Identification and enzymatic characterisation of digestive glucosidases from gut of red palm weevil,Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae)

Red palm weevil, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae) is a serious pest of date worldwide. Thus, damage to palms (almost exclusively to Phoenix canariensis Hort) has been recorded in various places. Thus, the aim of the current study was to investigate two major digestive enzymes of this insect, α-D-glucosidase and β-D-glucosidase. The results showed that α-D-glucosidase and β-D-glucosidase are present in the insect gut mainly in the midgut and hindgut but trace amounts of the both enzymes were found in the foregut. Optimum temperature for α- and β-glucosidases was found to be 50 and 40?°C, respectively, and pH values were 4.0. The activity of glucosidases were increased by NaCl and KCl and inhibited by some compounds such as MgCl₂ and CaCl₂. Kinetic parameters showed that K _m of α and β-D-glucosidases was 3.15 and 4.11?mM, respectively. Therefore, it is concluded that in this insect species, both α-glucosidase and β-glucosidase are active but with different amounts. Understanding of the digestive physiology and glucosidase activity of red palm weevil is important when new management strategies based on interfering in the gut physiology of the insects are devised.     

Characterization of a novel β-thioglucosidase CpTGG1 in Carica papaya and its substrate-dependent and ascorbic acid-independent O-β-glucosidase activity

Plant thioglucosidases are the only known S-glycosidases in the large superfamily of glycosidases.These enzymes evolved more recently and are distributed mainly in Brassicales.Thioglucosidase research has focused mainly on the cruciferous crops due to their economic importance and cancer preventive benefits.In this study,we cloned a novel myrosinase gene,CpTGG1,from Carica papaya Linnaeus.and showed that it was expressed in the aboveground tissues in planta.The recombinant CpTGG1 expressed in Pichia pastoris catalyzed the hydrolysis of both sinigrin and glucotropaeolin(the only thioglucoside present in papaya),showing that CpTGG1 was indeed a functional myrosinase gene.Sequence alignment analysis indicated that CpTGG1 contained all the motifs conserved in functional myrosinases from crucifers,except for two aglycon-binding motifs,suggesting substrate priority variation of the non-cruciferous myrosinases.Using sinigrin as substrate,the apparent Km and Vmax values of recombinant CpTGG1 were 2.82 mM and 59.9 μmol min-1 mg protein-1,respectively.The Kcat IKm value was 23 s-1 mM-1.O-β-glucosidase activity towards a variety of substrates were tested,CpTGG1 displayed substrate-dependent and ascorbic acid-independent O-β-glucosidase activity towards 2-nitrophenyl-βD-glucopyranoside and 4-nitrophenyl-β-D-glucopyranoside,but was inactive towards glucovanillin and n-octyl-β-D-glucopyranoside.Phylogenetic analysis indicated CpTGG1 belongs to the MYR II subfamily of myrosinases.     

Evidence for a single catalytic and two binding sites in the almond emulsin β-D-glucosidase molecule

An isoenzyme of glucosidase- isolated from sweet almond emulsin - and composed of a β-D-glucosidase, a β-D-galactosidase and a β-D-fucosidase, has been shown to possess β-D-xylosidase activity, as well. On the basis of the following results it has been concluded that the β-D-glucosidase and β-D-galactosidase activities reside in one catalytic site, but there are two kinetically distinst binding sites in the active center: 1./D-Glucono-1,5-lactone is shown to excert competitive inhibition on the hydrolysis of β-D-glucopyranoside and non-competitive inhibition on the hydrolysis of β-D-galactopyranoside. 2./ D-galactono-1,5-lactone competitively inhibits the hydrolysis of β-D-galactopyranoside, but possesses non-competitive inhibition on the hydrolysis of β-D-glucopyranoside. 3./ When the enzyme is incubated with two p-nitrophenyl glycoside substrates at or above their respective K_m values, the rate of p-nitrophenol formation is not additive but rather it is equal to the value calculated from the individual K_m values and relative maximum rates.     

Purification and properties of two β-glucosidases isolated from Aspergillus niger

The cellulase complex of the fungus Aspergillus niger (strain CBS 554.65 = ATCC 16 888) was fractionated by gel filtration yielding six pronounced peaks. Only proteins from the fraction corresponding to the first peak (96 kDa) showed β-glucosidase activity vs. the substrate 4-nitrophenyl-β-D-glucopyranoside (pNPG). These proteins have been fractionated by chromatofocusing, yielding two β-glucosidases (I and II) which are shown to be homogeneous in isoelectric focusing experiments (pI = 4.6 and 3.8, respectively). Kinetic experiments with pNPG, MU-glucopyranoside and cellobiose revealed that both types of β-glucosidases behave like aryl-β-glucosidases. β-Glucosidase-I acting on pNPG exhibits a split kinetics characterized by high and low substrateconcentration kinetics which are differentiated by different values of V and of K_m. In addition, β-glucosidase-II is shown to be an exo-glucohydrolase as deduced from experiments with MU-cellobiopyranoside. Experimental features should be emphasized; usual soft-gel ion-exchange materials did not work in the chromatofocusing separation of the two β-glucosidases, in contrast to the 10μ-Si 500 = DEAE exchange material (Serva) typically used in HPLC-experiments. Furthermore, protein content determinations based on different procedures yielded widely differing values.     

Overexpression and characterization of a novel cold-adapted and salt-tolerant GH1 β-glucosidase from the marine bacterium <Emphasis Type="Italic">Alteromonas</Emphasis> sp. L82

A novel gene (bgl) encoding a cold-adapted β-glucosidase was cloned from the marine bacterium Alteromonas sp. L82. Based on sequence analysis and its putative catalytic conserved region, Bgl belonged to the glycoside hydrolase family 1. Bgl was overexpressed in E. coli and purified by Ni²⁺ affinity chromatography. The purified recombinant β-glucosidase showed maximum activity at temperatures between 25°C to 45°C and over the pH range 6 to 8. The enzyme lost activity quickly after incubation at 40°C. Therefore, recombinant β-glucosidase appears to be a cold-adapted enzyme. The addition of reducing agent doubled its activity and 2 M NaCl did not influence its activity. Recombinant β-glucosidase was also tolerant of 700 mM glucose and some organic solvents. Bgl had a K_m of 0.55 mM, a V_max of 83.6 U/mg, a k_cat of 74.3 s^-1 and k_cat/K_m of 135.1 at 40°C, pH 7 with 4-nitrophenyl-β-D-glucopyranoside as a substrate. These properties indicate Bgl may be an interesting candidate for biotechnological and industrial applications.     

The beta-D-glucosidase system of an Actinoplanes sp

Actinoplanes sp. No. 1700, a sporangium-forming, filamentous, soil bacterium possesses a β-D-glucosidase (β-D-glucoside glucohydrolase, E.C. 3.2.1.21). The enzyme was induced to higher concentrations by addition of methyl or phenyl β-D-glucopyranoside, gentiobiose, or salicin to growing cultures. Addition of D-glucose, lactate, or acetate repressed enzyme induction back to the constitutive level, but never below it. The properties of this inducible system place it in the semi-constitutive category.Both the constitutive and the inducible enzyme were purified 60-fold; their properties were compared and found to be identical. Their pH optima lay between 5.8 and 6.0; the enzymes were stable for 2 h at 30° at pH 5.5 to 7.3. Rapid inactivation occurred at temperatures above 50°. The enzymes were inactivated by 100μM CU²⁺, Hg²⁺, Pb²⁺, and Ag⁺.Each of these β-D-glucosidases was inhibited by p-chloromercuribenzoate (100 μ/M); this effect was overcome by cysteine or 2-mercaptoethanol, indicating that the β-D-glucosidase is a sulfhydryl enzyme. Kinetic determinations with chromogenic p-nitrophenyl β-D-glucopyranoside established a K_m. of 2.5 x 10^-4 and an Arrhenius activation-energy of 8.5 kcal.mole^-1. The molecular weight of the induced enzyme was 165,000 as determined by elution from Sephadex G-200. Chromatographic studies showed the enzyme to be a hydrolase, not a transferase.     

Characterization of a β-glucosidase from Glycine max which hydrolyses coniferin and syringin

A β-glucosidase which rapidly hydrolyses the cinnamyl alcohol glucosides coniferin and syringin has been purified from cell cultures, hypocotyls and roots of Glycine max. Isoelectric focusing in a column separated the enzyme from several other β-glucosidases which were inactive against either substrate. Syringin and coniferin were the best substrates tested. Both exhibited identical V_max values, whereas the K_m of coniferin (0.6 mM) was twice that of syringin (0.3 mM). The widely used synthetic substrates 4-nitrophenyl-β-glucoside and 4-methyl-umbelliferyl-β-glucoside were poorly utilized. Glucono-1,5-lactone was an effective competitive inhibitor with a K_i of 0.01 mM. From the observed-substráte specificity, a role in the lignification process of higher plants may be predicted for this β-glucosidase.     

Purification and characterization of a novel and unique ginsenoside Rg1-hydrolyzing β-D-glucosidase from Penicillium sclerotiorum

In this paper, a novel and unique ginsenoside Rg(1)-hydrolyzing β-D-glucosidase from Penicillium sclerotiorum was isolated, characterized, and generally described. The β-glucosidase is an ~180 kDa glycoprotein with pI 6.5, and consists of four identical subunits of ~40 kDa. The β-glucosidase was active in a narrow pH range (4-5) and at relatively high temperature (60-70°C). The optimal activity against p-nitrophenyl-β-D-glucopyranoside (pNPG) was as follows: pH 4.5 and temperature 65°C. Under these conditions, the K(m) of the enzyme was 0.715 mM with a V(max) of 0.243 mmol nitrophenol/min mg. Metal ions such as Ba(2+), K(+), Fe(3+), and Co(2+) significantly promoted the enzymatic activity, while Ca(2+), Mg(2+), and Ag(+) inhibited its activity. Of the tested substrates, only ginsenoside Rg(1) could be specifically hydrolyzed by the β-glucosidase at the C6-glucoside to form the rare ginsenoside F(1). These properties were novel and different from those of other previously described glycosidases.     

Isolation and some properties of two extracellular β-glucosidases from Trichosporon adeninovorans

The yeast Trichosporon adeninovorans secretes two multiple forms of β-glucosidase at a high rate if grown in a medium containing cellobiose. Following mutagenesis a mutant strain resistant to 2-deoxy-D-glucose was selected. This strain produced more β-glucosidase activity and had acquired a strong resistance against repression by glucose. The β-glucosidases were separated one from each other by chromatography on hydroxylapatite and by gel filtration. Both enzymes have similar properties. The optimal temperature for their activity was 60 to 63°C and the enzymes displayed highest activity at pH of 4.5. The molecular weight of β-glucosidase I was found to be 570,000 and that for β-glucosidase II was 525,000. The K_m value for cellobiose was determined to be 4.1 mM for β-glucosidase I and 3.0 mM for β-glucosidase II.     

The inhibition of β-glucosidase activity in Trichoderma reesei C30 cellulase by derivatives and isomers of glucose

The inhibition of β-glucosidase in Trichoderma reesei C30 cellulase by D -glucose, its isomers, and derivatives was studied using cellobiose and ρ-nitrophenyl-β-glucoside (PNPG) as substrates for determining enzyme activity. The enzymatic hydrolysis of both substrates was inhibited competitively by glucose with approximate K_i values of 0.5mM and 8.7mM for cellobiose and PNPG as substrate, respectively. This inhibition by glucose was maximal at pH 4.8, and no inhibition was observed at pH 6.5 and above. The α anomer of glucose inhibited β-glucosidase to a greater extent than did the β form. Compared with D -glucose, L -glucose, D -glucose-6-phosphate, and D -glucose-1-phosphate inhibited the enzyme to a much lesser extent, unlike D -glucose-L -cysteine which was almost as inhibitory as glucose itself when cellobiose was used as substrate. Fructose (2?100mM) was found to be a poor inhibitor of the enzyme. It is suggested that high rates of cellobiose hydrolysis catalyzed by β-glucosidase may be prolonged by converting the reaction product glucose to fructose using a suitable preparation of glucose isomerase.     

New Pepsin Inhibitors (S-PI) from Streptomyces EF-44-201

Studies have been done on the inhibition and inactivation of the β-glucosidase and β-fucosidase enzyme from Thai Rosewood (Dalbergia cochinchinesis Pierre). The enzyme was inhibited by Tris with similar K_i of 11.7 mm and 14.3 mm for the hydrolysis of p/nitrophenyl β-d-glucoside (PNPG) and p/nitrophenyl β-d-fucoside (PNPF), respectively. Conduritol B epoxide inhibited both β-glucosidase and β/fucosidase activities to similar extents, with a pseudo-first-order rate constant (K_obs) of inactivation of 5.56 × 10^?3 s ^?1, and binding stoichiometry of 0.9 mol per subunit. Partially inactivated enzyme showed similar kinetics with PNPG and PNPF as substrates. Moreover, Tris at 300 mm protected both β-glucosidase and β-fucosidase activities from inactivation by 6mm CBE. The data support the idea that the Dalbergia cochinchinensis Pierre enzyme has a common active site for the hydrolysis of PNPG and PNPF.     

A glucose-tolerant β-glucosidase from Prunus domestica seeds: Purification and characterization

A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag⁺ and Hg²⁺. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a K_m of 3.09 mM and a V_max of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a K_m of 1.65 mM and a V_max of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with K_i values of 0.033 and 468 mM, respectively.     

Analysis of the <Emphasis Type="Italic">xynB5</Emphasis> gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-l-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (K_m 0.24 ± 0.0005 mM, V_max 0.041 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.27 mM^?1 s^?1), followed by β-xylosidase (K_m 0.64 ± 0.032 mM, V_max 0.055 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.14 mM^?1s^?1) and finally α-l-arabinosidase (K_m 1.45 ± 0.05 mM, V_max 0.091 ± 0.0004 µmol min^?1 mg^?1 and K_cat/K_m 0.1 mM^?1 s^?1). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.     

Properties of larval and imaginal membrane-bound digestive enzymes from Trichosia pubescens

Trichosia pubescens larval midgut ceca cells display in their plasma membranes α-glucosidases (M_r 95,000; pH_o 5.5; K_m 5.7 mM; K_i for TRIS 8.9 mM), trehalases (M_r 69,000; pH_o 5.3; K_m 0.92 mM; K_i for TRIS 57 mM), and aminopeptidases (M_r 95,000; pH_o 8.7; K_m 0.19 mM) which are solubilized by Triton X-100. The enzymes were purified by electrophoresis and used to raise antibodies in a rabbit. T. pubescens imaginal midgut cells display in their plasma membranes an α-glucosidase (M_r 156,000; pH_o 5.8; K_m 2.3 mM; K_i for TRIS 0.2 mM), a trehalase (M_r 93,000; pH_o 5.5; K_m 0.72 mM; K_i for TRIS 45.5 mM), and an aminopeptidase (M_r 210,000; pH_o 9.0; K_m 0.47 mM). Antiserum produced against the larval enzymes shows no precipitation arc when tested by double immunodiffusion or by immunoelectrophoresis with Triton X-100-solubilized membrane proteins from imaginal midguts. Otherwise, a similar test showed that larval midgut cecal enzymes and larval ventriculus enzymes display complete immunological identity. The data suggest that, despite the fact the larval and imaginal aminopeptidase, α-glucosidase, and trehalase probably have similar functions, the genes coding for them in larvae and imagoes must differ.     

β-Glucosidase production by Trichoderma reesei QM9414 on wheat straw. Location and some kinetic features

In this paper we studied the conditions for the production of β-glucosidase from T. reesei QM9414 in batch cultures using milled and sieved wheat straw as sole carbon source. High β-glucosidase production in the presence of wheat straw, a more realistic substrate than commercial cellulose, was obtained. The influence of particle size of wheat straw on β-glucosidase production in cell-free, cell and cell-wall extracts was studied. The particle size of wheat straw notably influenced enzyme production in cell and extramycelial extracts but it was less important with respect to the cell wall bound enzyme. β-glucosidase production was studied along of the fermentation. The results suggest a close relation between β-glucosidase from cell extract and extramycelial broth; geneticin levels of inhibition of β-glucosidase biosynthesis in both fractions were similar, a fact that suggests a common origin for the enzyme. Kinetic parameters for β-glucosidase from cell free and cell extracts were V_max = 0.28 μmol/min/mg, K_M = 0.91 mM and V_max = 0.095 μmol/min/mg, K_M = 0.39 mM respectively. Kinetic parameters for β-glucosidase from cell-wall could not be calculated because experimental data did not fit the different monosubstrate equations.     

Identity of β-alanine-oxo-glutarate aminotransferase and L-β-aminoisobutyrate aminotransferase in rat liver

L-β-Aminoisobutyrate served as an amino donor for purified β-alanine-oxo-glutarate aminotransferase from rat liver when 2-oxoglutarate was employed as an amino acceptor, but he D-isomer did not. L-β-Aminoisobutyrate acted as a competitive inhibitor with respect to β-alanine and had a K_i of approximately 2.6 mM, which is the same value as the K_m of 2.7 mM. When the crude extract was applied to a DEAE-Sepharose CL-6B column, L-β-aminoisobutyrate aminotransferase and β-alanine-oxo-glutarate aminotransferase activities were found in the same fractions with a single peak. Antiserum to rat liver β-alanine-oxo-glutarate aminotransferase inhibited L-β-aminoisobutyrate aminotransferase activity in rat liver in the same way as β-alanine-oxo-glutarate aminotransferase activity.     

Characterization of a novel β-glucosidase from a Stachybotrys strain

An improved mutant was isolated from the cellulolytic fungus Stachybotrys sp. after nitrous acid mutagenesis. It was fed-batch cultivated on cellulose and its extracellular cellulases (mainly the endoglucanases and β-glucosidases) were analyzed. One β-glucosidase was purified to homogeneity after two steps, MonoQ and gel filtration and shown to be a dimeric protein. The molecular weight of each monomer is 85 kDa. Besides its aryl β-glucosidase activity towards salicin, methyl-umbellypheryl-β-d-glucoside (MUG) and p-nitrophenyl-β-d-glucoside (pNPG), it showed a true β-glucosidase activity since it splits cellobiose into two glucose monomers. The V_max and the K_m kinetics parameters with pNPG as substrate were 78 U/mg and 0.27 mM, respectively. The enzyme shows more affinity to pNPG than cellobiose and salicin whose apparent values of K_m were, respectively, 2.22 and 37.14 mM. This enzyme exhibits its optimal activity at pH 5 and at 50 °C. Interestingly, this activity is not affected by denaturing gel conditions (SDS and β-mercaptoethanol) as long as it is not pre-heated. The N-terminal sequence of the purified enzyme showed a significant homology with the family 1 β-glucosidases of Trichoderma reesei and Humicola isolens even though these two enzymes are much smaller in size.     

Production of Cellulases by Aspergillus fumigatus and Characterization of One β-Glucosidase

Aspergillus fumigatus produces substantial extracellular cellulases on several cellulosic substrates including simple sugars. Low glucose potentiates enzyme production, but most cellulose-induced cellulases are repressed by high glucose. As production of cellulase in a wide substrate range is unusual, the cellulolytic complex of this thermophilic fungus was investigated. A β-glucosidase was separated by gel filtration and ion-exchange chromatography. It migrated in native polyacrylamide gel as a single protein (130 kDa), which split under denaturing conditions into two smaller proteins having molecular masses of 90 kDa and 45 kDa. However, only the 90-kDa protein was active. Conventional chromatographic procedures were unsuccessful for the separation of these two proteins. Therefore, the 130-kDa protein was studied for its kinetic properties. It hydrolyzed p-nitrophenyl-β-D-glucopyranoside (p-NPG) and cellobiose, but not β-glucans, laminarin, and p-nitrophenyl-β-D-xilopyranoside. The optimal pH and temperature of p-NPG and cellobiose hydrolysis were 5.0 and 4.0, and 65°C and 60°C, respectively. The K _m values, determined for cellobiose and p-NPG of hydrolysis, were 0.075 mM and 1.36 mM, respectively. Glucose competitively inhibited the hydrolysis of p-NPG. The K_i was 3.5 mM.     

Substrate and endproduct regulation of cellobiose uptake by Candida wickerhamii

Summary Cellobiose-grown cells of Candida wickerhamii transported cellobiose as glucose by a glucose-proton symport after previous hydrolysis of the disaccharide by an exocellular -glucosidase. Both the symport and the -glucosidase were subject to glucose-induced repression and inactivation while glucose also acted as a competitive inhibitor of the enzyme (K _i 0.3 mM). Under conditions of glucose repression glucose was transported by facilitated diffusion. Cellobiose acted as a competitive inhibitor of the latter (K _i 75 mM) and is possibly a low-affinity substrate, while it inhibited non-competitively the glucoseproton symport (K _i 80 mM). The affinity of cellobiose for the cell-bound -glucosidase was much higher (K _m 4.2 mM) than for the purified enzyme as reported by others (K _m 67–225 mM). Ethanol reversibly inhibited the two glucose transport systems with exponential non-competitive kinetics. The minimum inhibitory concentrations were about 3% and 4% (w/v) for facilitated diffusion and proton symport while the respective exponential inhibition constants were 0.58 l mol^-1 and 1.65 l mol^-1. Ethanol affected the -glucosidase in a complex way, a major effect was deviation from Michaelis-Menten kinetics for ethanol concentrations higher than 4% (w/v), the Hill coefficient increasing up to 1.8 at 6% (w/v) ethanol.