Total Β-glucosidase Research Articles

The effect of arsenic on soil intracellular and potential extracellular β-glucosidase differentiated by chloroform fumigation

Arsenic (As) contamination of soil is a global issue of serious ecological and human health concern. For better use of soil enzymes as biological indicators of As pollution, the response of soil β-glucosidase in different pools of soil (total, intracellular and potential extracellular) to As(V) stress was investigated. Chloroform fumigation method was employed to distinguish the intracellular and potential extracellular β-glucosidase in three soils. The intracellular and potential extracellular β-glucosidase accounted about 79% and 21% of the total β-glucosidase activity in the tested soils. Moreover, it was found that the response of these three enzyme pools to As(V) pollution was different. Under the stress of 400 mg kg−1 As(V), the β-glucosidase activities decreased by 69%, 79%, and 28% for the total, intracellular and potential extracellular pools, respectively. The calculated median ecological dose (ED50) showed the highest value for potential extracellular β-glucosidase (19.55–27.63 mg kg−1 for total, 18.49–27.42 mg kg−1 for intracellular, and 32.27–52.69 mg kg−1 for potential extracellular β-glucosidase). As(V) exhibited an uncompetitive inhibition for total and intracellular β-glucosidase and non-competitive inhibition for potential extracellular enzyme. The inhibition constant (Kiu) is biggest for potential extracellular β-glucosidase among the three enzyme pools (0.61–0.79 mmol L−1 for total, 0.34–0.36 mmol L−1 for intracellular, and 4.01–23.90 mmol L−1 for potential extracellular β-glucosidase). Thus, compared to potential extracellular β-glucosidase, the total and intracellular β-glucosidases are more suitable for their use as sensitive indicators of As(V) pollution.

Location and contribution of individual β-glucosidase from Neurospora crassa to total β-glucosidase activity

This study investigated the cellular location and the contribution of individual β-glucosidase (BGL) to total BGL activity in Neurospora crassa. Among the seven bgl genes, bgl3, bgl5, and bgl7 were transcribed at basal levels, whereas bgl1, bgl2, bgl4, and bgl6 were significantly up-regulated when the wild-type strain was induced with cellulose (Avicel). BGL1 and BGL4 were found to be contributors to intracellular BGL activity, whereas the activities of BGL2 and BGL6 were mainly extracellular. Sextuple bgl deletion strains expressing one of the three basally transcribed bgls did not produce any detectable BGL activity when they were grown on Avicel. BGL6 is the major contributor to overall BGL activity, and most of its activity resides cell-bound. The sextuple bgl deletion strain containing only bgl6 utilized cellobiose at a rate similar to that of the wild type, while the strain with only bgl6 deleted utilized cellobiose much slower than that of the wild type.

Plasma Membrane-Associated Glycohydrolases Along Differentiation of Murine Neural Stem Cells

The activities of plasma membrane associated sialidase Neu3, total β-glucosidase, CBE-sensitive β-glucosidase, non-lysosomal β-glucosyl ceramidase GBA2, β-galactosidase, β-hexosaminidase and sphingomyelinase were determined at three different stages of differentiation of murine neural stem cell cultures, corresponding to precursors, commited progenitors, and differentiated cells. Cell immunostaining for specific markers of the differentiation process, performed after 7days in culture in presence of differentiating agents, clearly showed the presence of oligodendrocytes, astrocytes and neurons. Glial cells were the most abundant. Sialidase Neu3 after a decrease from progenitors to precursors, showed an increase parallel to the differentiation process. All the other glycosidases increased their activity along differentiation. The activity of CBE-sensitive β-glucosidase and GBA2 were very similar at the precursor stage, but CBE-sensitive β-glucosidase increased 7 times while GBA2 only two in the differentiated cells. In addition, we analysed also sphingomyelinase as enzyme specifically associated to sphingolipids. The activity of this enzyme increased from precursors to differentiated cells.

A <i>β</i>-Glucosidase Activity Potentially Involved in Cell Division and Wall Development of <i>Phyllostachys</i> Bamboo Suspension Cells

We propose a novel Madake (Phyllostachys bambusoides) bamboo suspension culture model for investigation of key enzyme(s) activity involved in growth/differentiation. Sedimented Cell Volume (SCV) and fresh weight (FW) of the suspension cultured cells reached 34% (v/v) and 8.7 g in 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium while only 7% (v/v) SCV and 1.9 g FW of the cells in 10 μM gibberellic acid (GA3)-containing medium in 14 days. Proportion of mitotically active cells (S to G2/M phases) at a log phase was identified as 29.5% in the former cells with tiny cytoplasmic features while 5.4% in the latter cells with elongation, wall thickening, and lignification by using flow cytometry and laser scanning microscopic analysis. The total β-glucosidase (BGL) activity under the 2,4-D condition increased from 4.8 U in day 2 to 26.2 U in day 14 (ca. 5.5-fold) while a slight reduction, from 4.4 U in day 2 to 2.1 U in day 14 (ca. 0.5-fold), occurred when cell division was suppressed under the GA3 condition. Ratio of the BGL activity of the soluble fractions to the membrane-associated fractions varied depending of the culture condition. The ratio was stable (2 to 8) during the culture period under the 2,4-D condition. Interestingly, the activity of the soluble enzyme fractions increased up to ca. 65% under the GA3 condition in inverse proportion to the membrane-associated enzymes. All together, it was strongly suggested that the detected specificity/variability of BGL activity is potentially involved in cell division and lignification in Madake bamboo cells.

Increased total air pressure versus oxygen limitation for enhanced oxygen transfer and product formation in a Pichia pastoris recombinant protein process

Two strategies to increase the productivity of secreted Thai Rosewood β-glucosidase in Pichia pastoris processes were evaluated. Both methods were based on increasing the oxygen transfer rate (OTR) in the process by simple means. Increasing the driving force for the diffusion from the air bubbles to the medium by elevating the air pressure, from 1.2 to 1.9 bar increased the oxygen uptake rate (OUR) by 59% while increasing the driving force by accepting oxygen limitation increased the OUR by 35%. The OTR increased less than in proportion to the increased solubility in the high-pressure process, which indicates that air bubble compression reduces the volumetric oxygen transfer coefficient ( K L a). Even though the methanol consumption increased almost in proportion to the OTR in both processes the biomass production did not increase as much. This is explained as a higher maintenance demand for methanol in the oxygen limited (0.027 g g −1 g −1) and high-pressure processes (0.035 g g −1 g −1), compared to 0.022 g g −1 g −1 in the methanol limited reference process. However, in spite of the low effect of increasing OTR on the biomass production the total β-glucosidase yield increased almost in proportion to the increased methanol consumption and reached highest value in the high-pressure process, while the β-glucosidase purity was highest in the oxygen-limited process due to release of less contaminating proteins.

Purification and characterization of an intracellular β-glucosidase from Botrytis cinerea

The filamentous fungus Botrytis cinerea, grown on a cellobiose medium, was found to produce three intracellular β-glucosidases (GluI, II, and III). These enzymes were visualized by activity staining after separation by native polyacrylamide gel electrophoresis. GluI, which represents 95.5% of the total β-glucosidase activity, was purified to homogeneity by ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase estimated by gel filtration was 350 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate. The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 7 with citrate-phosphate buffer and 6.5 with phosphate buffer. The enzyme was active against glycosides with (1 → 4)-β, (1 → 2)-β, and (1 → 4)-α linkage configuration. The β-glucosidase was competitively inhibited by glucose and byd-gluconic-acid-lactone, and a glucosyl transferase activity was observed in the presence of ethanol.

Pattern of enzymes involved in cyanogenesis and HCN metabolism in cell cultures of Phaseolus lunatus L. varieties

Callus and cell suspension cultures were established from root and shoot tips of aseptically-grown seedlings of highly cyanogenic Phaseolus lunatus L. varieties. The content of cyanogenic glucosides in the explanted seedling sections decreased during storage, the derived callus cells were free of cyanogenic glycosides. In spite of the non-existence of cyanogenic glucosides, the cyanogen degrading linamarase, the cyanide detoxifying enzyme β-cyanoalanine synthase and also hydroxynitrile lyase were still present in suspension cultures. The linamarase activity equalled the total β-glucosidase activity, of which up to 80% was found in the culture medium. In contrast the β-cyanoalanine synthase and the hydroxy nitrile lyase were entirely localized in the cell biomass.

Formation and release of ?-glucosidase by aspergillus niger zimet 43 746 in correlation to process operations

The total formation of β-glucosidase by the wild strain of Aspergillus niger ZIMET 43 746 is non-growth-associated. In discontinuous culture the total β-glucosidase activity related to the mycelium is incresing with the age of the mycelium. The complete release of the remaining mycelial-associated β-glucosidase is dependent on the structure of the mycelium. In the cases of the mycelium forms pellets throughout the growth phase than the release of β-glucosidase is accelerated compared to the release from loosly branched mycelium. Incresing shear stress caused by increasing of the impeller speed promotes the formation of pellets.

The pH dependent distribution of β-glucosidase activity in isolated particulate fractions

Higher levels of β-glucosidase activity were found in particulate fractions isolated from corn roots under alkaline compared to acid conditions. The greatest pH effect was observed on the crude cell wall fraction (1000× g for 10 min) where 19% of the total β-glucosidase activity was recovered at pH 7.7 compared to 3% at pH 6.0. Further analysis indicated that the particulate activity recovered in the 1000× g pellet was dissociated over a narrow range of pH. To determine if the pH effect was on the cell wall or a subcellular component found in the crude cell wall fraction, cell walls were separated from organelles and membranes by sucrose density gradient centrifugation. Most of the β-glucosidase activity in the crude cell wall fraction was not associated with the cell wall but with an unidentified component which equilibrated at 51% (w/w) sucrose.

Influence of L-sorbose on the location of β-glucosidase in Trichoderma pseudokoningii

The effect of l-sorbose on growth, morphology, cell wall composition and β-glucosidase location has been examined with Trichoderma pseudokoningii. Sorbose-grown cultures exhibited a longer lag phase, a tendency to more frequent hyphal branching and showed a decreased cell wall content of β-1,3-glucan. In sorbose-containing cultures, a significant higher portion of total β-glucosidase was present in the culture fluid, whereas in sorbose-lacking control cultures the major part of activity was associated with the cell walls. The results support the previous hypothesis (Kubicek, C.P. (1982) Arch. Microbiol. 132, 349–354) that β-1.3-glucan is involved in cell wall binding of β-glucosidase in Trichoderma pseudokoningii.