Purification and characterization of piceid-β-d-glucosidase from Aspergillus oryzae

The piceid-β-d-glucosidase that hydrolyzes the β-d-glucopyranoside bond of piceid to release resveratrol was isolated from Aspergillus oryzae sp.100 strain, and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 77 kDa. The optimum temperature of the piceid-β-d-glucosidase was 60 °C, and the optimum pH was 5.0. The piceid-β-d-glucosidase was stable at less than 60 °C, and pH 4.0–5.0. Ca²⁺, Mg²⁺ and Zn²⁺ ions have no significant effect on enzyme activity, but Cu²⁺ ion inhibits enzyme activity strongly. The K_m value was 0.74 mM and the V_max value was 323 nkat mg⁻¹ for piceid.     

Kinetic properties of β-glucosidase from Aspergillus ornatus

Summary Kinetic properties of extracellular -glucosidase from Aspergillus ornatus were determined. The pH and temperature optima for the enzyme were found to be 4.6 and 60°C, respectively. Under these conditions, the enzyme exhibited a K _m (p-nitrophenyl--glucoside) value of 0.76±0.11 mM. The activation energy for the enzyme was 11.8 kcal/mol. Several divalent metal ions inhibited -glucosidase activity, some of which showed inhibition of enzyme activity only at higher concentrations. Ag²⁺ was the most potent inhibitor. A metal chelating agent, EDTA, also inhibited -glucosidase activity. Except for trehalose, glucose, glucono--lactone, cellobiose, gentiobiose, laminaribiose, maltose and isomaltose inhibited -glucosidase activity. Glucose was found to be a competitive inhibitor, whereas glucono--lactone and other -linked disaccharides were noncompetitive (mixed) inhibitors of the enzyme.     

The production of β-glucosidase in shake-flasks by Aspergillus wentii

Studies in shake-flasks showed that Aspergillus wentii produces the maximum activity of β-glucosidase among the cultures tested. The activity against cellobiose was about 2–3 fold that against 4NPG. Aspergillus wentii produced a maximum activity of 16.5 U/ml in 14 days on malt extract. It also produced a comparable amount on other simple soluble sugars, which indicates that it is constitutive and does not require an inducer. Peptone was found to the best nitrogen source for β-glucosidase production. Optimum C/N ratio was found to be 7.3. Phosphate, magnesium and trace metals did not play significant roles in the production of β-glucosidase when they were used with malt extract as a carbon source. An inoculum of 6% (v/v) of 20-h-old culture grown on malt extract produced the maxium β-glucosidase activity.     

Production and characterization of β-glucosidases from different Aspergillus strains

-D-Glucosidase enzymes (-D-glucoside glucohydrolase, EC 3.2.1.21) from different Aspergillus strains (Aspergillus phoenicis, A. niger and A. carbonarius) were examined with respect to the enzyme production of the different strains using different carbon sources and to the effect of the pH and temperature on the enzyme activity and stability. An efficient and rapid purification procedure was used for purifying the enzymes. Kinetic experiments were carried out using p-nitrophenyl -D-glucopyranoside (pNPG) and cellobiose as substrates. Two different fermentation methods were employed in which the carbon source was glucose or wheat bran. Aspergillus carbonarius proved to be the less effective strain in -glucosidase production. Aspergillus phoenicis produced the highest amount of -glucosidase on glucose as carbon source however on wheat bran A. niger was the best enzyme producer. Each Aspergillus strain produced one single acidic -glucosidase with pI values in the range of pH 3.52–4.2. There was no significant difference considering the effect of the pH and temperature on the activity and stability among the enzymes from different origins. The enzymes examined have only -glucosidase activity. The kinetic parameters showed that all enzymes hydrolysed pNPG with higher efficiency than cellobiose. This shows that hydrophobic interaction plays an important role in substrate binding. The kinetic parameters demonstrated that there was no significant difference among the enzymes from different origins in hydrolysing pNPG and cellobiose as the substrates.     

Purification and characterization of SDG-β-d-glucosidase hydrolyzing secoisolariciresinol diglucoside to secoisolariciresinol from Aspergillus oryzae

The SDG-β-d-glucosidase that hydrolyzes the glucopyranoside bond of secoisolariciresinol diglucoside (SDG) to release secoisolariciresinol (SECO) was isolated from Aspergillus oryzae 39 strain and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 64.9 kDa. The optimum temperature of the SDG-β-d-glucosidase was 40 °C, and the optimum pH was 5.0. The SDG-β-d-glucosidase was stable at less than 65 °C, and pH 4.0–6.0. Ca²⁺, K⁺, Mg²⁺ and Na⁺ ions have no significant effect on enzyme activity, Zn²⁺ and Cu²⁺ ions have weakly effect on enzyme activity, but Fe³⁺ ion inhibits enzyme activity strongly. The K_m value of SDG-β-d-glucosidase was 0.14 mM for SDG.     

Purification and characterization of an extracellular β-D-xylosidase from Aspergillus carbonarius

The production of an extracellular -D-xylosidase (-D-xyloside xylohydrolase, EC 3.2.1.37) by four Aspergillus strains (A. carbonarius, A. nidulans, A. niger and A. oryzae) grown on wheat bran medium was compared. The highest amount of the enzyme was found in the culture of A. carbonarius. The -D-xylosidase from A. carbonarius was purified to homogeneity by a rapid procedure, using hydrophobic interaction chromatography, chromatofocusing and affinity chromatography. The purified enzyme possessed not only -D-xylosidase activity, but also -L-arabinosidase activity. Mixed substrate experiments revealed that a single active centre was responsible for the splitting of the corresponding synthetic substrates. The molecular weight of the purified enzyme proved to be 100,000 Da, as estimated by SDS–PAGE. The isoelectric point was at pH 4.4. The pH and temperature optima were 4.0 and 60 °C, respectively. The enzyme remained stable over a pH range of 3.5–6.5 and up to 50 °C for 30 min. The Michaelis constant for p-nitrophenyl -D-xyloside was 0.198 mM. Kinetic studies demonstrated that the lack of the C-5 hydroxylmethyl group and the configuration of the C-4 hydroxyl group on the pyranoside ring play an important role in both substrate binding and splitting.     

Isolation and characterization of β-glucosidases from Aspergillus nidulans mutant USDB 1183

Two extracellular -glucosidases (cellobiase, EC 3.2.1.21), I and II, from Aspergillus nidulans USDB 1183 were purified to homogeneity with molecular weights of 240,000 and 78,000, respectively. Both hydrolysed laminaribiose, -gentiobiose, cellobiose, p-nitrophenyl--L-glucoside, phenyl--L-glucoside, o-nitrophenyl--L-glucoside, salicin and methyl--L-glucoside but not -linked disaccharides. Both were competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-1,5-lactone. -Glucosidase I was more susceptible to inhibition by Ag⁺ and less inhibited by Fe²⁺ and Fe³⁺ than -glucosidase II.     

Purification and biochemical characterization of a novel α-glucosidase from Aspergillus niveus

An extracellular α-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS–PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical α-glucosidase activity, hydrolyzing p-nitrophenyl α-d-glucopyranoside and presented an optimum temperature and pH of 65°C and 6.0, respectively. In the absence of substrate the purified α-glucosidase was stable for 60 min at 60°C, presenting t ₅₀ of 90 min at 65°C. Hydrolysis of polysaccharide substrates by α-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and β-ciclodextrin were poor substrates, and sucrose and α-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an α-helical content of 31% and a β-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.     

Purification and characterization of a novel β-glucosidase from Aspergillus flavus and its application in saccharification of soybean meal

Abstract

Aspergillus flavus has been regarded as a potential candidate for its production of industrial enzymes, but the details of β-glucosidase from this strain is very limited. In herein, we first reported a novel β-glucosidase (AfBglA) with the molecular mass of 94.2?kDa from A. flavus. AfBglA was optimally active at pH 4.5 and 60?°C and is stable between pH 3.5 and 9.0 and at a temperature of up to 55?°C for 30?min remaining more than 90% of its initial activity. It showed an excellent tolerance to Trypsin, Pepsin, Compound Protease, and Flavourzyme and its activity was not inhibited by specific certain cations. AfBglA displayed broad substrate specificity, it acted on all tested pNP-glycosides and barley glucan, indicating this novel β-glucosidase exhibited a β-1, 3-1, 4-glucanase activity. Moreover, the AfBglA could effectively hydrolyze the soybean meal suspension into glucose and exhibit a strong tolerance to the inhibition of glucose at a concentration of 20.0?g/L during the saccharification. The maximum amount of the glucose obtained by AfBglA corresponded to 67.0?g/kg soybean meal. All of these properties mentioned above indicated that the AfBglA possibly attractive for food and feed industry and saccharification of cellulolytic materials.     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.     

Optimization of β-glucosidase production from Aspergillus niger

本研究对Aspergillus niger Glu05生产β-葡萄糖苷酶的培养基组分及培养条件进行了优化.优化后的培养基组成和培养条件分别为:麸皮4％,tryptone 4％,1μmol MnSO4,1μmol NaCl,KH2PO40.2％,oH自然,摇床转速250 r/min,培养温度30℃,培养周期5d.优化后发酵液中酶活力达到44.11 IU/mL,与初始的产酶水平32.87 IU/mL相比,提高了36％.     

Biochemical properties of a native β-1,4-mannanase from Aspergillus aculeatus QH1 and partial characterization of its N-glycosylation

N-glycosylation plays critical roles in protein secretion, sorting, stability, activity modulation, and interactions to other molecules in the eukaryotic organisms. Fungal β-1,4-mannanases have been widely used in the agri-food industry and contribute to the pathogenesis on plants. However, the information on N-glycosylation of a specific fungal carbohydrate-active enzyme (CAZyme) is currently limited. Herein, a cDNA was cloned from Aspergillus aculeatus QH1, displaying a full length of 1302 bp with an open reading frame of 1134 bp encoding for a GH5 subfamily 7 β-1, 4-mannanase, namely AacMan5_7A. The enzyme was purified and exhibited an optimal activity at pH 4.6 and 60 °C, hydrolyzing glucomannan and galactomannan, but not yeast mannan. AacMan5_7A is an N-glycosylated protein decorated with a high-mannose type glycan. Further through UPLC-ESI-MS/MS analysis, one of the four predicted N-glycosylation sites at N255 position was experimentally verified. The present study expands the information of N-glycosylation in fungal CAZymes, providing scientific bases for enhancing the production of fungal enzymes and their applications in food, feed, and plant biomass conversions.     

Characterization of a β-xylosidase from Aspergillus terreus (IJIRA 6.2)

Aspergillus terreus (IJIRA 6.2), a common soil microorganism, produces an extracellular β-xylosidase during its growth on wheat bran. The enzyme has been purified 328 fold (with a sp act of 4233 units/mg protein) by chromatography on DEAE-Sephadex A-25, hydroxyapatite, ConA-Sepharose and gel filtration on Sephacryl-S-300. Molecular mass of β-xylosidase by gel filtration was estimated to be about 95,000 and sedimentation coefficient of 5.6S was determined by glycerol density gradient centrifugation. The enzyme displayed maximum activity at pH 5.0 and 40°C; and in the absence of substrate, the β-xylosidase was stable up to 50°C and between pH 4.5 to 6.5. The purified enzyme hydrolysed p-nitrophenyl-β-Dxylopyranoside (PNPX) and xylooligosaccharides but not xylan, carboxymethyl cellulose or cellobiose. With PNPX as the substrate, the purified β-xylosidase exhibited a Km of 1.0 mM and D(+) xylose served as a competitive inhibitor with a K₁ of 10.5 mM.     

Purification and characterization of β-galactosidase from a strain of Bacillus coagulans

-Galactosidase from B. coagulans strain L4 is produced constitutively, has a mol. wt. of 4.3×10⁵ and an optimal temperature of 55°C. The optimal pH at 30°C is 6.0 whereas at 55°C it is 6.5. The energy of activation of enzyme activity is 41.9 kJ/mol (10 kcal/mol). No cations are required. The K_m with ONPG as substrate is 4.2–5.6mm and with lactose is 50mm. The K_i for inhibition by galactose is 11.7–13.4mm and for dextrose is 50mm. Galactose inhibited competitively while dextrose inhibited noncompetitively. The purified and unprotected enzyme is 70% destroyed in 30 min at 55°C whereas in the presence of 2 mg/ml of BSA 42% of the activity is destroyed in 30 min at 55°C. An overall purification of 75.3-fold was achieved.     

Purification and characterization of α-glucosidase from Aspergillus carbonarious

Summary An -glucosidase was purified from Aspergillus carbonarious CCRC 30414 over 20 fold with 37 % recovery. Its molecular mass was estimated to be 328 kDa by gel filtration with an optimum pH from 4.2 to 5.0, and pI=5.0. The optimum temperature is at 60°C over 40 min. The enzyme was partially inhibited by 5 mM Ag⁺, Hg²⁺, Ba²⁺, Pb²⁺, and Aso₄ ⁺.     

Molecular characterization of β-thalassemia intermedia: a report from Iran

Thalassemia intermedia is a clinical definition applied to patients whose clinical phenotype is milder than thalassemia major. To characterize different common mechanisms involving in pathogenesis of moderate to severe β-thalassemia intermedia, we have studied four factors in 38 Iranian patients with thalassemia intermedia: β-globin gene mutation, deletion in α-globin genes, presence of XmnI polymprphism and RFLP haplotype at β-globin gene cluster. The results showed that 84.4% of patients were associated with severe mutations in β-globin gene, mainly IVSII-1(G to A) (56.4%). The positive XmnI polymorphism was seen in 76.9% of the studied alleles which showed strong linkage to β° mutations and high level of fetal hemoglobin. Co-existence of α-globin gene deletions, β⁺ mutation and the most frequent of RFLP haplotype (−/−, +/+, −/+, +/+, +/+, +/+, −/−) were seen in 7.7, 12.8 and 17.9%, respectively. In this group of our study it seems the main ameliorating factor in the patients was co-inheritance of a positive XmnI polymorphism with β° mutation especially IVSII-1, which were associated with increased production of fetal hemoglobin. However, the other probable genetic factors should be investigated to describe genotype-phenotype correlation in thalassemia intermedia patients.     

Mixed inhibition of β-d-glucosidase from Stachybotrys atra by substrate analogues

The binding of series of alkyl and aryl β-d-glycopyranosides and their 1-thio analogues to the active site of β-d-glucosidase from Stachybotrys atra has been investigated. The binding constants for competitive and uncompetitive inhibition clearly demonstrated the existence of a hydrophobic aglycon-binding-site. The correlations found between competitive and uncompetitive inhibition suggest that the latter type of inhibition originates from the unspecific binding of the aglycon group to the aglycon binding-site of the intermediary enzyme-glycosyl complex.