The celA Gene, Encoding a Glycosyl Hydrolase Family 3 β-Glucosidase in Azospirillum irakense, Is Required for Optimal Growth on Cellobiosides

The CelA β-glucosidase of Azospirillum irakense, belonging to glycosyl hydrolase family 3 (GHF3), preferentially hydrolyzes cellobiose and releases glucose units from the C₃, C₄, and C₅ oligosaccharides. The growth of a ΔcelA mutant on these cellobiosides was affected. In A. irakense, the GHF3 β-glucosidases appear to be functional alternatives for the GHF1 β-glucosidases in the assimilation of β-glucosides by other bacteria.     

Degradation of Native Starch Granules by Barley alpha-Glucosidases

The initial hydrolysis of native (unboiled) starch granules in germinating cereal kernels is considered to be due to α-amylases. We report that barley (Hordeum vulgare L.) seed α-glucosidases (EC 3.2.1.20) can hydrolyze native starch granules isolated from barley kernels and can do so at rates comparable to those of the predominant α-amylase isozymes. Two α-glucosidase charge isoforms were used individually and in combination with purified barley α-amylases to study in vitro starch digestion. Dramatic synergism, as much as 10.7-fold, of native starch granule hydrolysis, as determined by reducing sugar production, occurred when high pl α-glucosidase was combined with either high or low pl α-amylase. Synergism was also found when low pl α-glucosidase was combined with α-amylases. Scanning electron micrographs revealed that starch granule degradation by α-amylases alone occurred specifically at the equatorial grooves of lenticular granules. Granules hydrolyzed by combinations of α-glucosidases and α-amylases exhibited larger and more numerous holes on granule surfaces than did those granules attacked by α-amylase alone. As the presence of α-glucosidases resulted in more areas being susceptible to hydrolysis, we propose that this synergism is due, in part, to the ability of the α-glucosidases to hydrolyze glucosidic bonds other than α-1,4- and α-1,6- that are present at the granule surface, thereby eliminating bonds which were barriers to hydrolysis by α-amylases. Since both α-glucosidase and α-amylase are synthesized in aleurone cells during germination and secreted to the endosperm, the synergism documented here may function in vivo as well as in vitro.     

Novel α-Glucosidase from Aspergillus nidulans with Strong Transglycosylation Activity

Aspergillus nidulans possessed an α-glucosidase with strong transglycosylation activity. The enzyme, designated α-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to α-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other α-glucosidases. We propose here that AgdB is a novel α-glucosidase with unusually strong transglycosylation activity.     

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.     

Phanerochaete chrysosporium β-Glucosidases: Induction, Cellular Localization, and Physical Characterization

Phanerochaete chrysosporium produces intracellular soluble and particulate β-glucosidases and an extracellular β-glucosidase. The extracellular enzyme is induced by cellulose but repressed in the presence of glucose. The molecular weight of this enzyme is 90,000. The K_m for p-nitrophenyl-β-glucoside is 1.6 × 10⁻⁴ M; the K_i for glucose, a competitive inhibitor, is 5.0 × 10⁻⁴ M. The K_m for cellobiose is 5.3 × 10⁻⁴ M. The intracellular soluble enzyme is induced by cellobiose; this induction is prevented by cycloheximide. The presence of 300 mM glucose in the medium, however, had no effect on induction. The K_m for p-nitrophenyl-β-glucoside is 1.1 × 10⁻⁴ M. The molecular weight of this enzyme is ~410,000. Both enzymes have an optimal temperature of 45°C and an E_act of 9.15 kcal (ca. 3.83 × 10⁴ J). The pH optima, however, were ~7.0 and 5.5 for the intracellular and extracellular enzymes, respectively.     

Purification of an acid α-glucosidase by dextran-gel filtration

1. The behaviour of rat liver α-glucosidases on dextran gel (Sephadex G-100) columns was studied. A `retardation' of an acid α-glucosidase activity was observed. This activity was identified as lysosome α-(1→4)-glucosidase. A single gel-filtration step resulted in a 700-fold purification of the enzyme. The same technique was also used to purify the acid α-glucosidase of human kidney. 2. The acid α-glucosidases of both tissues show very similar pH optima when tested with maltose or glycogen as substrate.     

Endoplasmic reticulum alpha-glycosidases of Candida albicans are required for N glycosylation, cell wall integrity, and normal host-fungus interaction

The cell surface of Candida albicans is enriched in highly glycosylated mannoproteins that are involved in the interaction with the host tissues. N glycosylation is a posttranslational modification that is initiated in the endoplasmic reticulum (ER), where the Glc₃Man₉GlcNAc₂ N-glycan is processed by α-glucosidases I and II and α1,2-mannosidase to generate Man₈GlcNAc₂. This N-oligosaccharide is then elaborated in the Golgi to form N-glycans with highly branched outer chains rich in mannose. In Saccharomyces cerevisiae, CWH41, ROT2, and MNS1 encode for α-glucosidase I, α-glucosidase II catalytic subunit, and α1,2-mannosidase, respectively. We disrupted the C. albicans CWH41, ROT2, and MNS1 homologs to determine the importance of N-oligosaccharide processing on the N-glycan outer-chain elongation and the host-fungus interaction. Yeast cells of Cacwh41Δ, Carot2Δ, and Camns1Δ null mutants tended to aggregate, displayed reduced growth rates, had a lower content of cell wall phosphomannan and other changes in cell wall composition, underglycosylated β-N-acetylhexosaminidase, and had a constitutively activated PKC-Mkc1 cell wall integrity pathway. They were also attenuated in virulence in a murine model of systemic infection and stimulated an altered pro- and anti-inflammatory cytokine profile from human monocytes. Therefore, N-oligosaccharide processing by ER glycosidases is required for cell wall integrity and for host-fungus interactions.     

Genetic Screening Identifies Cyanogenesis-Deficient Mutants of Lotus japonicus and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism

Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid–derived cyanogenic glucosides (α-hydroxynitrile glucosides) by specific β-glucosidases. Release of cyanide functions as a defense mechanism against generalist herbivores. We developed a high-throughput screening method and used it to identify cyanogenesis deficient (cyd) mutants in the model legume Lotus japonicus. Mutants in both biosynthesis and catabolism of cyanogenic glucosides were isolated and classified following metabolic profiling of cyanogenic glucoside content. L. japonicus produces two cyanogenic glucosides: linamarin (derived from Val) and lotaustralin (derived from Ile). Their biosynthesis may involve the same set of enzymes for both amino acid precursors. However, in one class of mutants, accumulation of lotaustralin and linamarin was uncoupled. Catabolic mutants could be placed in two complementation groups, one of which, cyd2, encoded the β-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related β-glucosidase, BGD4, were identified. This indicated that BGD4 plays no role in cyanogenesis in L. japonicus in vivo. Biochemical analysis confirmed that BGD4 cannot hydrolyze linamarin or lotaustralin and in L. japonicus is specific for breakdown of related hydroxynitrile glucosides, such as rhodiocyanoside A. By contrast, BGD2 can hydrolyze both cyanogenic glucosides and rhodiocyanosides. Our genetic analysis demonstrated specificity in the catabolic pathways for hydroxynitrile glucosides and implied specificity in their biosynthetic pathways as well. In addition, it has provided important tools for elucidating and potentially modifying cyanogenesis pathways in plants.     

Characterization of Five β-Glycoside Hydrolases from Cellulomonas fimi ATCC 484

The Gram-positive bacterium Cellulomonas fimi produces a large array of carbohydrate-active enzymes. Analysis of the collection of carbohydrate-active enzymes from the recent genome sequence of C. fimi ATCC 484 shows a large number of uncharacterized genes for glycoside hydrolase (GH) enzymes potentially involved in biomass utilization. To investigate the enzymatic activity of potential β-glucosidases in C. fimi, genes encoding several GH3 enzymes and one GH1 enzyme were cloned and recombinant proteins were expressed in Escherichia coli. Biochemical analysis of these proteins revealed that the enzymes exhibited different substrate specificities for para-nitrophenol-linked substrates (pNP), disaccharides, and oligosaccharides. Celf_2726 encoded a bifunctional enzyme with β-d-xylopyranosidase and α-l-arabinofuranosidase activities, based on pNP-linked substrates (CfXyl3A). Celf_0140 encoded a β-d-glucosidase with activity on β-1,3- and β-1,6-linked glucosyl disaccharides as well as pNP-β-Glc (CfBgl3A). Celf_0468 encoded a β-d-glucosidase with hydrolysis of pNP-β-Glc and hydrolysis/transglycosylation activities only on β-1,6-linked glucosyl disaccharide (CfBgl3B). Celf_3372 encoded a GH3 family member with broad aryl-β-d-glycosidase substrate specificity. Celf_2783 encoded the GH1 family member (CfBgl1), which was found to hydrolyze pNP-β-Glc/Fuc/Gal, as well as cellotetraose and cellopentaose. CfBgl1 also had good activity on β-1,2- and β-1,3-linked disaccharides but had only very weak activity on β-1,4/6-linked glucose.     

A Versatile Family 3 Glycoside Hydrolase from Bifidobacterium adolescentis Hydrolyzes β-Glucosides of the Fusarium Mycotoxins Deoxynivalenol,Nivalenol, and HT-2 Toxin in Cereal Matrices

Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A K_m of 5.4 mM and a V_max of 16 μmol min⁻¹ mg⁻¹ were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples.     

The StpA Protein Functions as a Molecular Adapter To Mediate Repression of the bgl Operon by Truncated H-NS in Escherichia coli

The mechanism of repression of the β-glucoside utilization (bgl) operon of Escherichia coli by a carboxy-terminally truncated derivative of the nucleoid-associated protein H-NS which is defective in DNA binding was investigated. The DNA-binding function of the H-NS-like protein StpA was found to be necessary for repression, which is consistent with a role for StpA as a DNA-binding adapter for mutant derivatives of H-NS.     

Alpha-Glucosidase Promotes Hemozoin Formation in a Blood-Sucking Bug: An Evolutionary History

Background

Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated.

Methodology/Principal Findings

Hz formation activity of an α-glucosidase was investigated. Hz formation was inhibited by specific α-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect α-glucosidase was able to inhibit Hz formation. The α-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that α-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of α-glucosidase gene, was injected into R. prolixus females'' hemocoel. Gene silencing was accomplished by reduction of both α-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of α-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of α-glucosidase shows a high similarity to the insect α-glucosidases, with critical histidine and aspartic residues conserved among the enzymes.

Conclusions/Significance

Herein the Hz formation is shown to be associated to an α-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that α-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.     

Comparison of β-Glucosidase Activities in Different Streptomyces Strains

Cellobiase (β-glucosidase) production was compared for two streptomycetes: Streptomyces flavogriseus, a known producer of cellulase complex, and Streptomyces sp. strain CB-12, a strain isolated for its rapid growth on cellobiose. The optimal conditions for enzyme activity were established in relation to pH, temperature, enzyme stability, and substrate affinity. The production of β-glucosidase by the two strains depended on the carbon substrate in the medium. Cellobiose was found to repress the biosynthesis of the enzyme in S. flavogriseus and to stimulate its production in strain CB-12. The biosynthesis of the enzyme correlated well with the accumulation of glucose in the culture filtrates. The combined action of the β-glucosidases produced by the two Streptomyces strains might allow a better utilization of the reaction products which arise during the biodegradation of cellulose.     

An Inducible System for the Hydrolysis and Transport of β-Glucosides in Yeast : I. Characteristics of the β-glucosidase activity of intact and of lysed cells

A strain of bakers'' yeast was isolated which could utilize cellobiose and other β-D-glucosides quantitatively as carbon and energy sources for growth. Cellobiose-grown cells contained a largely cryptic enzyme active against the chromogenic substrate p-nitrophenyl-β-D-glucoside. The patent (intact cell) activity of such cells was inhibited by azide and, competitively, by cellobiose; neither agent inhibited the β-glucosidase activity of lysed cells or of extracts. The enzyme induced by growth in cellobiose medium had no affinity for cellobiose as either substrate or inhibitor; its substrate specificity classifies it as an aryl-β-glucosidase. It was concluded that growth in cellobiose also induced the formation of a stereospecific and energy-dependent system whose function determined the rate at which intact cells could hydrolyze substrates of the intracellular β-glucosidase.     

Sets of Covariant Residues Modulate the Activity and Thermal Stability of GH1 β-Glucosidases

The statistical coupling analysis of 768 β-glucosidases from the GH1 family revealed 23 positions in which the amino acid frequencies are coupled. The roles of these covariant positions in terms of the properties of β-glucosidases were investigated by alanine-screening mutagenesis using the fall armyworm Spodoptera frugiperda β-glycosidase (Sfβgly) as a model. The effects of the mutations on the Sfβgly kinetic parameters (k _cat/K _m) for the hydrolysis of three different p-nitrophenyl β-glycosides and structural comparisons of several β-glucosidases showed that eleven covariant positions (54, 98, 143, 188, 195, 196, 203, 398, 451, 452 and 460 in Sfβgly numbering) form a layer surrounding the active site of the β-glucosidases, which modulates their catalytic activity and substrate specificity via direct contact with the active site residues. Moreover, the influence of the mutations on the transition temperature (T _m) of Sfβgly indicated that nine of the coupled positions (49, 62, 143, 188, 223, 278, 309, 452 and 460 in Sfβgly numbering) are related to thermal stability. In addition to being preferentially occupied by prolines, structural comparisons indicated that these positions are concentrated at loop segments of the β-glucosidases. Therefore, due to these common biochemical and structural properties, these nine covariant positions, even without physical contacts among them, seem to jointly modulate the thermal stability of β-glucosidases.     

Fate of the H-NS–Repressed bgl Operon in Evolution of Escherichia coli

In the enterobacterial species Escherichia coli and Salmonella enterica, expression of horizontally acquired genes with a higher than average AT content is repressed by the nucleoid-associated protein H-NS. A classical example of an H-NS–repressed locus is the bgl (aryl-β,D-glucoside) operon of E. coli. This locus is “cryptic,” as no laboratory growth conditions are known to relieve repression of bgl by H-NS in E. coli K12. However, repression can be relieved by spontaneous mutations. Here, we investigated the phylogeny of the bgl operon. Typing of bgl in a representative collection of E. coli demonstrated that it evolved clonally and that it is present in strains of the phylogenetic groups A, B1, and B2, while it is presumably replaced by a cluster of ORFans in the phylogenetic group D. Interestingly, the bgl operon is mutated in 20% of the strains of phylogenetic groups A and B1, suggesting erosion of bgl in these groups. However, bgl is functional in almost all B2 isolates and, in approximately 50% of them, it is weakly expressed at laboratory growth conditions. Homologs of bgl genes exist in Klebsiella, Enterobacter, and Erwinia species and also in low GC-content Gram-positive bacteria, while absent in E. albertii and Salmonella sp. This suggests horizontal transfer of bgl genes to an ancestral Enterobacterium. Conservation and weak expression of bgl in isolates of phylogenetic group B2 may indicate a functional role of bgl in extraintestinal pathogenic E. coli.