The Characterization of the Endoglucanase Cel12A from Gloeophyllum trabeum Reveals an Enzyme Highly Active on β-Glucan

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50°C on β-glucan. Under these conditions specific activity was 239.2±9.1 U mg⁻¹ and the half-life of the enzyme was 84.6±3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the K_m was 3.2±0.5 mg mL⁻¹ and the V_max was 0.41±0.02 µmol min⁻¹. Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.     

Effects of a non-conservative sequence on the properties of β-glucuronidase from Aspergillus terreus Li-20

We cloned the β-glucuronidase gene (AtGUS) from Aspergillus terreus Li-20 encoding 657 amino acids (aa), which can transform glycyrrhizin into glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). Based on sequence alignment, the C-terminal non-conservative sequence showed low identity with those of other species; thus, the partial sequence AtGUS(-3t) (1-592 aa) was amplified to determine the effects of the non-conservative sequence on the enzymatic properties. AtGUS and AtGUS(-3t) were expressed in E. coli BL21, producing AtGUS-E and AtGUS(-3t)-E, respectively. At the similar optimum temperature (55°C) and pH (AtGUS-E, 6.6; AtGUS(-3t)-E, 7.0) conditions, the thermal stability of AtGUS(-3t)-E was enhanced at 65°C, and the metal ions Co(2+), Ca(2+) and Ni(2+) showed opposite effects on AtGUS-E and AtGUS(-3t)-E, respectively. Furthermore, Km of AtGUS(-3t)-E (1.95 mM) was just nearly one-seventh that of AtGUS-E (12.9 mM), whereas the catalytic efficiency of AtGUS(-3t)-E was 3.2 fold higher than that of AtGUS-E (7.16 vs. 2.24 mM s(-1)), revealing that the truncation of non-conservative sequence can significantly improve the catalytic efficiency of AtGUS. Conformational analysis illustrated significant difference in the secondary structure between AtGUS-E and AtGUS(-3t)-E by circular dichroism (CD). The results showed that the truncation of the non-conservative sequence could preferably alter and influence the stability and catalytic efficiency of enzyme.     

Purification,Characterization, and Substrate Specificity of a Novel Highly Glucose-Tolerant β-Glucosidase from Aspergillus oryzae

Aspergillus oryzae was found to secrete two distinct β-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3′,4′,5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total β-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant β-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-β-d-glucoside as the substrate, we found that the enzyme was optimally active at 50°C and pH 5.0 and had a specific activity of 1,066 μmol min⁻¹ mg of protein⁻¹ and a K_m of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (K_i, 1.36 M) or glucono-δ-lactone (K_i, 12.5 mM), another powerful β-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal β-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1→3)- and (1→6)-β-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-β-d-glucosides in a grape must (pH 2.9, 90 g of glucose liter⁻¹). Other flavor precursors (benzyl- and 2-phenylethyl-β-d-glucosides) and prunin (4′,5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel β-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.β-Glucoside glucohydrolases, commonly called β-glucosidases, catalyze the hydrolysis of alkyl- and aryl-β-glucosides, as well as diglucosides and oligosaccharides. These enzymes are widely used in various biotechnological processes, including the production of fuel ethanol from cellulosic agricultural residues (4, 27, 48) and the synthesis of useful β-glucosides (21, 38). In the flavor industry, β-glucosidases are also key enzymes in the enzymatic release of aromatic compounds from glucosidic precursors present in fruits and fermentating products (13, 39). Indeed, many natural flavor compounds, such as monoterpenols, C-13 norisoprenoids, and shikimate-derived compounds, accumulate in fruits as flavorless precursors linked to mono- or diglycosides and require enzymatic or acidic hydrolysis for the liberation of their fragrances (41, 45). Finally, β-glucosidases can also improve the organoleptic properties of citrus fruit juices, in which the bitterness is in part due to a glucosidic compound, naringin (4′,5,7-trihydroxyflavanone-7-rhamnoglucoside), whose hydrolysis requires, in succession, an α-rhamnosidase and a β-glucosidase (33).It is now well-established that certain monoterpenols of grapes (e.g., linalol, geraniol, nerol, citronelol, α-terpineol, and linalol oxide), which are linked to diglycosides, such as 6-O-α-l-rhamnopyranosyl-, 6-O-α-l-arabinofuranosyl-, and 6-O-β-d-apiofuranosyl-β-d-glucosides, contribute significantly to the flavor of wine (15, 44). The enzymatic hydrolysis of these compounds requires a sequential reaction; first, an α-l-rhamnosidase, an α-l-arabinofuranosidase, or a β-d-apiofuranosidase cleaves the (1→6) osidic linkage, and then, the flavor compounds are liberated from the monoglucosides by the action of a β-glucosidase (18, 19). Unlike acidic hydrolysis, enzymatic hydrolysis is highly efficient and does not result in modifications of the aromatic character (16). However, grape and yeast glucosidases exhibit limited activity on monoterpenyl-glucosides during winemaking, and a large fraction of the aromatic precursors remains unprocessed (9, 16, 35). The addition of exogenous β-glucosidase during or following fermentation has been found to be the most effective way to improve the hydrolysis of the glycoconjugated aroma compounds in order to enhance wine flavor (2, 14, 39, 40). The ideal β-glucosidase should function and be stable at a low pH value (pH 2.5 to 3.8) and should be active at a high concentration of glucose (10 to 20%) and in the presence of 10 to 15% ethanol. However, most microbial β-glucosidases are very sensitive to glucose inhibition (4, 12, 47), as well as to inhibition by glucono-δ-lactone, another powerful β-glucosidase inhibitor produced by grape-attacking fungi which can be found in wine must at concentrations up to 2 g/liter (10).The need for more suitable enzymes has led us and other workers to search for novel β-glucosidases with the desired properties. Recently, we showed that an extracellular glucose-tolerant and pH-stable β-glucosidase can be produced by Aspergillus strains (17). However, the enzyme of interest represented only a minor fraction of total β-glucosidase activity, and the major form was highly sensitive to glucose inhibition. Aspergillus oryzae appeared to be the best producer of the minor form when it was grown on quercetin (3,3′,4′,5,7-pentahydroxyflavone), a phenolic flavonoid found in plant cell walls. This paper presents further data on the production and characterization of this novel highly glucose-tolerant β-glucosidase (designated HGT-BG) purified from the extracellular culture filtrate of A. oryzae grown on quercetin.     

Effects of galactose and glucose on the hydrolysis reaction of a thermostable β-galactosidase from Caldicellulosiruptor saccharolyticus

A recombinant β-galactosidase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 211 U mg^?1 by using heat treatment and His-trap affinity chromatography. The native enzyme was an 80-kDa trimer with a molecular mass of 240 kDa. Maximum activity was observed at pH 6.0 and 80ºC, and the half-life at 70ºC was 48 h. The enzyme exhibited hydrolytic activity for p-nitrophenyl-β-d-galactopyranoside (pNPGal), oNPGal, or lactose, whereas no activity for p-nitrophenyl-β-d-glucopyranoside (pNPGlu), oNPGlu, or cellobiose. The catalytic residues E150 and E311 of β-galactosidase from C. saccharolyticus were completely conserved in all aligned glycoside hydrolase family 42 β-galactosidases. The results indicated that the enzyme was a β-galactosidase. Galactose uncompetitively inhibited the enzyme. Glucose inhibition of the enzyme was the lowest among β-galactosidases. When 50 g l^?1 galactose was added, the enzyme activity for pNPGal was reduced to 26%. When 400 g l^?1 glucose instead of galactose was added, the activity was reduced to 82%. When adding galactose (200 g l^?1), only 14% of the lactose was hydrolyzed after 180 min. In contrast, the addition of glucose (400 g l^?1) did not affect lactose hydrolysis, and more than 99% of the lactose was hydrolyzed after 120 min.     

The Synergistic Effects among β-,3-Glucanases from Oerskovia sp. CK on Lysis of Viable Yeast Cells

Oerskovia sp. CK produced three types of β-1,3-glucanases designated as F-L, F-0 and F-2. F-L showed high lytic activity to viable yeast cells and weak activity to yeast glucan. F-0 and F-2 had little or no lytic activity and strong β-1,3-gIucanase activity.

F-0 or F-2 showed high lytic activities to yeast cells pretreated with small amounts of F-L which did not lysed the cells. Lytic activity of F-0 or F-2 also increased when cells were treated with alkaline pH or with both reducing agents and pH.

From these results, it is supposed that the ineffectiveness of F-0 or F-2 on the lysis of yeast cells might be attributed to a spatial inaccessibility of enzymes to the yeast glucan layer. However, the treatment of F-L, alkaline pH and reducing agents would bring about a modification of cells to give F-0 or F-2 access to the wall glucan and consequently the lysis of cells would occur.     

Effects of Secondary Metabolite Extract from Phomopsis occulta on β-Amyloid Aggregation

Inhibition of β-amyloid (Aβ) aggregation is an attractive therapeutic and preventive strategy for the discovery of disease-modifying agents in Alzheimer''s disease (AD). Phomopsis occulta is a new, salt-tolerant fungus isolated from mangrove Pongamia pinnata (L.) Pierre. We report here the inhibitory effects of secondary metabolites from Ph. occulta on the aggregation of Aβ42. It was found that mycelia extracts (MEs) from Ph. occulta cultured with 0, 2, and 3 M NaCl exhibited inhibitory activity in an E. coli model of Aβ aggregation. A water-soluble fraction, ME0-W-F1, composed of mainly small peptides, was able to reduce aggregation of an Aβ42-EGFP fusion protein and an early onset familial mutation Aβ42E22G-mCherry fusion protein in transfected HEK293 cells. ME0-W-F1 also antagonized the cytotoxicity of Aβ42 in the neural cell line SH-SY5Y in dose-dependent manner. Moreover, SDS-PAGE and FT-IR analysis confirmed an inhibitory effect of ME0-W-F1 on the aggregation of Aβ42 in vitro. ME0-W-F1 blocked the conformational transition of Aβ42 from α-helix/random coil to β-sheet, and thereby inhibited formation of Aβ42 tetramers and high molecular weight oligomers. ME0-W-F1 and other water-soluble secondary metabolites from Ph. occulta therefore represent new candidate natural products against aggregation of Aβ42, and illustrate the potential of salt tolerant fungi from mangrove as resources for the treatment of AD and other diseases.     

Characterization of a Highly Purified, Fully Active, Crystallizable RC–LH1–PufX Core Complex from Rhodobacter sphaeroides

Photosynthetic complexes in bacteria absorb light and undergo photochemistry with high quantum efficiency. We describe the isolation of a highly purified, active, reaction center-light-harvesting 1–PufX complex (RC–LH1–PufX core complex) from a strain of the photosynthetic bacterium, Rhodobacter sphaeroides, which lacks the light-harvesting 2 (LH2) and contains a 6 histidine tag on the H subunit of the RC. The complex was solubilized with diheptanoyl-sn-glycero-3-phosphocholine (DHPC), and purified by Ni-affinity, size-exclusion and ion-exchange chromatography in dodecyl maltoside. SDS-PAGE analysis shows the complex to be highly purified. The quantum efficiency was determined by measuring the charge separation (DQ_A → D⁺Q_A^-) in the RC as a function of light intensity. The RC–LH1–PufX complex had a quantum efficiency of 0.95 ± 0.05, indicating full activity. The stoichiometry of LH1 subunits per RC was determined by two independent methods: (i) solvent extraction and absorbance spectroscopy of bacteriochlorophyll, and (ii) density scanning of the SDS-PAGE bands. The average stoichiometry from the two measurements was 13.3 ± 0.9 LH1/RC. The presence of PufX was observed in SDS-PAGE gels at a stoichiometry of 1.1 ± 0.1/RC. Crystals of the core complex have been obtained which diffract X-rays to 12 Å. A preliminary analysis of the space group and unit cell analysis indicated a P1 space group with unit cell dimensions of a = 76.3 Å, b = 137.2 Å, c = 137.5 Å; α = 60.0°, β = 89.95°, γ =90.02°.     

The β-Glucanase ZgLamA from Zobellia galactanivorans Evolved a Bent Active Site Adapted for Efficient Degradation of Algal Laminarin

Laminarinase is commonly used to describe β-1,3-glucanases widespread throughout Archaea, bacteria, and several eukaryotic lineages. Some β-1,3-glucanases have already been structurally and biochemically characterized, but very few from organisms that are in contact with genuine laminarin, the storage polysaccharide of brown algae. Here we report the heterologous expression and subsequent biochemical and structural characterization of ZgLamA_GH16 from Zobellia galactanivorans, the first GH16 laminarinase from a marine bacterium associated with seaweeds. ZgLamA_GH16 contains a unique additional loop, compared with other GH16 laminarinases, which is composed of 17 amino acids and gives a bent shape to the active site cleft of the enzyme. This particular topology is perfectly adapted to the U-shaped conformation of laminarin chains in solution and thus explains the predominant specificity of ZgLamA_GH16 for this substrate. The three-dimensional structure of the enzyme and two enzyme-substrate complexes, one with laminaritetraose and the other with a trisaccharide of 1,3–1,4-β-d-glucan, have been determined at 1.5, 1.35, and 1.13 Å resolution, respectively. The structural comparison of substrate recognition pattern between these complexes allows the proposition that ZgLamA_GH16 likely diverged from an ancestral broad specificity GH16 β-glucanase and evolved toward a bent active site topology adapted to efficient degradation of algal laminarin.