Histochemical investigation of β-glucuronidase in culture cells and regenerated plants of Scutellaria baicalensis Georgi

 Strong activity of β-glucuronidase first appeared in the epidermal and glandular hair cells of leaf primordia regenerated from callus of Scutellaria baicalensis Georgi. Leaf primordia matured rapidly in culture to form shoots within 1 month in which both the mesophyll cells and the glandular hairs were deeply stained. Leaves predominantly accumulated β-glucuronidase in both glandular hair cells and mesophyll cells. β-Glucronidase activity in leaves was higher in the summer and decreased in the winter. The stem section collected in the summer had a different β-glucuronidase distribution pattern from that of the root in that in the former strong activity appeared in the periderm cells and collenchyma cells which was decreasingly dispersed into the phloem layer cells. In the winter, β-glucronidase activity decreased compared to that in summer. It can be argued that the distribution of β-glucuronidase in this plant is closely linked with the defense against pathogens: it is a starting key enzyme which may act together with the flavonoids, which play an important role as a proton donor for the detoxification metabolism of H₂O₂. Received: 1 December 1998 / Revision received: 10 March 1999 / Accepted: 14 June 1999     

Purification and properties of a β-glucuronidase from pig kidney

A β-glucuronidase has been isolated from pig kidney and purified 1600-fold using sodium desoxycholate precipitation, ammonium sulphate fractionation, heat treatment and chromatography on Sephadex G200, DEAE-cellulose (DE-52) and hydroxyapatite. The enzyme activity was assayed using oestrone 3-glucuronide as substrate; the final specific activity was 254 nmol oestrone/min/mg of protein. The purified enzyme showed apparent homogeneity in gel filtration and polyacrylamide gel electrophoresis. The pig kidney β-glucuronidase has a single pH optimum of 4.0–4.4 in acetate- and 5.4 in citrate-buffer; an activation energy of 16,800 cal/mol and a molecular weight of 275,000 were estimated. The K_M for oestrone 3-glucuronide was 22.6 μM. The enzyme was not inhibited by N-ethylmaleimide nor by dithioerythritol, however, it was strongly inhibited by Hg²⁺. Oestradiol-17β 3-glucuronide and oestriol 3-glucuronide acted as competitive inhibitors, whereas oestradiol-17β 17β-glucuronide, oestriol 16α-glucuronide, testosterone 17-glucuronide and cholesteryl 3-glucuronide were uncompetitive, pregnanediol 3-glucuronide was noncompetitive, and Cortisol 21-glucuronide gave a mixed type inhibition. The synthetic β-d-glucuronides of phenolphthalein, p-nitrophenol, naphthol, 6-bromo-naphthol and methylumbelliferone all inhibited the hydrolysis of oestrone 3-glucuronide; the inhibition was of a more complex type than simple competitive inhibition.     

Purification and characterization of a highly selective glycyrrhizin-hydrolyzing β-glucuronidase from Penicillium purpurogenum Li-3

A novel β-glucuronidase from filamentous fungus Penicillium purpurogenum Li-3 was purified to electrophoretic homogeneity by ultrafiltration, ammonium sulfate precipitation, DEAE-cellulose ion exchange chromatography, and Sephadex G-100 gel filtration with an 80.7-fold increase in specific activity. The purified β-glucuronidase is a dimeric protein with an apparent molecular mass of 69.72 kDa (m/z = 69,717), determined by MALDI/TOF-MS. The optimal temperature and pH of the purified enzyme are 40 °C and 6.0, respectively. The enzyme is stable within pH 5.0–8.0, and the temperature up to 45 °C. Mg²⁺ ions enhanced the activity of the enzyme, Ca²⁺ and Al³⁺ showed no effect, while Mn²⁺, Zn²⁺, Hg²⁺ and Cu²⁺ substantially inhibited the enzymatic activity. The K_m and V_max values of the purified enzyme for glycyrrhizin (GL) were evaluated as 0.33 mM and 59.0 mmol mg^?1 min^?1, respectively. The purified enzyme displayed a highly selective glycyrrhizin-hydrolyzing property and converted GL directly to glycyrrhetic acid mono-glucuronide (GAMG), without producing byproduct glycyrrhetic acid (GA). The results suggest that the purified enzyme may have potential applications in bio-pharmaceutical and biotechnological industry.     

Purification and characterization of β-xylosidase from Aureobasidium pullulans

Summary -Xylosidase was obtained from Aureobasidium pullulans CBS 58475 with an activity of 0.35 units/ml culture filtrate. The production of the enzyme was strongly inducible. -Xylosidase was purified in two steps by anion exchange and gel-permeation chromatography to high purity. The enzyme is a glycoprotein with an apparent molecular mass of 224 kDa in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and separates into two subunits of equal molecular mass. After SDS-PAGE -xylosidase could be renatured and stained with methylumbelliferyl--xylopyranoside. The enzyme was able to split substrates of other glycosidases. The maximum activity was reached at pH 4.5 and 80° C. -Xylosidase showed high stability over a broad pH range from pH 2.0 to 9.5 and up to 70° C. Analysis of cleavage patterns revealed that the enzyme was a typical glycosidase. Larger oligosaccharides consisting of xylose were degraded by an exomechanism together with a transxylosylation reaction.     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.     

Purification and partial characterization of β-glucosidase from papaya fruit

β-Glucosidase (I) was isolated from Carica papaya fruit pulp and purified ca 1000-fold to electrophoretic homogeneity. The procedure used ammonium sulphate fractionation followed by chromatography on Phenyl-Sepharose CL-4B and Sephacryl S-200 to separate α-mannosidase (II) and, in part, β-galactosidase (III) from (I). Final separation of (III) from (I) was achieved by preparative isoelectric focusing (PIEF). The glycosidases had pI of 5.2 (I), 4.9 (II) and 6.9 (III). M,s of 54 000 (I), 260 000 (II) and 67 000 (III) were determined by gel filtration. The M, of (I) estimated by SDS-PAGE was 27 000 suggesting that (I) consisted of two subunits. The optimum pH and optimum temperature of (I) were 5.0 and 50°, respectively, and the enzyme followed typical Michaelis kinetics with K_m and V_max of 1.1 × 10⁻⁴ M and 1.8 × 10⁻⁶ mol/hr, respectively, for p-nitrophenyl-β-d-glucoside (40°).     

Purification and characterization of extracellular β-glucosidase from Myceliophthora thermophila

The extracellular -glucosidase has been purified from culture broth of Myceliophthora thermophila ATCC 48104 grown on crystalline cellulose. The enzyme was purified approximately 30-fold by (NH₄)₂SO₄ precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G-200 and DEAE-Sephadex A-50. The molecular mass of the enzyme was estimated to be about 120 kD by both sodium dodecyl sulphate gel electrophoresis and gel filtration chromatography. It displayed optimal activity at pH 4.8 and 60°C. The purified enzyme in the absence of substrate was stable up to 60°C and pH between 4.5 and 5.5. The enzyme hydrolysed p-nitrophenyl--d-glucoside, cellobiose and salicin but not carboxymethyl cellulose or crystalline cellulose. The K _m of the enzyme was 1.6mm for p-nitrophenyl--d-glucoside and 8.0mm for cellobiose. d-Glucose was a competitive inhibitor of the enzyme with a K of 22.5mm. Enzyme K activity was inhibited by HgCl₂, FeSO₄, CuSO₄, EDTA, sodium dodecyl sulphate, p-chloromercurobenzoate and iodoacetamide and was stimulated by 2-mercaptoethanol, dithiothreitol and glutathione. Ethanol up to 1.7 m had no effect on the enzyme activity.The authors are with the Department of Microbiology, Bose Institute, 93/1, A.P.C. Road, Calcutta 700 009, India. S.K. Raha is presently with the Department of Medicine, University of Saskatchewan, Saskatoon, Canada S7N OXO.     

Purification and immunological characterization of acid β-galactosidase from dog liver

• 1.1. Dog liver acid β-galactosidase was isolated in high yield and purified to homogeneity using a series of chromatographies on Con A-Sepharose, decyl-agarose, anion-exchange HPLC and gel-filtration HPLC.
• 2.2. Non-denaturing gel filtration by HPLC gave a single homogeneous peak corresponding to molecular mass of 180–190 kDa. During SDS-PAGE analysis, the single peak dissociated into a major band corresponding to molecular mass of 32 kDa with minor bands at 18 and 13 kDa.
• 3.3. Polyclonal antibodies raised against the purified enzyme immunoprecipitated β-galactosidase activity specifically from dog liver extracts and recognized a single 32 kDa band in Western blot analysis of dog tissue homogenates. This antibody did not crossreact with any protein band in tissue homogenates from other species examined except cat.
• 4.4. Western blot analysis of tissue extracts from dogs affected with G_MI-gangliosidosis showed the presence of a 32 kDa band similar to that of controls.

     

Purification and characterization of β-galactosidase from a strain of Bacillus coagulans

-Galactosidase from B. coagulans strain L4 is produced constitutively, has a mol. wt. of 4.3×10⁵ and an optimal temperature of 55°C. The optimal pH at 30°C is 6.0 whereas at 55°C it is 6.5. The energy of activation of enzyme activity is 41.9 kJ/mol (10 kcal/mol). No cations are required. The K_m with ONPG as substrate is 4.2–5.6mm and with lactose is 50mm. The K_i for inhibition by galactose is 11.7–13.4mm and for dextrose is 50mm. Galactose inhibited competitively while dextrose inhibited noncompetitively. The purified and unprotected enzyme is 70% destroyed in 30 min at 55°C whereas in the presence of 2 mg/ml of BSA 42% of the activity is destroyed in 30 min at 55°C. An overall purification of 75.3-fold was achieved.     

Purification and characterization of an extracellular β-D-xylosidase from Aspergillus carbonarius

The production of an extracellular -D-xylosidase (-D-xyloside xylohydrolase, EC 3.2.1.37) by four Aspergillus strains (A. carbonarius, A. nidulans, A. niger and A. oryzae) grown on wheat bran medium was compared. The highest amount of the enzyme was found in the culture of A. carbonarius. The -D-xylosidase from A. carbonarius was purified to homogeneity by a rapid procedure, using hydrophobic interaction chromatography, chromatofocusing and affinity chromatography. The purified enzyme possessed not only -D-xylosidase activity, but also -L-arabinosidase activity. Mixed substrate experiments revealed that a single active centre was responsible for the splitting of the corresponding synthetic substrates. The molecular weight of the purified enzyme proved to be 100,000 Da, as estimated by SDS–PAGE. The isoelectric point was at pH 4.4. The pH and temperature optima were 4.0 and 60 °C, respectively. The enzyme remained stable over a pH range of 3.5–6.5 and up to 50 °C for 30 min. The Michaelis constant for p-nitrophenyl -D-xyloside was 0.198 mM. Kinetic studies demonstrated that the lack of the C-5 hydroxylmethyl group and the configuration of the C-4 hydroxyl group on the pyranoside ring play an important role in both substrate binding and splitting.     

Purification and characterization of cyclodextrin β-glucanotransferase from novel alkalophilic bacilli

The discovery of novel bacterial cyclodextrin glucanotransferase (CGTase) enzyme could provide advantages in terms of its production and relative activity. In this study, eight bacterial strains isolated from soils of a biodiversity-rich vegetation in Egypt based on their hydrolyzing activity of starch, were screened for CGTase activity, where the most active strain was identified as Bacillus lehensis. Optimization process revealed that the using of rice starch (25 %) and a mixture of peptone/yeast extract (1 %) at pH 10.5 and 37 °C for 24 h improved the bacterial growth and enzyme activity. The bacterial CGTase was successively purified by acetone precipitation, gel filtration chromatography in a Sephadex G-100 column and ion exchange chromatography in a DEAE-cellulose column. The specific activity of the CGTase was increased approximately 274-fold, from 0.21 U/mg protein in crude broth to 57.7 U/mg protein after applying the DEAE-cellulose column chromatography. SDS-PAGE showed that the purified CGTase was homogeneous with a molecular weight of 74.1 kDa. Characterization of the enzyme exhibited optimum pH and temperature of 7 and 60 °C, respectively. CGTase relative activity was strongly inhibited by Mg²⁺, Zn²⁺, Al³⁺ and K⁺, while it was slightly enhanced by 5 and 9 % with Cu²⁺ and Fe²⁺ metal ions, respectively.     

Purification and characterization of β-mannanase from Bacillus licheniformis for industrial use

An easily scaled-up technique has been designed to purify -mannanase from Bacillus licheniformis. Using flocculation, ultrafiltration and ion-exchange chromatography, the enzyme was purified 33-fold with a final recovery of 47% and a specific activity of 4341 U mg^–1protein. The enzyme had maximum activity at 60 °C and pH 7.0. It was stable at 50 °C and pH 6.0 for 6 h, but lost all of its activity when held at 70 °C and pH 6.0 for 1 h.     

Purification and characterization of a β-galactoside-binding soluble lectin from rat and bovine brain

A β-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggragation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the β-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30 000 for the bovine brain lectin and 32 000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15 000 and 16 000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for β-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.     

Purification and characterization of β-agarase from marine bacterium Bacillus cereus ASK202

Extracellular agarase of Bacillus cereus ASK202 was purified 32-fold, giving a single band on PAGE with activity staining. The M_r of purified agarase was determined as 90 kDa by SDS-PAGE. The N-terminal amino acid was sequenced and the sequence did not show homology to any other known agarases. The optimum pH and temperature were 7.0 and 40 °C, respectively. This enzyme was found to be a -agarase which catalyzed the hydrolysis of the -1,4 linkage of agarose to yield neoagarohexaose, neoagarotetraose and neoagarobiose.     

Purification and characterization of a sodium-stimulated β-galactosidase from Bifidobacterium longum CCRC 15708

Summary β-galactosidase from Bifidobacterium longum CCRC 15708 was first extracted by ultrasonication then purified by Q Fast-Flow chromatography and gel chromatography on a Superose 6 HR column. These steps resulted in a purification of 15.7-fold, a yield of 29.3%, and a specific activity of 168.6 U mg⁻¹ protein. The molecular weight was 357 kDa as determined from Native-PAGE. Using o-nitrophenyl-β-d-galactopyranoside (ONPG) as a substrate, the pH and temperature optima of the purified β-galactosidase were 7.0 and 50 °C, respectively. The enzyme was stable at a temperature up to 40 °C and at pH values of 6.5–7.0. K _m and V _max for this purified enzyme were noted to be 0.85 mM and 70.67 U/mg, respectively. Na⁺ and K⁺ stimulated the enzyme up to 10-fold, while Fe³⁺, Fe²⁺, Co²⁺, Cu²⁺, Ca²⁺, Zn²⁺, Mn²⁺ and Mg²⁺ inhibited the activity of β-galactosidase. Furthermore, although glucose, galactose, maltose, or raffinose exerted little or no effect on the β-galactosidase activity, lactose and fructose inhibited the enzyme activity. The effect of lactose on the enzyme activity for ONPG is probably a case of competitive inhibition. A relatively high specific activity of β-galactosidase from B. longum CCRC 15708 could be obtained by Q Fast-Flow chromatography and gel chromatography on a Superose 6 HR column. In some aspects, particularly the activation by monovalent cations, the properties of β-galactosidase of B. longum CCRC 15708 are different from those obtained from other sources. Data collected in the present study are of value and indispensable when β-galactosidase from B. longum CCRC 15708 is employed in practical application.     

Purification and characterization of a novel β-galactosidase from Bacillus sp MTCC 3088

An extracellular β-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was harvested from the late stationary-phase of Bacillus sp MTCC 3088. The enzyme was purified 36.2-fold by ZnCl₂ precipitation, ion exchange, hydrophobic interaction and gel filtration chromatography with an overall recovery of 12.7%. The molecular mass of the purified enzyme was estimated to be about 484 kDa by gel filtration on a Sephadex G-200 packed column and the molecular masses of the subunits were estimated to be 115, 86.5, 72.5, 45.7 and 41.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the native enzyme, determined by polyacrylamide gel electrofocusing, was 6.2. The optimum pH and temperature were 8 and 60°C, respectively. The Michaelis–Menten constants determined with respect to o-NO₂-phenyl-β-D-galactopyranoside and lactose were 6.34 and 6.18 mM, respectively. The enzyme activity was strongly inhibited (68%) by galactose, the end product of lactose hydrolysis reaction. The β-galactosidase was specific for β-D anomeric linkages. Enzyme activity was significantly inhibited by metal ions (Hg²⁺, Cu²⁺ and Ag⁺) in the 1–2.5 mM range. Mg²⁺ was a good activator. Catalytic activity was not affected by the chelating agent EDTA. Journal of Industrial Microbiology & Biotechnology (2000) 24, 58–63. Received 09 February 1999/ Accepted in revised form 24 September 1999     

Purification and characterization of a β-glucosidase fromAspergillus niger

The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation, hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110 kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheK _m andk _cat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively, with half-lives of 73 min.     

Purification and characterization of the intracellular β-glucosidase from Aureobasidium sp ATCC 20524

β-Glucosidase hydrolyzing cellobiose was extracted from Aureobasidium sp ATCC 20524 and purified to homogeneity. The molecular mass was estimated to be about 331 kDa. The enzyme contained 26.5% (w/w) carbohydrate. The optimum pH and temperature for the enzyme reaction were pH 4 and 80°C, respectively. The enzyme was stable at a wide range of pH, 2.2–9.8, after 3 h and at 75°C for 15 min. The kinetic parameters were determined. The enzyme was relatively stable against typical organic enzyme inhibitors. The enzyme also hydrolyzed gentiobiose, p-nitrophenyl-β-glucoside and salicin. Received 05 November 1998/ Accepted in revised form 14 February 1999     

Structural and biochemical characterization of glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum

We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)(8)-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.