The interaction between genistein and beta-glucosidase was studied using fluorescence quenching method and synchronous fluorimetry. The binding reaction was simultaneously studied by the AutoDock 4.2 molecular docking model. Data from fluorescence spectroscopy indicated that these interactions resulted in the endogenous fluorescence quenching of beta-glucosidase, which belongs to a static quenching mechanism. The calculated binding constants were 3.69 x 10(4), 3.06 x 10(4) and 2.36 x 10(4) L x mol(-1) at 17, 27 and 37 degrees C, respectively. The evidences from synchronous fluorescence showed the effect of genistein on the microenvironment around beta-glucosidase in aqueous solution. The inhibition test showed that the activity of beta-glucosidase could be inhibited by genistein. The determined bimolecular rate constant (k(i)) was 1.2 x 10(3) (mol x L(-1)(-1) x min(-1). Molecular docking was performed to reveal the possible binding mode or mechanism and suggested that genistein could bind strongly to beta-glucosidase. The results revealed that genistein tended to bind with beta-glucosidase mainly by hydrogen bond and hydrophobic interaction as well as electrostatic forces.
Genistein ; metabolism ; Molecular Docking Simulation ; Protein Binding ; Protein Kinase Inhibitors ; metabolism ; Spectrometry, Fluorescence ; beta-Glucosidase ; metabolism

In order to explore the influence of reaction temperature on the product composition, the effect of continuous temperature change (22 degrees C-60 degrees C, +/-0.1 degree C) on hydrolysis of yeast beta-glucan by endo-beta-1,3-glucanase was determined by using self-developed Biochem-temperature Characteristic Apparatus. The activation energy of enzymatic hydrolysis of yeast beta-glucan was 84.17 kJ/mol. The optimum temperature represented by accumulation of products decreased exponentially within a certain period of time. The components of the products were changed with reaction temperature. The length of oligosaccharides decreased with the increase of temperature. The main products were laminaribiose and laminaritriose at the temperature higher than 46 degrees C, while the main products were laminaripentaose and larger molecular weight components at the temperature lower than 30 degrees C. The results can provide precise parameters to control the reaction temperature of the production of 1,3-beta-D-glucooligosaccharides.
Enzyme Activation ; Glucan Endo-1,3-beta-D-Glucosidase ; chemistry ; metabolism ; Hydrolysis ; Oligosaccharides ; chemistry ; metabolism ; Temperature ; Yeasts ; metabolism ; beta-Glucans ; metabolism

Constructing ethanologenic strains with cellulose activity is important to achieve consolidated bioprocessing of lignocellulose for ethanol production. In this study, we integrated the pyruvate decarboxylase gene pdc and alcohol dehydrogenase gene adhB from Zymomonas mobilis ZM4 into Escherichia coli JM109 by Red recombination method to generatea recombinant strain E. coli P81 that could produce ethanol from glucose. Abeta-glucosidase gene bglB from Bacillus polymyxa 1.794 was cloned into the recombinant E. coli P81 and beta-glucosidase was expressed to give a new recombinant strain E. coli P81 (pUC19-bglB) with dual functions of cellobiose degradation and ethanol production. The extracellular beta-glucosidaseactivity was 84.78 mU/mL broth and the extracellular cellobiase activity of E. coli P81 (pUC19-bglB) was 32.32 mU/mL broth. E. coli P81 (pUC19-bglB) fermented cellobiose to ethanol with a yield of 55.8% of the theoretical value, and when glucose and cellobiose were co-fermented, the ethanol yield reached 46.5% of thetheoretical value. The construction of consolidated bioprocessing strain opens the possibility to convert cellobiose to ethanol in a single bioprocess.
Bacterial Secretion Systems ; Cellulose ; metabolism ; Escherichia coli ; genetics ; metabolism ; Ethanol ; metabolism ; Fermentation ; Recombinant Proteins ; biosynthesis ; genetics ; beta-Glucosidase ; biosynthesis ; genetics

Cellulases are relatively costly enzymes that are sold in large volumes for use in different industrial applications, and a significant reduction in cost will be important for their commercial use in biorefineries. The production of cellulase is a major factor in the hydrolysis of cellulosic materials. Hence it is essential to make the process economically viable. A strain (L-06) with high cellulase activity was screened from rice straw compost and classified as Penicillium decumbens by the analysis of its morphology and 18S rRNA gene sequences. Different conditions of liquid fermentation medium including nitrogen source, carbon source, surfactant, temperature, initial pH, inoculation quantity for the production of cellulase had been studied. The maximal beta-1, 4-glucosidase(BGL) activity was 1662 u/mL which is 1.49 times of the previous and the maximal exo-beta-1, 4-glucanases(CBH) activity was 2770 u/mL which is 1.36 times of the previous, cultured in the optimal condition for three days. And the maximal endo-beta-1, 4-glucanases (EG) activity was 18064 u/mL which is 1.87 times of the previous and the maximal filter paper enzyme(FPase) activity was 4035 u/mL which is 1.47 times of the previous, cultured in the optimal condition for four days. In the optimization experiments, the EG and CBH in the culture condition (pH10) maintained 70% and 43% activity. In the culture condition (50 degrees C) EG and CBH maintained 59% and 68% activity, which showed heat and alkali resistant characteristics.
Cell Culture Techniques ; Cellulase ; biosynthesis ; isolation & purification ; metabolism ; Cellulose ; metabolism ; Glucan 1,4-beta-Glucosidase ; biosynthesis ; metabolism ; Oryza ; Penicillium ; cytology ; enzymology ; isolation & purification ; Plant Stems ; microbiology ; Temperature ; beta-Glucosidase ; biosynthesis ; metabolism

Cellulosic material is the most abundant renewable carbon source in the world. Cellulose may be hydrolyzed using cellulase to produce glucose, which can be used for production of ethanol, organic acids, and other chemicals. Cellulase is a complex enzyme containing endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1.91) and cellobiase (EC 3.2.1.21). The hydrolysis of natural cellulose to glucose depends on the synergism of these three components. The mostly used cellulase produced by Trichoderma reesei has high activity of endoglucanase and exoglucanase, but the activity of cellobiase is relatively low. Therefore, improving the activity of cellobiase in cellulase reaction system is the key to enhance the sacchrification yield of cellulosic resources. Aspergillus niger LORRE 012 was a high productivity strain for cellobiase production. It was found that the spores of this strain were rich in cellobiase. In this work, the cellobiase was immobilized efficiently by simply entrapping the spores into calcium alginate gels instead of immobilizing the pure cellobiase proteins. The immobilized cellobiase was quite stable, and its half-life was 38 days under pH 4.8, 50 degrees C. The thermal stability of the immobilized cellobiase was improved, and it was stable below 70 degrees C. The suitable pH range of the immobilized cellobiase was pH 3.0 - 5.0, with the optimal pH value 4.8. The Km and Vmax value of the immobilized cellobiase were 6.01 mmol/L and 7.06 mmol/min x L, respectively. In repeated batch hydrolysis processes, 50 mL of substrate (10 g/L cellobiose) and 10 mL of immobilized beads containing cellobiase were added into a 150 ml flask. After reacting at pH 4.8, 50 degrees C for several hours, the hydrolysate was harvested for assay, and the immobilized beads were used for the next batch hydrolysis with the fresh substrate. This process was repeated, and the yield of enzymatic hydrolysis kept higher than 97% during 10 batches. The continuous hydrolysis process was carried out in a column reactor (inside diameter 2.8 cm, inside height 40 cm) packed with the immobilized beads. Using 10 g/L cellobiose as substrate, the hydrolysis yield reached 98% under 0.4 h (-1) dilution rate and pH 4.8, 50 degrees C. After corncob was treated by 1% dilute acid, the cellulosic residue (100 g/L) was used as substrate, and hydrolyzed by the cellulase (15 IFPU/g substrate) from Trichoderma reesei, at pH 4.8, 50 degrees C for 48 h. The concentration of reducing sugar in the hydrolysate was only 48.50 g/L (hydrolysis yield 69.5%). When the hydrolysate was further treated by the immobilized cellobiase, the cellobiose was hydrolyzed into glucose, and the feedback inhibition caused by the cellobiose accumulation disappeared sharply. By the synergism of immobilized cellobiase and the cellulase from T. reesei left in the hydrolysate, other oligosaccharides were mostly converted to monosaccharides. At 48 h, the reducing sugar concentration was increased to 58.78 g/L, the hydrolysis yield of the corncob residue was improved to 84.2%, and the ratio of the glucose in the total reducing sugar was increased from 53.6% to 89.5%. The reducing sugars converted from corncob could be used further in the fermentation of valuable industrial products. This research results were meaningful in the conversion and utilization of renewable biomass.
Aspergillus niger ; enzymology ; Biotechnology ; Cellobiose ; metabolism ; Enzyme Stability ; Hydrogen-Ion Concentration ; Kinetics ; Temperature ; beta-Glucosidase ; chemistry ; metabolism

β-glucosidase has important applications in food, medicine, biomass conversion and other fields. Therefore, exploring β-glucosidase with strong stability and excellent properties is a research hotspot. In this study, a GH3 family β-glucosidase gene named Iubgl3 was successfully cloned from Infirmifilum uzonense. Sequence analysis showed that the full length of Iubgl3 was 2 106 bp, encoding 702 amino acids, with a theoretical molecular weight of 77.0 kDa. The gene was cloned and expressed in E. coli and the enzymatic properties of purified IuBgl3 were studied. The results showed that the optimal pH and temperature for pNPG hydrolysis were 5.0 and 85 ℃, respectively. The enzyme has good thermal stability, and more than 85% of enzyme activity can be retained after being treated at 80 ℃ for2 h. This enzyme has good pH stability and more than 85% of its activity can be retained after being treated at pH 4.0-11.0 for 1 h. It was found that the enzyme had high hydrolysis ability to p-nitrophenyl β-d-glucoside (pNPG) and p-nitrophenyl β-d-xylopyranoside (pNPX). When pNPG was used as the substrate, the kinetic parameters K_m and V_max were 0.38 mmol and 248.55 μmol/(mg·min), respectively, and the catalytic efficiency k_cat/K_m was 6 149.20 s^-1mmol^-1. Most metal ions had no significant effect on the enzyme activity of IuBgl3. SDS completely inactivated the enzyme, while EDTA increased the enzyme activity by 30%. This study expanded the β-glucosidase gene diversity of the thermophilic archaea GH3 family and obtained a thermostable acid bifunctional enzyme with good industrial application potential.
beta-Glucosidase/chemistry* ; Archaea/metabolism* ; Escherichia coli/metabolism* ; Hydrogen-Ion Concentration ; Temperature ; Glucosides ; Enzyme Stability ; Substrate Specificity ; Kinetics

Selective markers used in yeast vector for gene manipulation were usually drug resistance or autotrophy. Unfortunately, drug resistance selective marker requires drug sensitive host and most industrial strains were not autotrophy. In this paper, flocculation gene (FLO1) from Saccharomyces cerevisiae ABXL-1D was amplified by PCR, sequenced and cloned to construct an expression vector. The new vector was easy to manipulate and suitable for broad host of yeasts without either autotrophy or drugs. beta-glucosidase gene from Bacillus polymyxa was cloned with the vector and expressed in Saccharomyces cerevisiae. The specific activity of beta-glucosidase of the recombinant yeast cell-free extract was 3.91 u/mg protein. The residue glucose of the recombinant yeast was considerably reduced in mixed fermentation of glucose and cellobiose. It should be favorable for ethanol fermentation when utilize lignocellulosic biomass as raw material.
Bacillus ; enzymology ; genetics ; Flocculation ; Genetic Vectors ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; beta-Glucosidase ; biosynthesis ; genetics

The beta-glucosidase encoding gene bglA was cloned from Bacillus polymyxa 1.794. The bglA gene was inserted in expression vector pET28a(+) and transformed into Escherichia coli BL21 (DE3), finally the recombinant strain BL1979 was obtained. Induced by IPTG, the expression P-glucosidase activity reached to 24.7 IU/mL. The optimum temperature and optimum pH of the recombinant expression P-glucosidase in BL1979 were 37 degrees C and 7.0 respectively,the purity can reach to 92.7%. Analysis of the fusion protein by nondenaturing gradient gel electrophoresis, we found the fusion protein exists in dimmer, tetramer,hexamer and octamer, they all have hydrolase activity.
Bacillus ; enzymology ; Escherichia coli ; genetics ; Plasmids ; Recombinant Proteins ; biosynthesis ; isolation & purification ; beta-Glucosidase ; genetics ; isolation & purification ; metabolism

OBJECTIVEThis study was conducted to compare serum cytosolic β-glucosidase (CBG) levels of age-matched control patients with those of infants with necrotizing enterocolitis (NEC) thereby to determine the eventual association between serum CBG levels with extensive disease in infants with NEC.

METHODA total of 96 premature infants were divided into the early NEC group (n = 25), confirmed NEC group (n = 23) and the control group (n = 48). Serum CBG concentration, C-reactive protein (CRP) and peripheral blood white blood cells (WBC) were measured at the onset of the disease in patients in early NEC or confirmed NEC groups and at weeks 2-3 in control infants. Data were analyzed using descriptive statistics, non-parametric tests, Student's t-test, linear correlation, Spearman correlation analysis, receiver operating characteristic (ROC) curve were used for statistical analysis.

RESULTSThe median birth weights (mean ± SE) in the three groups were not statistically significant (P > 0.05). Serum CBG concentration in the 3 groups were (112.369 ± 108.539) nmol/L, (693.013 ± 211.614) nmol/L and (36.478 ± 28.31) nmol/L, respectively. The infants in the confirmed NEC group had highest CBG levels, compared with the other 2 groups (P < 0.05). When the levels of CBG ≥ 65 ng/ml, CRP ≥ 2 mg/L and WBC < 5 × 10(9)/L within 3 days after birth or > 20 × 10(9)/L 3 days after birth were considered as positive parameters, the sensitivity of CBG and CRP was higher than that of WBC (P < 0.05). Among these indices, CBG had the highest specificity (87.4%), positive predictive (95.6%) and Youden's index (81.3%). CBG is correlated with CRP (the Spearman correlation coefficient was 0.379, P < 0.01).

CONCLUSIONSerum CBG concentration increases early in NEC. Serum CBG level was associated with extensive disease in infants with NEC. Therefore CBG can be used as a marker in the early diagnosis of NEC.

Case-Control Studies ; Enterocolitis, Necrotizing ; blood ; diagnosis ; Humans ; Infant, Newborn ; Infant, Premature ; Leukocyte Count ; Serum ; metabolism ; beta-Glucosidase ; blood

Six new prenylated flavonoid glycosides, including four new furan-flavonoid glycosides wushepimedoside A-D (1-4) and two new prenyl flavonoid derivatives wushepimedoside E-F (5-6), and one know analog epimedkoreside B (7) were isolated from biotransformation products of the aerial parts of Epimedium wushanense. Their structures were elucidated according to comprehensive analysis of HR-MS and NMR spectroscopic data, and the absolute configurations were assigned using experimental and calculated electronic circular dichroism (ECD) data. The regulatory activity of compounds 1-7 on the production of testosterone in primary rat Leydig cells were investigated, and 4 and 5 exhibited testosterone production-promoting activities. Molecular docking analysis suggested that bioactive compounds 4 and 5 showed the stable binding with 3β-HSD and 4 also had good affinity with Cyp17A1, which suggested that these compounds may regulate testosterone production through stimulating the expression of the above two key proteins.
Animals ; Epimedium/chemistry* ; Flavonoids/chemistry* ; Furans ; Glycosides/chemistry* ; Hydrolysis ; Male ; Molecular Docking Simulation ; Molecular Structure ; Rats ; Testosterone ; beta-Glucosidase/metabolism*