization), glyoxyl agarose did not reach a good thermal stability; it seems that the enzyme surface is very poor in amino groups from lysine residues. Better results were obtained with amine-epoxy agarose supports. β-Glucosidase immobilized on that support kept 80% of its activity and was ca. 200 times more stable than the soluble enzyme.

Production of glucose and bioactive aglycone by chemical and enzymatic hydrolysis of purified oleuropein fromOlea Europea

Pure-grade oleuropein, a bitter, hypotensive, phenolic glucoside, was obtained from organic extracts of olive plant leaves by two Chromatographic steps. The purified compound was characterized by spectroscopic NMR and FAB-MS methods. The glucoside underwent chemical and enzymatic hydrolysis. Aglycone was characterized by spectroscopic methods (1H-NMR and FAB-MS). Glucose was measured by enzymatic methods. The enzymatic hydrolysis of oleuropein was carried out by a soluble β-glucosidase. The reaction was characterized in terms of kinetic parameters, optimal pH value, activation energy, inhibition constant by glucose, and thermal stability. Preliminary experiments were also performed in a continuousflow ultrafiltration membrane reactor. The cut-off of the membrane was lower than the molecular-weight of the enzyme, thus determining β-glucosidase confinement within the reactor. Under these conditions, β-glucosidase had a good long-term stability. This is an encouraging result in view of possible industrial applications.

Purification and characterization of a broad specificity β-glucosidase from sheep liver

1. 1. A soluble β-glucosidase from sheep liver has been isolated and purified 114-fold by conventional enzyme fractionation procedures. The specific activity of the purified enzyme was 5910 mU/mg of protein. 2. 2. The enzyme has a broad specificity and hydrolyzes the p-nitrophenyl derivatives of β- d-glucose, β- d-galactose, β- d-fucose, β- d-xylose and α- l-arabinose. The best V max K m value corresponds to the β-glucosidase activity. 3. 3. The enzyme has a pH optimum between 4.5–5.5 for all the activities, and mol. wt 95,000. 4. 4. A variety of chemicals was tested as possible activators or inhibitors. 5. 5. The enyme is strongly inhibited by aldono 1–5 lactones and DMDP. 6. 6. The kinetic evidences suggest a substrate activation model and the existence of two active sites (a “gluco-fuco” site and a “galacto” site). 7. 7. The activation energies were calculated from β-glucosidase and β-galactosidase activities.

Hydrolysis of Conjugated Gibberellins by β-Glucosidases from Dwarf Rice ( Oryza sativa L. cv. «Tan-ginbozu»)

Dwarf rice (Oryza sativa L. cv. «Tan-ginbozu») is widely used for gibberellin conjugate bioassay. Soluble and particulate fractions from seeds and seedlings of this variety showed hydrolytic activity toward [(3)H]GA-O-Glc. The soluble fraction from mature seeds exhibited the highest [(3)H]GA-O-Glc/pNP-β-Glc hydrolysis ratio which decreased during the early germination phase. The soluble β-glucosidases from mature seeds were efficiently separated by CMSephadex C-50 column chromatography. It was found that the most active β-glucosidase CM 1 from seeds hydrolyses GA(8)-O-2-Glc 8 times faster than GA(3)-O-3-Glc and GA(3) Glc ester. In shoots of 4-d-old etiolated seedlings a soluble β-glucosidase (CM 4) is present which showed a 200 times faster hydrolysis of GA(8)-O-2-Glc as compared to GA(3)-O-3-Glc. Both the β-glucosidase CM 4 and the main β-glucosidase component CM 5 possessed relatively high GA(3) Glc ester hydrolysing activity. Our enzymatic findings demonstrate that in dwarf rice bioassay (leaf application) the GA-OGlc with equatorially arranged glucosyl residues (O-2-glucosides of GA(8), GA(26), GA(27), and GA(29)) and GA Glc esters are hydrolysed by the concerted action of the endogenous a-glucosidases CM 4 and CM 5 from shoots.

Enzymology of lignification. Cell-wall bound beta-glucosidase for coniferin from spruce (Picea abies) seedlings.

Spruce (Picea abies) seeds contained a soluble β-glucosidase which was active with 4-nitrophenyl β-D-glucoside but did not catalyse hydrolysis of coniferin (conifery β-D-glucoside). In contrast, a cell wall fraction from hypocotyls and roots of spruce seedlings showed good glucosidase activity towards coniferin with a pH optinum Between 4.5 and 5.5. A number of other aryl β-glucosides were also hydrolysed by these particulate fractions. Michaelis constants and maximal velocities were determined for coniferin (Km =380 and 1300 μ for roots and hypocotyls, respectively) and other aryl glycosides. The cell wall fractions probably contain more than one glycosidase. For 4-nitrophenyl β-D-glucoside and picein (4-hydroxyacetophenone β-D-glucoside) two separate and distinct lines could be drawn for high and low substrate concintrations in Lineweaver-Burk of Eadie-Hofstee plots. Attempts to solublize the β-glucohydrolase activity could be extracted from the hypocotyl particulate fraction by treatment with 0.6 M Nacl. The solublized enzyme was separated on Sepharose 6 B into was fractions with glucosidase activity (glucosidase I and II). Glucosidase I was Purified to apparent homogeneity by repeated gel filtration on Sepharose 6 B. Dodecylsulfate-gel electrophoris and sedimentation equilibrium measurements showed that glucosidase I is composed of only one polypeptide chain with Mr 58 570 SS2400. The properties of glucodidase I with respect to pH and temperature optimum and to substrate specificity towards coniferin and other aryl β-glucosides are similar to those of the hypocotyol particulate fraction. In contrast, glucosidase II has a 10 times smaller ratio for the relative maximal velocities with coniferin and 4-nitrophenyl β-glucosidase than glucosidase I. The presince of cell-well-bound β-glucosidase for coniferin in spruce seedlings is consistent with the view that coniferin participates in lignification.