Purification and characterization of cyanogenic β-glucosidase from wild apricot (Prunus armeniaca L.)

β-Glucosidase catalyzes the sequential breakdown of cyanogenic glycosides in cyanogenic plants. The β-glucosidase from Prunus armeniaca L. was purified to 8-fold, and 20% yield was obtained, with a specific activity of 281 U/mg protein. The enzyme showed maximum activity in 0.15 M sodium citrate buffer, pH 6, at 35 °C with p-nitrophenylglucopyranoside as substrate. The β-glucosidase from wild apricot was used successfully for the saccharification of cellobiose into D-glucose. This enzyme has a V_max of 131.6 μmol min⁻¹ mg⁻¹ protein, K_m of 0.158 mM, K_cat of 144.8 s⁻¹, K_cat/K_m of 917.4 mM⁻¹ s⁻¹, and K_m/V_max of 0.0012 mM min mg μmole⁻¹, using cellobiose as substrate. The half-life, deactivation rate coefficient, and activation energy of this β-glucosidase were 12.76 h, 1.509 × 10⁻⁵ s⁻¹, and 37.55 kJ/mol, respectively. These results showed that P. armeniaca is a potential source of β-glucosidase, with high affinity and catalytic capability for the saccharification of cellulosic material.     

Diversity and seasonal occurrence of mites (Acari) in a rubber tree crop (Hevea brasiliensis, Muell. Arg.) in northwestern São Paulo, Brazil

Brazilian southeastern region has soil and climate conditions suitable for the growing of rubber trees, and most part of national yield arises from S?o Paulo State. The aims proposed for this work were to determine the diversity, the richness and the seasonal occurrence of mites found in a rubber tree crop in a triennial survey with monthly samplings, as well as to estimate the populational density of the major phytophagous species. This study found 74,407 mites from 26 species belonging to 10 families. The phytophagous and predators represented 95.4% and 3.9% of the total abundance, respectively. Twelve species were rare, six accessories and eight constant. The families Phytoseiidae and Tydeidae had the greatest richness (five and four species, respectively). The most numerous species was Calacarus heveae Feres (50,573), with great abundance at the end of rainy season until the beginning of dry season. Among predators, the most abundant were Zetzellia quasagistemas Hernandes & Feres (1,345), Pronematus sp. (455), Zetzellia agistzellia Hernandes & Feres (409) and Euseius citrifolius Denmark & Muma (243). C. heveae had greatest densities on March and April 2003, and Lorryia formosa Cooreman and Tenuipalpus heveae Baker on March and May 2001, respectively. Many stigmaeids were observed in association with colonies of L. formosa preying their eggs and immatures.     

Cloning and characterization of a putative β-glucosidase (NfBGL595) from Neosartorya fischeri

Whole genome sequence of Neosartorya fischeri NRRL181 revealed four putative GH1 β-glucosidases (BGLs). One BGL, NfBGL595 was successfully expressed and characterized. DNA sequence analysis revealed an open reading frame of 1590 bp, encoding a polypeptide of 529 amino acid residues. The gene was cloned in pET28a and overexpressed in Escherichia coli. The purified recombinant BGL showed high levels of catalytic activity, with V_max of 1693 U mg-protein⁻¹ and a K_m of 2.8 mM for p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimal temperature and pH for enzyme activity were 40 °C and 6.0, respectively. The enzyme exhibited broad substrate specificity towards aryl glycosides including pNP-mannose, pNP-galactose, pNP-xylose, and pNP-cellobioside. A homology model of NfBGL595 was constructed based on the X-ray crystal structure of Trichoderma reesei BGL2. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose, shed light on the substrate specificity of N. fischeri BGL595 only towards aryl glycoside.     

Purification and biochemical characterization of a mycelial glucose- and xylose-stimulated β-glucosidase from the thermophilic fungus Humicola insolens

A mycelial β-glucosidase from the thermophilic mold Humicola insolens was purified and biochemically characterized. The enzyme showed carbohydrate content of 21% and apparent molecular mass of 94 kDa, as estimated by gel filtration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a single polypeptide band of 55 kDa, suggesting that the native enzyme was a homodimer. Mass spectrometry analysis showed amino acid sequence similarity with a β-glucosidase from Humicola grisea var. thermoidea, with about 22% coverage. Optima of temperature and pH were 60 °C and 6.0–6.5, respectively. The enzyme was stable up to 1 h at 50 °C and showed a half-life of approximately 44 min at 55 °C. The β-glucosidase hydrolyzed cellobiose, lactose, p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl-β-d-fucopyranoside, p-nitrophenyl-β-d-xylopyranoside, p-nitrophenyl-β-d-galactopyranoside, o-nitrophenyl-β-d-galactopyranoside, and salicin. Kinetic studies showed that p-nitrophenyl-β-d-fucopyranoside and cellobiose were the best enzyme substrates. Enzyme activity was stimulated by glucose or xylose at concentrations up to 400 mM, with maximal stimulatory effect (about 2-fold) around 40 mM. The high catalytic efficiency for the natural substrate, good thermal stability, strong stimulation by glucose or xylose, and tolerance to elevated concentrations of these monosaccharides qualify this enzyme for application in the hydrolysis of cellulosic materials.     

Characterization of cyanogenic glucosides and β-glucosidases in Triglochin maritima seedlings

In addition to triglochinin, taxiphyllin has been detected as a cyanogenic glucoside in seedlings of Triglochin maritima. Taxiphyllin at first increases during seedling development and then decreases, whereas tri-glochinin increases to a level higher than that ever reached by taxiphyllin and remains there during further seedling development. Two β-glucosidases have also been characterized in these seedlings. One of these shows a distinct specificity for triglochinin, whereas taxiphyllin appears to be the preferred substrate of the other.     

Molecular cloning and biochemical characterization of α- and β-tubulin from potato plants (Solanum tuberosum L.)

Few studies have investigated microtubules from plants that host pathogenic fungi. Considerable efforts are underway to find an antimitotic agent against plant pathogens like Phytophthora infestans. However, screening the effects of antifungal agents on plant tubulin in vivo or using purified native microtubule in vitro is a time consuming process. A recombinant, correctly folded, microtubule-like structure forming tubulin could accelerate research in this area. In this study, we cloned full length cDNAs isolated from potato leaves using reverse-transcribed polymerase chain reaction (RT-PCR). Solanum tuberosum (Stub) α-tubulin and β-tubulin were predicted to encode 449 and 451 amino acid long proteins with molecular masses of 57 kDa and 60 kDa, respectively. Average yields of α- and β-tubulin were 2.0–3.5 mg l^?1 and 1.3–3.0 mg l^?1 of culture, respectively. The amino acids, His6, Glu198, and Phe170 involved in benomyl sensitivity were conserved in Stub tubulin. The dimerization of tubulin monomers was confirmed by western blot analysis. When combined under appropriate conditions, these recombinant α- and β-tubulins were capable of polymerizing into microtubules. Accessibility of cysteine residues of tubulin revealed that important ligand binding sites were folded correctly. This recombinant tubulin could serve as a control of phytotoxicity of selected antimitotic fungicide compounds during in vitro screening experiments.     

Cloning,expression and biochemical characterization of a GH1 β-glucosidase from Cellulosimicrobium cellulans

β-Glucosidase plays an important role in the degradation of cellulose. In this study, a novel β-glucosidase ccbgl1b gene for a glycosyl hydrolase (GH) family 1 enzyme was cloned from the genome of Cellulosimicrobium cellulans and expressed in Escherichia coli BL21 cells. The sequence contained an open reading frame of 1494?bp, encoded a polypeptide of 497?amino acid residues. The recombinant protein CcBgl1B was purified by Ni sepharose fastflow affinity chromatography and had a molecular weight of 57?kDa, as judged by SDS-PAGE. The optimum β-glucosidase activity was observed at 55?°C and pH 6.0. Recombinant CcBgl1B was found to be most active against aryl-glycosides p-nitrophenyl-β-D-glucopyranoside (pNPβGlc), followed by p-nitrophenyl-β-D-galactopyranoside (pNPβGal). Using disaccharides as substrates, the enzyme efficiently cleaved β-linked glucosyl-disaccharides, including sophorose (β-1,2-), laminaribiose (β-1,3-) and cellobiose (β-1,4-). In addition, a range of cello-oligosaccharides including cellotriose, cellotetraose and cellopentaose were hydrolysed by CcBgl1B to produce glucose. The interaction mode between the enzyme and the substrates driving the reaction was modelled using a molecular docking approach. Understanding how the GH1 enzyme CcBgl1B from C. cellulans works, particularly its activity against cello-oligosaccharides, would be potentially useful for biotechnological applications of cellulose degradation.     

Purification and biochemical characterization of a soluble α-glucosidase from the parasite Entamoeba histolytica

A soluble α-glucosidase presumably involved in the general carbohydrate metabolism was purified from E. histolytica trophozoites by a three-step procedure consisting of ion exchange, size exclusion and adsorption chromatographies in columns of Mono Q, Sepharose CL-6B and hydroxyapatite, respectively. After the last step, the enzyme was enriched about 673-fold over the starting material with a yield of 18%. SDS-PAGE revealed the presence in the purified preparations of two polypeptides of comparable intensity exhibiting molecular weights of 43 and 68 kDa. These results and the molecular weight of 243 kDa determined by gel filtration, suggest that the native enzyme is a heterotetramer consisting of two copies of each subunit. Some properties were investigated to determine the role of this activity in glycoprotein processing. Analysis of linkage specificity using a number of substrates indicated a preferential hydrolysis of isomaltose (α1,6) with much less activity on nigerose (α1,3) and maltose (α1,4). Trehalose (α1,1), kojibiose (α1,2) and cellobiose (β1,4) were not cleaved at all. As expected, isomaltose competed away hydrolysis of 4-methylumbelliferyl-α-D-glucoside with a higher efficiency than nigerose and maltose. Hydrolysis of the fluorogenic substrate was competitively inhibited by glucose and 6-deoxy-D-glucose with comparable K_i values of 0.23 and 0.22 mM, respectively. Sensitivity of the enzyme to the α-glucosidase inhibitors 1-deoxynojirimycin, castanospermine and australine largely depended on the substrate utilized to determine activity. 1-Deoxynojirimycin and castanospermine inhibited isomaltose hydrolysis in a competitive manner with K_i values of 1.2 and 1.5 μM, respectively. The properties of the purified enzyme are consistent with a general glycosidase probably involved in glycogen metabolism. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biochemical characterisation of the Li locus,which controls the activity of the cyanogenic β-glucosidase in Trifolium repens L.

The cyanogenic -glucosidase (linamarase) was purified from white clover leaf tissue. The enzyme is a homodimer with a molecular weight of 105 300–103 400 daltons estimated from molecular exclusion chromatography. The effect of buffer ions on the pH optimum and charge properties of the enzyme are presented. A combination of molecular exclusion chromatography and CM cellulose ion exchange chromatography purified linamarase 16 fold to a single 62 000 dalton polypeptide on SDS polyacrylamide gel electrophoresis. This polypeptide represented about 5% of the total soluble leaf protein and can be seen as a prominent band in SDS polyacrylamide gel electrophoresis of crude leaf extracts from Li Li plants. Screening backcross progeny showed that extracts from li li plants, which have no linamarase activity, lack this 62 000 dalton polypeptide. Linamarase is the major glycoprotein in white clover leaf extracts which binds to Concanavalin A-Sepharose.     

Nucleotide and derived amino acid sequence of the cyanogenic β-glucosidase (linamarase) from white clover (Trifolium repens L.)

The nucleotide sequence and derived amino acid sequence of two different -glucosidase cDNA clones were determined. One clone (TRE104) was identified as the cyanogenic -glucosidase by homology with the N-terminal and internal peptide amino acid sequence of the purified enzyme. The biological function of the other -glycosidase (TRE361) is not known. Co-segregation of genomic restriction fragments uniquely identified by each cDNA clone shows that these two genes are linked in the white clover genome. Both TRE104 and TRE361 fragments co-segregate with cyanogenic -glucosidase activity. Extensive homology was found between the white clover -glucosidase sequences and a group of prokaryote and mammalian -glycosidases. This group of sequences has no homology with a separate set of -glucosidase genes isolated from fungi and the thermophilic bacterium Clostridium thermocellum.     

Purification and characterization of a hydroxamic acid glucoside β-glucosidase from wheat (Triticum aestivum L.) seedlings

A beta-glucosidase (EC 3.2.1.21) with a high affinity for cyclic hydroxamic acid beta-D-glucosides was purified from 48-h-old wheat (Triticum aestivum L.) seedlings. The activity occurred transiently at a high level during the non-autotrophic stage of growth, and the nature of the transient occurrence was correlated with that of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). The glucosidase had maximum activity at an acidic pH (pH 5.5) and the purified enzyme showed a high affinity for DIMBOA-Glc, Vmax and Km being 4100 nkat/mg protein and 0.27 mM, respectively. It also hydrolyzed p-nitrophenol beta-glycosides, as well as flavone and isoflavone glucosides, but to a lesser extent. The results indicated that the primary natural substrate for the glucosidase is DIMBOA-Glc and that the enzyme is involved in defense against pathogens and herbivores in non-autotrophic wheat. The glucosidase was found to be present as oligomeric forms with a molecular mass of 260-300 kDa comprising 60- and 58-kDa monomers. The N-terminal 12-amino-acid sequences of the two monomers were identical (Gly-Thr-Pro-(Ser?)-Lys-Pro-Ala-Glu-Pro-Ile-Gly-Pro), and showed no similarity to those of other plant glucosidases. Polyacrylamide gel electrophoresis under nondenaturing condition indicated the existence of at least eight isozymes. Three cultivars of Triticum aestivum had the same zone of glucosidase activity on zymograms, but the activity zones of the Triticum species, T. aestivum L., T. spelta L. and T. turgidum L., had different mobilities.     

Cloning and expression of a β-glycosidase gene from Thermus thermophilus. Sequence and biochemical characterization of the encoded enzyme

A 3.2 kilobase pair DNA fragment from Thermus thermophilus HB27 coding for a -galactosidase activity was cloned and sequenced. A gene and a truncated open reading frame orf1 encoding respectively a -glycosidase (tt-gly) and probably a sugar permease were located directly adjacent to each other. The deduced aminoacid sequence of the enzyme Tt-gly showed strong identity with those of -glycosidases belonging to the glycosyl hydrolase family 1. The enzyme was overexpressed in Escherichia coli and was purified by a two-step purification procedure. The recombinant enzyme is monomeric with a molecular mass of 49-kDa. It catalyzes the hydrolysis of -D-galactoside, -D-glucoside and -D-fucoside derivatives. However, the kcat/Km ratio is much higher for p-nitrophenyl--D-glucoside and p-nitrophenyl--D-fucoside than for p-nitrophenyl--D-galactoside. The specificity towards linkage positions of the disaccharides tested decreased in the following order: 1-3 (100%) < 1-2 (71%) < 1-4 (40%) < 1-6 (10%). Tt-gly is a thermostable enzyme displaying an optimum temperature of 88°C and a half life of 10 min at 90°C. It performs transglycosylation reactions at high temperature with a yield exceeding 63% for transfucosylation reactions. On the basis of this work, the enzyme appears to be an attractive tool in the synthesis of fucosyl adducts and fucosyl sugars.     

Heterologous production and biochemical characterization of a new highly glucose tolerant GH1 β-glucosidase from Anoxybacillus thermarum

The enzymatic lignocellulosic biomass conversion into value-added products requires the use of enzyme-rich cocktails, including β-glucosidases that hydrolyze cellobiose and cellooligosaccharides to glucose. During hydrolysis occurs accumulation of monomers causing inhibition of some enzymes; thus, glucose/xylose tolerant β-glucosidases could overcome this drawback. The search of new tolerant enzymes showing additional properties, such as high activity, wide-pH range, and thermal stability is very relevant to improve the bioprocess. We describe a novel β-glucosidase GH1 from the thermophilic Anoxybacillus thermarum (BgAt), which stood out by the robustness combination of great glucose/xylose tolerance, thermal stability, and high Vmax. The recombinant his-tagged-BgAt was overexpressed in Escherichia coli, was purified in one step, showed a high glucose/xylose tolerance, and activity stimulation (presence of 0.4 M glucose/1.0 M xylose). The optimal activity was at 65 °C - pH 7.0. BgAt presented an extraordinary temperature stability (48 h – 50 °C), and pH stability (5.5–8.0). The novel enzyme showed outstanding Vmax values compared to other β-glucosidases. Using p-nitrophenyl-β-d-glucopyranoside as substrate the values were Vmax (7614 U/mg), and K_M (0.360 mM). These values suffer a displacement in Vmax to 14,026 U/mg (glucose), 14,886 U/mg (xylose), and K_M 0.877 mM (glucose), and 1.410 mM (xylose).     

Cloning and characterization of a new broadspecific β-glucosidase from Lactococcus sp. FSJ4

A β-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure–function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this β-glucosidase of great interest for further study on physiological and catalytic reaction processes.     

A method of purification, identification and characterization of β-glucosidase from Trichoderma koningii AS3.2774

In this study, we used native gradient-polyacrylamide gel electrophoresis and electroelution (NGGEE) to purify enzymatic proteins from Trichoderma koningii AS3.2774. With this method, we purified eight enzymatic proteins and classified them to the cellulase system by comparing secretions of T. koningii in inductive medium and in repressive medium. It resulted in 24-fold β-glucosidase (BG) purification with a recovery rate of 5.5%, and a specific activity of 994.6 IU mg^− 1 protein. The final yield of BG reached 8 μg under purifying procedure of NGGEE. We also identified BG using the enzyme assay with thin-layer chromatography and MALDI-TOFMS. This BG had one subunit with a molecular mass of 69.1 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The hydrolytic activity of the BG had an optimal pH of 5.0, an optimal temperature of 50 °C, an isoelectric point of 5.68 and a K_m for p-nitrophenyl-β-d-glucopyranoside of 2.67 mM. Taken together, we show that NGGEE is a reliable method through which μg grade of active proteins can be purified.     

Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli

A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl--glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the -glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl--D-glucoside were 0.19mM and 0.25mM, respectively.     

Expression of a secretory β-glucosidase from Trichoderma reesei in Pichia pastoris and its characterization

A β-glucosidase gene (bglI) from Trichoderma reesei was cloned into the pPIC9 vector and integrated into the genome of Pichia pastoris GS115. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter and using Saccharomyces cerevisiae secretory signal peptide (α-factor), the recombinant β-glucosidase was expressed and secreted into the culture medium. The maximum recombinant β-glucosidase activity achieved was 60 U/ml, and β-glucosidase expression reached 0.3 mg/ml. The recombinant 76 kDa β-glucosidase was purified 1.8-fold with 26% yield and a specific activity of 197 U/mg. It was optimally active at 70°C and pH 5.0.