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β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

Effective and low-cost inducer is a prerequisite for efficiency improvement in cellulase production by Trichoderma reesei. In this study, spores of Aspergillus niger enriched with cellobiase were embedded in Ca-alginate microcapsules, attaining a sustained cellobiase-release system (named CRMA) which was then used to catalyze the conversion of glucose into glucose-sophorose sugar mixture via transglycosylation. Further, the process and optimal parameters of transglycosylation reaction were studied in a 5 L reactor and the sophorose concentration reached 70.55 g/L at 50 °C, pH 4.0 with CRMA loading of 200 mL after 72 h of reaction. The obtained sugar mixture was subsequently used as both carbon source and inducer in cellulase production by Trichoderma reesei under fed-batch process. The filter paper activity (FPA) reached 90.28 IU/mL at 144 h fermentation time, 1.85-fold higher than that induced by lactose. These results indicate that the newly established strategy for soluble inducer synthesis has the potential for efficiency improvement and cost reduction in cellulase production., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Xylanase gene xynB, cellulase genes egxA and bgl4 were subjected to codon optimisation using two opposing strategies. One was designated the 'one amino acid-one codon' approach, which employs only the codon most used by humans for each amino acid. The other one is referred to as the "humanised" codon usage method, which selects synonymous codons for each amino acid according to the human codon usage table to mimic patterns used in humans. Protein expression levels in mammalian cell lines from each sequence were measured using fluorescence-activated cell sorting, western blotting and enzymatic activity assay. The results indicate that compared with the humanised codon usage method, the relatively simple 'one amino acid-one codon' approach could enhance heterologous protein expression in mammalian cells without apparent drawbacks.

This study reports differential expression of endoglucanase (EG) and beta-glucosidase (betaG) isoforms of Aspergillus terreus. Expression of multiple isoforms was observed, in presence of different carbon sources and culture conditions, by activity staining of poly acrylamide gel electrophoresis gels. Maximal expression of four EG isoforms was observed in presence of rice straw (28 U/g DW substrate) and corn cobs (1.147 U/ml) under solid substrate and shake flask culture, respectively. Furthermore, the sequential induction of EG isoforms was found to be associated with the presence of distinct metabolites (monosaccharides/oligosaccharides) i.e., xylose (X), G(1), G(3) and G(4) as well as putative positional isomers (G(1)/G(2), G(2)/G(3)) in the culture extracts sampled at different time intervals, indicating specific role of these metabolites in the sequential expression of multiple EGs. Addition of fructose and cellobiose to corn cobs containing medium during shake flask culture resulted in up-regulation of EG activity, whereas addition of mannitol, ethanol and glycerol selectively repressed the expression of three EG isoforms (Ia, Ic and Id). The observed regulation profile of betaG isoforms was distinct when compared to EG isoforms, and addition of glucose, fructose, sucrose, cellobiose, mannitol and glycerol resulted in down-regulation of one or more of the four betaG isoforms.

Aims: Ceriporiopsis subvermispora produces endoglucanase and beta-glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major beta-glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood., Methods and Results: Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid-state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l(-1), pH 5.4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S-300 gel filtration. The retained fraction attained 76% of beta-glucosidase activity with 3.7-fold purification. Two beta-glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the beta-glucosidase of 110 kDa. The optimum pH and temperature were 3.5 and 60 degrees C, respectively. The K(m) and V(max) values were respectively 3.29 mmol l(-1) and 0.113 micromol min(-1) for the hydrolysis of p-nitrophenyl-beta-glucopyranoside (pNPG) and 2.63 mmol l(-1) and 0.103 micromol min(-1), towards cellobiose. beta-Glucosidase activity was strongly increased by Mn(2+) and Fe(3+), while Cu(2+) severely inhibited it., Conclusions: Ceriporiopsis subvermispora produces small amounts of beta-glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two beta-glucosidases with 110 and 53 kDa. The 110 kDa beta-glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical beta-glucosidase., Significance and Impact of the Study: Ceriporiopsis subvermispora produces beta-glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of beta-glucosidases during biopulping.

Two suggestions can be found in the literature to improve the reproducibility of the Mandels' filter paper assay: add supplemental cellobiase and increase the boiling time for color development. Here we provide data that strongly supports adding supplemental cellobiase. Adding supplemental cellobiase increased assay response by 56%. Cellulases from different sources have different cellobiase activities, which would cause significant variation in the assay response. There is no need for additional boiling time-5 minutes is sufficient. For maximum reproducibility, it is essential that the water bath vigorously boil so that temperature excursions are minimized., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 745-749, 2003.)

Activity of supplemental enzymes in a barley-soybean-maize based diet at 60, 75 and 90 degrees C pelleting temperatures was studied using feed viscosity, in-vitro enzyme activity and broiler performance data. High pelleting temperatures increased feed viscosity but supplemented enzymes reduced the viscosity at all three temperatures levels by 11, 14 and 17%, respectively. Water intake and losses in excreta of birds were found to be affected by feed viscosity. Activity of cellulase enzyme, measured using the radial diffusion method, was unaffected at 60 and 75 degrees C, but reduced by 73% in feed processed at 90 degrees C. Enzymes increased the weight gain of broilers by 11.1% at 90 degrees C, but no effect could be seen at low pelleting temperatures possibly due to high dietary protein and energy contents. Feed intake was unaffected by enzymes. Birds consumed 6% more feed and grew 9% faster when the pelleting temperature was increased from 60 to 75 degrees C. Reduced feed intake and daily weight gain observed at 90 degrees C could be fully compensated by the enzyme supplementation. High pelleting temperature reduced energy metabolizability (3.2%) and nitrogen utilization (4%) but enzyme almost compensated them (by 3.3% and 2.6%, respectively). No interaction could be detected between the pelleting temperatures and enzymes. It is concluded that pelleting temperatures as high as 90 degrees C drastically reduce cellulase activity, energy and nitrogen utilization thus lowering broiler performance. Either the remaining activity of cellulase or other thermostable enzymes can prevent the losses.

Prevotella ruminicola B(1)4 is a gram-negative, anaerobic gastrointestinal bacterium. A 2.4-kbp chromosomal fragment from P. ruminicola encoding an 87-kDa aryl-glucosidase (CdxA) with cellodextrinase activity was cloned into Escherichia coli DH5 alpha and sequenced. CdxA activity was found predominantly in the membrane fraction of both P. ruminicola and E. coli, but P. ruminicola localized the protein extracellularly while E. coli did not. The hydrolase had the highest activity on cellodextrins (3.43 to 4.13 mumol of glucose released min-1 mg of protein-1) and p-nitrophenyl-beta-D-glucoside (3.54 mumol min-1 mg of protein-1). Significant activity (70% of p-nitrophenyl-beta-D-glucoside activity) was also detected on arbutin and prunasin. Less activity was obtained with cellobiose, amygdalin, or gentiobiose. CdxA attacks cellodextrins from the nonreducing end, releasing glucose units, and appears to be an exo-1,4-beta-glucosidase (EC 3.2.1.74) which also is able to attack beta-1,6 linkages. Comparison of the deduced amino acid sequence with other glycosyl-hydrolases suggests that this enzyme belongs to family 3 (B. Henrissat, Biochem. J. 280:309-316, 1991). On the basis of this sequence alignment, the catalytic residues are believed to be Asp-275 and Glu-265. This is the first report of a cloned ruminal bacterial enzyme which can cleave cyanogenic plant compounds and which may therefore contribute to cyanide toxicity in ruminants.

A new method that uses nonradioactive active site-directed enzyme inactivators and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESIMS/MS) to identify labeled peptides in a proteolytic digest is described. This method relies upon the fragmentation of labeled peptides in a predictable and reproducible manner in the collision cell of a tandem mass spectrometer. The exoglycanase from Cellulomonas fimi, endoglucanase C from Clostridium thermocellum, and the beta-glucosidase from Agrobacterium faecalis were labeled using 2-deoxy-2-halo-beta-glycosides, digested with pepsin, and subjected to HPLC-ESIMS/MS analysis, scanning in the neutral loss mode. Under these conditions only peptides that lose the (known) mass of the label are detected. Preliminary identification of candidate peptides can be achieved from the mass measured, in combination with the known sequence of the protein. Peptide identity can be confirmed through subsequent sequencing, either via further tandem MS experiments or via the Edman degradation. In all cases the peptides identified in this manner were consistent with those identified by the standard radioactive method. This mass spectrometric method represents a rapid, nonradioisotopic solution to the problem of identifying a modified peptide in a complex mixture. The technique is also sensitive, requiring only picomole amounts of protein.

Using electroncytochemical and biochemical methods, differences between the cytochemical reaction intensity and activity of the cellulosolytic enzymes in Funaria hygrometrica moss cells grown for 30 days in the horizontal clinostat (2 rev/min) and in control have been studied. It has been shown that on clinostating the precipitate amount and size increases with the cellulase activity enhancement in the periplasmic space and protonema cell walls, when compared to control. Using biochemical methods it has been found that the activity of both endo-1,4-beta-glucanase and exo-1,4-beta-glucanase was higher under these conditions. A decrease of cellulose total content, its crystalline form, and pectic substances as well as an increase of hemicellulose content have been revealed in the clinostated material compared to control. Data obtained are discussed regarding the possible mechanism of cellulase activation and synthesis inhibition and cellulose crystallization in plant cell walls at clinostating.

Endoglucanase and cellobiohydrolase components of thermophilic cellulases can be detected in situ after gel electrophoresis in the presence of sodium dodecyl sulfate by incorporating a mixed linkage beta-glucan (barley beta-glucan, lichenan) in the separation gel. Zymograms are prepared after a renaturation treatment and incubation by staining the gel with Congo red. This method is suitable for the detection of beta-glucanases with different substrate specificities cleaving beta-1,4-, beta-1,4-1,3-, or beta-1,3-glucans. Cellobiohydrolase activities can be detected by adding 4-methylumbelliferyl-beta-D-cellobioside to the incubation buffer. The gels are subsequently stained with Coomassie blue to establish identical molecular weights of beta-glucanase and protein bands. Applications of this technique for the comparison of cellulases and for the identification of cellulase components expressed from recombinant clones are presented.

We demonstrate homology between the catalytic domains of exoglucanase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) from Cellulomonas fimi and those of endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8) from Bacillus sp. strain C-125 and the fungus Cryptococcus albidus; and between the catalytic domains of endoglucanase (1,4-(1,3:1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) from Cellulomonas fimi and exoglucanase II from Trichoderma reesei. These five enzymes apparently evolved by reshuffling of two catalytic domains and several substrate-binding domains.

A selective procedure using synthetic substrates for determination of exo-1,4,-beta-glucanases in a mixture of exoglucanases , endoglucanases , and beta-glucosidases is formulated. The heterobiosides , p- nithrophenyl -beta-D- cellobioside ( pNPC ) or p-nitrophenyl-beta-D-lactoside ( pNPL ), were used as selective substrates for the measurement of exoglucanase activity. The exoglucanases (especially cellobiohydrolases , which split off cellobiose units from the nonreducing end of the cellulose chain) specifically act on the agluconic bond (between p-nitrophenyl and the disaccharide moiety) and not on the holosidic bond (between the two glucose units of cellobiose). The interfering effect of beta-glucosidase, which acts on both agluconic and holosidic bonds, is overcome by the addition of D-glucono-1,5-delta-lactone, a specific inhibitor of beta-glucosidases. The interference of endoglucanases , which also act on both agluconic and holosidic bonds, can be compensated for by prior standardization of the assay procedure with a purified endoglucanase from the studied mixture of cellulases.