Klotho-related protein is a novel cytosolic neutral beta-glycosylceramidase

Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the activity of a conduritol B epoxide-insensitive neutral glycosylceramidase in cytosolic fractions of zebrafish embryos, mouse and rat brains, and human fibroblasts. The candidates for the enzyme were assigned to the Klotho (KL), whose family members share a beta-glucosidase-like domain but whose natural substrates are unknown. Among this family, only the KL-related protein (KLrP) is capable of degrading C6-NBD-GlcCer when expressed in CHOP cells, in which Myc-tagged KLrP was exclusively distributed in the cytosol. In addition, knockdown of the endogenous KLrP by small interfering RNA increased the cellular level of GlcCer. The purified recombinant KLrP hydrolyzed 4-methylumbelliferyl-glucose, C6-NBD-GlcCer, and authentic GlcCer at pH 6.0. The enzyme also hydrolyzed the corresponding galactosyl derivatives, but each k(cat)/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6A resolution revealed that KLrP is a (beta/alpha)8 TIM barrel, in which Glu(165) and Glu(373) at the carboxyl termini of beta-strands 4 and 7 could function as an acid/base catalyst and nucleophile, respectively. The substrate-binding cleft of the enzyme was occupied with palmitic acid and oleic acid when the recombinant protein was crystallized in a complex with glucose. GlcCer was found to fit well the cleft of the crystal structure of KLrP. Collectively, KLrP was identified as a cytosolic neutral glycosylceramidase that could be involved in a novel nonlysosomal catabolic pathway of GlcCer.     

Extracellular expression of glucose inhibition-resistant Cellulomonas flavigena PN-120 β-glucosidase by a diploid strain of Saccharomyces cerevisiae

The catalytic fraction of the Cellulomonas flavigena PN-120 oligomeric β-glucosidase (BGLA) was expressed both intra- and extracellularly in a recombinant diploid of Saccharomyces cerevisiae, under limited nutrient conditions. The recombinant enzyme (BGLA¹⁵) expressed in the supernatant of a rich medium showed 582 IU/L and 99.4 IU/g dry cell, with p-nitrophenyl-β-d-glucopyranoside as substrate. BGLA¹⁵ displayed activity against cello-oligosaccharides with 2–5 glucose monomers, demonstrating that the protein is not specific for cellobiose and that the oligomeric structure is not essential for β-d-1,4-bond hydrolysis. Native β-glucosidase is inhibited almost completely at 160 mM glucose, thus limiting cellobiose hydrolysis. At 200 mM glucose concentration, BGLA¹⁵ retained more than 50 % of its maximal activity, and even at 500 mM glucose concentration, more than 30 % of its activity was preserved. Due to these characteristics of BGLA¹⁵ activity, recombinant S. cerevisiae is able to utilize cellulosic materials (cello-oligosaccharides) to produce bioethanol.     

A novel high molecular weight thermo-acidoactive β-glucosidase from <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

An extracellular high molecular weight β-glucosidase was secreted by a local strain P1 of Beauveria bassiana. The enzyme was produced in the presence of various carbon sources, namely glucose, maltose, lactose, glycerol, starch, wheat bran and gruel. The highest level of β-glucosidase activity was produced with wheat bran at the concentration of 3%. Glucose caused a repressor effect on the β-glucosidase expression in a dose-dependent manner. The highest enzyme production level was obtained at initial pH of 6.0 and 7.0 in the culture medium. The zymography analysis revealed that B. bassiana secreted a β-glucosidase with high molecular weight between 400 and 600 kDa. The enzymatic preparation was characterized and showed temperature and pH optima of 55°C and 5.0, respectively. The enzyme was stable at 40 and 50°C but its stability declined at 60°C. Interestingly, this β-glucosidase had high stability at acid and basic pH saving its initial activity after 24 h incubation at pH from 3.0 to 11.0. It was stable also in presence of monovalent Na⁺ and K⁺ ions saving 60% of its initial activity at 2 M salts. Bivalent metal ions preserved totally or partially the enzymatic activity; in addition, Ba²⁺ was revealed as an activator. This is the first report that focuses on the production and the biochemical characterization of a β-glucosidase from the entomopathogenic fungus, B. bassiana.     

Cellobiose metabolism and cellobiohydrolase I biosynthesis by Trichoderma reesei

The relationship between β-linked disaccharide (cellobiose, sophorose) utilization and cellulase, particularly cellobiohydrolase I (CBH I) synthesis by Trichoderma reesei, was investigated. During growth on cellobiose and sophorose as carbon sources in batch as well as resting-cell culture, only sophorose induced cellulase formation. In the latter experiments, sophorose was utilized at a much lower rate than cellobiose, and the more cellulase produced, the lower its rate of utilization. Cellobiose and sophorose were utilized by the fungus mainly via hydrolysis by the cell wall- and cell membrane-bound β-glucosidase. Addition of sophorose to T. reesei growing on cellulose did not further stimulate cellulase synthesis, and addition of cellobiose was inhibitory. Cellobiose, however, promoted cellulase formation in both batch and resting cell cultures, when its hydrolysis by β-glucosidase was inhibited by nojirimycin. No cellulase formation was observed when the uptake of glucose (produced from cellobiose by β-glucosidase) was inhibited by 3-O-methylglucoside. Cellodextrins (C₂ to C₆) promoted formation of low levels of cellobiohydrolase I in indirect proportion to their rate of hydrolysis by β-glucosidase. Studies on the uptake of [³H]cellobiose, [³H]sophorose, and [¹⁴C]glucose in the presence of inhibitors of β-glucosidase (nojirimycin) and glucose transport (3-O-methylglucoside) show that glucose transport occurs at a much higher rate than disaccharide hydrolysis. Extracellular disaccharide hydrolysis accounts for at least 95% of their metabolism. The presence of an uptake system for cellobiose was established by demonstrating the presence of intracellular labeled [³H]cellobiose in T. reesei after its extracellular supply. The data are consistent with induction of cellulase and particularly CBH I formation in T. reesei by β-linked disaccharides under conditions where their uptake is favored at the expense of extracellular hydrolysis.     

Characterization of a membrane bound β-glucosidase responsible for the activation of oat leaf saponins

Oat leaves contain a β-glucosidase (= avenacosidase) specific for the cleavage of the C-26 bound glucose moiety of the oat saponins avenacosides A and B. This transformation activates the fungitoxicities of the avenacosides. Evidence is presented that this enzyme is bound to the tonoplast membrane. The solubilized enzyme showed a pH optimum of 6.0–7.0, a temperature optimum around 40°, a molecular weight of 68 000±3000 and a K_m of 183 (±16) μM. The enzyme is inhibited by Hg²⁺ (10^-2 M) but not by Cu²⁺ (10^-2 M).     

Coupling of glucose oxidase and Fenton's reaction for a simple and inexpensive assay of β-glucosidase

A simple and inexpensive assay for β-glucosidase, based on the coupling of glucose oxidase and Fenton's reagent has been described. Hydrogen peroxide formed as a result of the action of glucose oxidase on glucose (derived from the action of β-glucosidase on cellobiose) oxidizes ferrous sulphate, resulting in an increase in absorbance. The oxidation products produced a peak of maximum absorbance at 340 nm. Using this assay system, a linear relationship between glucose concentration in the range 5.55–27.78 mmol l^?1(100–500 mg dl^?1) and absorbance was obtained, indicating conformity to Beer's law. The preciseness of the glucose oxidase/Fenton's reagent for the assay of glucose was shown to be satisfactory. β-Glucosidase was assayed using the hexokinase assay reagent and the glucose oxidase/ferrous sulphate reagent. The values obtained using both reagents did not differ significantly. Although 2.6 times less sensitive than the hexokinase reagent when absorbance is measured at 340 nm, the glucose oxidase/Fenton's reagent is 10 times cheaper and could be used satisfactorily for routine assays of β-glucosidase and other carbohydrases including cellulase and amylase. In this respect, fructose, mannose, xylose, sucrose and cellobiose did not affect the sensitivity of the reagent. Of several metals tested, only aluminium interfered with the reagent, decreasing its sensitivity.     

Immobilization of β-glucosidase from Scytalidium lignicola on Chitosan

Chitosan was found to be a better support than alginate beads for immobilization of β-glucosidase from Scytalidium lignicola. The optimum concentration of glutaraldehyde for enzyme immobilization was 0.2%. Immobolized β-glucosidase was more able in the pH range of 3–6. Immobilized β-glucosidase retained about 70% of its activity at 50%C after 72 h of incubation while free enzyme lost most of its activity. The log of activity retained vs time was a straight line with free enzyme but was curved for immnobilized enzyme. Lineweaver-Burk plots of free and immoblized β-glucosidase gave K_m values of 2 × 10⁻⁴ M and 5.5 × 10⁻⁴ M for p-nitrophenyl β-d-glucopyranoside, respectively. Addition of immobilized β-glucosidase to a saccharification system gave a 30% increase in reducing sugar availability compared to free enzyme addition and was at least 4 times reusable without appreciable loss in enzyme activity.     

The role of the oligosaccharide binding cleft of rice BGlu1 in hydrolysis of cellooligosaccharides and in their synthesis by rice BGlu1 glycosynthase

Rice BGlu1 β-glucosidase nucleophile mutant E386G is a glycosynthase that can synthesize p-nitrophenyl (pNP)-cellooligosaccharides of up to 11 residues. The X-ray crystal structures of the E386G glycosynthase with and without α-glucosyl fluoride were solved and the α-glucosyl fluoride complex was found to contain an ordered water molecule near the position of the nucleophile of the BGlu1 native structure, which is likely to stabilize the departing fluoride. The structures of E386G glycosynthase in complexes with cellotetraose and cellopentaose confirmed that the side chains of N245, S334, and Y341 interact with glucosyl residues in cellooligosaccharide binding subsites +2, +3, and +4. Mutants in which these residues were replaced in BGlu1 β-glucosidase hydrolyzed cellotetraose and cellopentaose with k(cat) /K(m) values similar to those of the wild type enzyme. However, the Y341A, Y341L, and N245V mutants of the E386G glycosynthase synthesize shorter pNP-cellooligosaccharides than do the E386G glycosynthase and its S334A mutant, suggesting that Y341 and N245 play important roles in the synthesis of long oligosaccharides. X-ray structural studies revealed that cellotetraose binds to the Y341A mutant of the glycosynthase in a very different, alternative mode not seen in complexes with the E386G glycosynthase, possibly explaining the similar hydrolysis, but poorer synthesis of longer oligosaccharides by Y341 mutants.     

Purification and Characterization of two Extracellular β-Glucosidases from Trichoderma viride ITCC 1433

T. viride ITCC 1433 synthesizes a two component system for the hydrolysis of cellobiose and cellooligodextrins. 80% of the total activity are solubilized during growth. The large protein (A), mol. weight 98 000 d, is glycosylated and slightly acidic (pH = 6.1). The smaller protein (B), mol. weight 70 000 d, is unglycosylated and neutral (pH = 7.2). Both proteins form a two-step system where β-glucosidase A is active at low substrate concentrations (K_M = 2.3 × 10^?4 M cellobiose) while β-glucosidase B covers the range of 10-fold higher cellobiose concentrations (K_M = 1.8 × 10^?3 M). The enzymes are fairly stable with a residual activity of 70% at 50°C after 24 h.     

Trans-glycosylation capacity of a highly glycosylated multi-specific β-glucosidase from Fusarium solani

An extracellular β-glucosidase from Fusaruim solani cultivated on wheat bran was purified by only two chromatographic steps. The purified enzyme exhibited optimal temperature and pH at 60 °C and pH 5, respectively. The purified β-glucosidase behaves as a very large protein due to its high degree of glycosylation. More interestingly, the endoglycosidase H (Endo H) treatment led to 97.55% loss of its initial activity after 24 h of treatment. Besides, the addition of Tunicamycin (nucleoside antibiotic blocking the N-glycosylation first step) during the culture of the fungus affected seriously the glycosylation of the enzyme. Both treatments (endo H and Tunicamycin) strengthened the idea that the hyperglycosylation is involved in the β-glucosidase activity and thermostability. This enzyme was also shown to belong to class III of β-glucosidases (multi-specific) since it was able to act on either cellobiose, gentiobiose or sophorose which are disaccharide composed of two units of d-glucose connected by β1–4, β1–6 and β1–2 linkage, respectively. The β-glucosidase activity was strongly enhanced by ferrous ion (Fe²⁺) and high ionic strength (1 M KCl). The purified enzyme exhibited an efficient transglycosylation capacity allowing the synthesis of cellotriose and cellotetraose using cellobiose as donor.

     

Fluorometric determination of carbohydrates

Fluorometric methods are described for the determination of the enzymes hexokinase and β-glucosidase, and the carbohydrates glucose, fructose, maltose, cellobiose, lactose, glycogen, and salicin Glucose and fructose are determined fluorometrically using hexokinase and the resazurin-resorufin indicator reaction. Maltose, cellobiose, lactose, glycogen, and salicin are enzymically hydrolyzed to glucose, which is then determined fluorometrically using glucose oxidase, p-hydroxyphenylacetic acid, and peroxidase. All the carbohydrates were assayed in the range 0.01–50 μg/ml and the enzymes hexokinase and β-glucosidase in the range 10^?3 to 10^?1 unit/ml with an accuracy and precision of about 1.5%.     

Cloning, Molecular Characterization, and mRNA Expression of the Thermostable Family 3 β-Glucosidase from the Rare Fungus Stachybotrys microspora

The filamentous fungus Stachybotrys microspora possess a rich β-glucosidase system composed of five β-glucosidases. Three of them were already purified to homogeneity and characterized. In order to isolate the β-glucosidase genes from S. microspora and study their regulation, a PCR strategy using consensus primers was used as a first step. This approach enabled the isolation of three different fragments of family 3 β-glucosidase gene. A representative genomic library was constructed and probed with one amplified fragment gene belonging to family 3 of β-glucosidase. After two rounds of hybridization, seven clones were obtained and the analysis of DNA plasmids leads to the isolation of one clone (CF3) with the largest insert of 7 kb. The regulatory region shows multiple TC-rich elements characteristic of constitutive promoter, explaining the expression of this gene under glucose condition, as shown by zymogram and RT-PCR analysis. The tertiary structure of the deduced amino acid sequence of Smbgl3 was predicted and has shown three conserved domains: an (α/β)₈ triose phosphate isomerase (TIM) barrel, (α/β)₅ sandwich, and fibronectin type III domain involved in protein thermostability. Zymogram analysis highlighted such thermostable character of this novel β-glucosidase.     

Enhanced Formation of β-Glucosidase by Aspergillus niger VKMF-2092 in Fed-Batch Operation with Frequently Intermittent Glucose Addition

The wild strain Aspergillus niger VKMF-2092 forms β-glucosidase on the basis of glucose as the sole carbon source. The formation of β-glucosidase is initiated after the glucose concentration in the medium has reached a low level since the formation is subjected to catabolite repression. The β-glucosidase is mainly cell-bound and only released after a longer period. Under non-repressed conditions the total formation of β-glucosidase is not associated with growth. By supplying glucose in a fed-batch-technique, maintaining a low actual glucose concentration in the medium, the formation of β-glucosidase is enhanced in comparison to simple batch fermentation. Using a fed-batch-technique with a frequently intermittent addition of glucose it is possible to increase the formation of β-glucosidase with regard to both productivity and the activity related to the mycelium and the fermentation broth as well. The increase of productivity is about two to four times greater than at constant feed rate of the same overall amount of glucose. The reason for this increase will be discussed below. A method is presented which permits to investigate the influence of the substrate concentration and other parameters on the enzyme formation in short periods of one and the same fermentation run.     

Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies

Solids resulting from pretreatment of corn stover by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) technologies were hydrolyzed by enzyme cocktails based on cellulase supplemented with β-glucosidase at an activity ratio of 1:2, respectively, and augmented with up to 11.0 g xylanase protein/g cellulase protein for combined cellulase and β-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose. It was found that glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments despite substantial differences in their relative yields. The ratio of the fraction of glucan removed by enzymes to that for xylose was defined as leverage and correlated statistically at two combined cellulase and β-glucosidase mass loadings with pretreatment type. However, no direct relationship was found between leverage and solid features following different pretreatments such as residual xylan or acetyl content. However, acetyl content not only affected how xylanase impacted cellulase action but also enhanced accessibility of cellulose and/or cellulase effectiveness, as determined by hydrolysis with purified CBHI (Cel7A). Statistical modeling showed that cellulose crystallinity, among the main substrate features, played a vital role in cellulase–xylanase interactions, and a mechanism is suggested to explain the incremental increase in glucose release with xylanase supplementation.     

G6P-capturing molecules in the periplasm of Escherichia coli accelerate the shikimate pathway

Escherichia coli, the most studied prokaryote, is an excellent host for producing valuable chemicals from renewable resources as it is easy to manipulate genetically. Since the periplasmic environment can be easily controlled externally, elucidating how the localization of specific proteins or small molecules in the periplasm affects metabolism may lead to bioproduction development using E. coli. We investigated metabolic changes and its mechanisms occurring when specific proteins are localized to the E. coli periplasm. We found that the periplasmic localization of β-glucosidase promoted the shikimate pathway involved in the synthesis of aromatic chemicals. The periplasmic localization of other proteins with an affinity for glucose-6-phosphate (G6P), such as inactivated mutants of Pgi, Zwf, and PhoA, similarly accelerated the shikimate pathway. Our results indicate that G6P is transported from the cytoplasm to the periplasm by the glucose transporter protein EIICB^Glc, and then captured by β-glucosidase.     

Isolation and Characterization of a β-Glucosidase from a Clavispora Strain with Potential Applications in Bioethanol Production from Cellulosic Materials

We previously reported on a new yeast strain of Clavispora sp. NRRL Y-50464 that is capable of utilizing cellobiose as sole source of carbon and energy by producing sufficient native β-glucosidase enzyme activity without further enzyme supplementation for cellulosic ethanol production using simultaneous saccharification and fermentation. Eliminating the addition of external β-glucosidase reduces the cost of cellulosic ethanol production. In this study, we present results on the isolation and identification of a β-glucosidase protein from strain Y-50464. Using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and blast search of the NCBInr database (National Center for Biotechnology Information nonredundant), the protein from Y-50464 was identified as a β-glucosidase (BGL1) with a molecular weight of 93.3 kDa. The BGL1 protein was purified through multiple chromatographic steps to a 26-fold purity (K _m?=?0.355 mM [pNPG]; K _i?=?15.2 mM [glucose]), which has a specific activity of 18.4 U/mg of protein with an optimal performance temperature at 45 °C and pH of 6.0. This protein appears to be intracellular although other forms of the enzyme may exist. The fast growth rate of Y-50464 and its capability to produce sufficient β-glucosidase activity for ethanol conversion from cellobiose provide a promising means for low-cost cellulosic ethanol production through a consolidated bioprocessing development.     

Enzymatic hydrolysis of cellulose to glucose lung immobilized β-glucosidase

The production of sugars by enzymatic hydrolysis of cellulose is a multistep process which includes conversion of the intermediate cellobiose to glucose by β-glucosidase. Aside from its role as an intermediate, cellobiose inhibits the endoglucanase components of typical cellulase enzyme systems. Because these enzyme systems often contain insufficient concentrations of β-glucosidase to prevent accumulation of inhibitory cellobiose, this research investigated the use of supplemental immobilized β-glucosidase to increase yield of glucose. Immobilized β-glucosidase from Aspergillus phoenicis was produced by sorption at controlled-pore alumina with about 90% activity retention. The product lost only about 10% of the original activity during an on-stream reaction period of 500 hr with cellobiose as substrate; maximum activity occurred near pH 3.5 and the apparent activation energy was about 11 kcal/mol. The immobilized β-glucosidase was used together with Trichoderma reesei cellulase to hydrolyze cellulosic materials, such as Solka Floc, corn stove and exploded wood. Increased yields of glucose and greater conversions of cellobiose of glucose were observed when the reaction systems contained supplemental immobilized β-glucosidase.     

Identification of a β-glucosidase from the Mucor circinelloides genome by peptide pattern recognition

Mucor circinelloides produces plant cell wall degrading enzymes that allow it to grow on complex polysaccharides. Although the genome of M. circinelloides has been sequenced, only few plant cell wall degrading enzymes are annotated in this species. We applied peptide pattern recognition, which is a non-alignment based method for sequence analysis to map conserved sequences in glycoside hydrolase families. The conserved sequences were used to identify similar genes in the M. circinelloides genome. We found 12 different novel genes encoding members of the GH3, GH5, GH9, GH16, GH38, GH47 and GH125 families in M. circinelloides. One of the two GH3-encoding genes was predicted to encode a β-glucosidase (EC 3.2.1.21). We expressed this gene in Pichia pastoris KM71H and found that the purified recombinant protein had relative high β-glucosidase activity (1.73 U/mg) at pH5 and 50 °C. The K_m and V_max with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.20 mM and 2.41 U/mg, respectively. The enzyme was not inhibited by glucose and retained 84% activity at glucose concentrations up to 140 mM. Although zygomycetes are not considered to be important degraders of lignocellulosic biomass in nature, the present finding of an active β-glucosidase in M. circinelloides demonstrates that enzymes from this group of fungi have a potential for cellulose degradation.     

Involvement of Ca2+ signalling in the sugar-inducible expression of genes coding for sporamin and β-amylase of sweet potato

Genes coding for sporamin and β-amylase of sweet potato are inducible not only by high levels of metabolizable sugars, such as sucrose, but also by a low concentration of polygalacturonic acid (PGA). Calmodulin inhibitors and EGTA inhibited both the PGA-inducible and the sucrose-inducible accumulation of mRNAs for sporamin and β-amylase in sweet potato. Calmodulin inhibitors, EGTA and La³⁺, also inhibited the sucrose-inducible expression, in leaves of transgenic tobacco, of a fusion gene, β-Amy:GUS, which consists of the promoter of the β-amylase gene and the coding sequence for β-glucuronidase. The sucrose-inducible expression of the β-Amy:GUS fusion gene was also inhibited by two inhibitors of Ca²⁺ channels, diltiazem and nicardipine. These results suggest that the sugar-inducible expression of genes for sporamin and β-amylase involves, at least in part, Ca²⁺-mediated signalling, and that the cytosolic free Ca²⁺ may mediate cross-talk between signals related to carbohydrate metabolism and other stimuli. Treatment of coelenterazine-loaded leaf discs of tobacco expressing a Ca²⁺-binding photoprotein, aequorin, with 0.2 M sucrose for 24 h significantly reduced the level of luminescence that could be induced by cold shock, as compared to cold shock-induced luminescence in coelenterazine-loaded leaf discs treated with water. Repression of cold shock-induced luminescence was due to the conversion of holoaequorin to apoaequorin during the treatment with sucrose. Treatment of coelenterazine-loaded leaf discs with a 0.2 M solution of glucose or fructose, but not of mannitol or sorbitol, also reduced the cold shock-induced luminescence. It is suggested that non-synchronous increases in cytosolic level of free Ca²⁺ occur in leaf discs during treatment with high levels of metabolizable sugars.     

A highly glucose-tolerant GH1 β-glucosidase with greater conversion rate of soybean isoflavones in monogastric animals

In the feed industry, β-glucosidase has been widely used in the conversion of inactive and bounded soybean isoflavones into active aglycones. However, the conversion is frequently inhibited by the high concentration of intestinal glucose in monogastric animals. In this study, a GH1 β-glucosidase (AsBG1) with high specific activity, thermostability and glucose tolerance (IC₅₀ = 800 mM) was identified. It showed great glucose tolerance against substrates with hydrophobic aryl ligands (such as pNPG and soy isoflavones). Using soybean meal as the substrate, AsBG1 exhibited higher hydrolysis efficiency than the GH3 counterpart Bgl3A with or without the presence of glucose in the reaction system. Furthermore, it is the first time to find that the endogenous β-glucosidase of soybean meal, mostly belonging to GH3, plays a role in the hydrolysis of soybean isoflavones and is highly sensitive to glucose. These findings lead to a conclusion that the GH1 rather than GH3 β-glucosidase has prosperous application advantages in the conversion of soybean isoflavones in the feed industry.