Copurification and separation of β-galactosidase and sialidase from porcine testis

• 1.1. A soluble sialidase was copurified apparently as an enzyme complex with acid β-galactosidase from porcine testis.
• 2.2. The sialidase exhibited its maximum activity at acidic pH. It was efficiently active towards 4-methylumbelliferyl-α-d-N-acetyl-neuraminic acid and sialyllactose, relatively inactive towards glycoproteins, and had little activity towards glycolipids.
• 3.3. The complex could be separated by sucrose gradient centrifugation or isoelectric focusing.
• 4.4. The separated enzymes had molecular weights about 600,000 for β-galactosidase and more than about 1,000,000 for sialidase by Sepharose 4B gel filtration.
• 5.5. SDS-polyacrylamide gel electrophoresis of the β-galactosidase showed three protein bands with molecular weights of 63,000, 31,000 and 20,000.

     

Generation of superoxide and singlet oxygen from α-tocopherolquinone and analogues

Three potential routes to generation of reactive oxygen species (ROS) from α-tocopherolquinone (α-TQ) have been identified. The quinone of the water-soluble vitamin E analogue Trolox C (Trol-Q) is reduced by hydrated electron and isopropanol α-hydroxyalkyl radical, and the resulting semiquinone reacts with molecular oxygen to form superoxide with a second order rate constant of 1.3 × 10⁸ dm³/mol/s, illustrating the potential for redox cycling. Illumination (UV-A, 355 nm) of the quinone of 2,2,5,7,8-pentamethyl-6-hydroxychromanol (PMHC-Q) leads to a reactive short-lived (ca. 10^{? 6} s) triplet state, able to oxidise tryptophan with a second order rate constant greater than 10⁹ dm³/mol/s. The triplet states of these quinones sensitize singlet oxygen formation with quantum yields of about 0.8. Such potentially damaging reactions of α-TQ may in part account for the recent findings that high levels of dietary vitamin E supplementation lack any beneficial effect and may lead to slightly enhanced levels of overall mortality.     

Separation and purification of α-ketoglutarate and pyruvate from the fermentation broth of Yarrowia lipolytica

Bioprocess and Biosystems Engineering - A strategy to achieve the efficient co-production of α-ketoglutarate (KGA) and pyruvate (PYR) via Yarrowia lipolytica fermentation was established in...     

Purification and properties ofβ-fructofuranosidase from Aspergillus japonicus

-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The K_m value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.     

Purification and properties of α-d-mannosidase from rat epididymis

1. alpha-d-Mannosidase from rat epididymis was purified 300-fold. beta-N-Acetyl-glucosaminidase and beta-galactosidase were removed from the preparation by treatment with pyridine. Zn(2+) was added during the purification to stabilize the alpha-mannosidase. 2. Mammalian alpha-mannosidase is most stable at pH6. At lower pH values it undergoes reversible spontaneous inactivation. The enzyme is also subject to irreversible inactivation, which is delayed by the addition of albumin. 3. Reversible inactivation of alpha-mannosidase is accelerated by EDTA and reversed or prevented by Zn(2+). Other cations, such as Co(2+), Cd(2+) and Cu(2+), accelerate inactivation and the action of a toxic cation can be prevented by Zn(2+) or by EDTA in suitable concentration. 4. The enzyme is stabilized by substrate and neither Zn(2+), EDTA nor a toxic cation has more than a small effect in the assay of an untreated preparation. The addition of Zn(2+) is necessary, however, for a constant rate of hydrolysis during prolonged incubation of the enzyme with substrate. In an EDTA-treated preparation, Zn(2+) reactivates the enzyme during the assay. 5. Evidence is presented that alpha-mannosidase is a dissociable Zn(2+)-protein complex, in which Zn(2+) is essential for enzyme activity.     

Purification and Properties of β-Galactosidases from Bacillus circulans

Six compounds, Z- and E-fadyenolide (3, 4), 1-ally1-2,3-(methylenedioxy)-4,5-dimethoxy-benzene (5), 4-methoxy-3,5-bis (3′-methyl-2′-butenyl)-benzoic acid (6), 2,6-dihydroxy-4-methoxy-dihydrochalcone (7), and 5-hydroxy-7-methoxyflavanone (8) were isolated from three species of Jamaican Piper, Piper fadyenii, C.D.C., Piper aduncum L. and Piper hispidum Sw. Three amides (9 ~ 11) of 3,5-dimethoxy-4-oxo-5-phenylpent-2-enoic acid using piperidine, pyrrolidine and morpholine, respectively, were synthesized from compounds 3 and 4, and tested for insecticidal activity against the tick Boophilus microplus (Canestrini) and the flour feetle, Tribolium confusum Duval. In our experiment, compounds 9 ~ 11 inhibited ovogenesis of B. microplus and were toxic to T. confusum. Compounds 3 ~ 8 were found to have no activity.     

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 Properties of α-Glucosidase from Bacillus cereus

α-Glucosidase has been isolated from Bacillus cereus in ultracentrifugally and electrophoretically homogeneous form, and its properties have been investigated. The enzyme has a sedimentation constant of 1.4 S and a molecular weight of 12,000. The highly purified enzyme splits α-d-(1→4)-glucosidic linkages in maltose, maltotriose, and phenyl α-maltoside, but shows little or no activity toward polysaccharides, such as amylose, amylopectin, glycogen and soluble starch. The enzyme has α-glucosyltransferase activity, the main transfer product from maltose being maltotriose. The enzyme can also catalyze the transfer of α-glucosyl residue from maltose to riboflavin. On the basis of inhibition studies with diazonium-1-H-tetrazole, rose bengal and p-chloromercuribenzoate, it is assumed that the enzyme contains both histidine and cysteine residues in the active center.     

Isolation and Identification of α-Methyloctanylcarnitines from Human Urine

Medium-chain acylcarnitines were isolated from human urine using a combination of chloroform-methanol extraction, silicic acid column and molecular sieving chromatography and preparative HPLC. Three purified acylcarnitines were analyzed by fast atom bombardment mass spectrometry and were also saponified and the free fatty acids analyzed by gas chromatography and mass spectrometry. Combined electron inpact mass spectrometry and fast atom bombardment mass spectrometry and periodate oxidation for location of double bonds, demonstrated the occurrence of δ⁶-octenylcarnitine, 2-methyloct-anylcarnitine and 2-methyl-δ⁶-octenylcarnitine. These acylcarnitines were present in the thirteen urines obtained from normal humans, but were not detected in urines from three individuals who had been on total parenteral nutrition for more than a year. The occurrence of α-methyl medium-chain acylcarnitines in human urine indicates a role for carnitine in excretion (detoxification) of these acyl derivatives.     

Kinetics and properties of β-ketothiolase from Clostridium pasteurianum

1. Beta-Ketothiolase of Clostridium pasteurianum was purified 130-fold by ammonium sulphate fractionation and by column chromatography using DEAE-Sephadex A-50 and hydroxylapatite. Subjected to gel electrophoresis beta-ketothiolase revealed two distinct bands; by isoelectric focusing two enzymes with isoelectric points at pH 4.5 and 7.6 were separated. As established by sucrose density gradient centrifugation the molecular weight of both enzymes was found to be 158000. 2. The condensation reaction was measured by a coupled optical test using beta-hydroxybutyryl-CoA dehydrogenase as auxiliary enzyme and either acetyl-CoA or free coenzyme A plus acetyl-phosphate and phosphotransacetylase (regenerating system) or acetyl-CoA plus regenerating system as substrates. Beta-Ketothiolase from C. pasteurianum used only 20% of the chemically synthesized acetyl-CoA; the enzyme from Alcaligenes eutrophus H 16 used 25%. When the regenerating system was added the condensation reaction continued. The enzyme from C. pasteurianum was inactivated by free coenzyme A, while the enzyme from A. eutrophus was inhibited. When acetyl-CoA was added as the substrate the initial velocity determination was impeded by the lack of linearity. With acetyl-CoA as the substrate the Km-value was found to be 2.5 mM acetyl-CoA. If free CoASH (or acetyl-CoA) plus regenerating system was added the Km was 0.44 mM (0.42 mM) acetyl-CoA. 3. The beta-ketothiolase activity was measured in the direction of acetoacetyl-CoA cleavage by an optical assay following the decrease of the enol and chelate form of acetoacetyl-CoA by absorption measurement at 305 nm. The activity was maximal at 24 nM MgCl2. The apparent Km values for acetoacetyl-CoA were 0.133 mM and 0.105 mM with 0.065 and 0.016 mM CoASH, respectively. The Km-values as calculated for only the keto form of acetoacetyl-CoA were 0.0471 and 0.0372 mM, respectively. The cleavage reaction was inhibited by high acetoacetyl-CoA concentrations; the inihibition was partially relieved by CoASH. In the range of low concentrations of acetoacetyl-CoA only a slight inhibition by CoASH was observed. The Km for CoASH was found to be 0.0288 and 0.0189 mM with 0.09 and 0.045 mM acetoacetyl-CoA, respectively. High concentrations of CoASH exerted an inhibitory effect on the cleavage reaction. With respect to enzyme kinetics and sensitivity to inhibitors and metabolites the beta-ketothiolases of C. pasteurianum and A. eutrophus were rather similar.     

Purification and properties of β-ketothiolase from Zoogloea ramigera

-Ketothiolase from Zoogloea ramigera I-16-M was purified 140-fold to electrophoretic homogeneity. The bacterium appeared to contain a single isoenzyme of -ketothiolase with a molecular weight of 190000, as determined by Sephadex G-200 gel filtration. The monomer molecular weight was 44000, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme thus appeared to be a tetramer with identical subunits.The enzyme showed a pH optimum of 7.5 in the condensation reaction, and 8.5 in the thiolysis reaction. The enzyme employed a Bi Bi ping pong mechanism for the forward thiolysis reaction. The apparent K _m value for acetoacetyl coenzyme A in the thiolysis reaction was 10 M, and that for coenzyme A was 8.5 M. The apparent K _m value for acetyl coenzyme A in the condensation reaction was 0.33 mM. The condensation reaction was inhibited by coenzyme A concentrations lower than 0.1 mM.The enzyme was stable in the presence of dithiothreitol and other SH-compounds, but was strongly inhibited by 0.4 mM p-chloromercuribenzoate.Non-Standard Abbreviation PHB poly--hydroxybutyrate     

Production of d(−)-lactate from sucrose and molasses

Escherichia coli W3110 derivatives, strains SZ63 and SZ85, were previously engineered to produce optically pure D(-) and L(+)-lactate from hexose and pentose sugars. To expand the substrate range, a cluster of sucrose genes (cscR' cscA cscKB) was cloned and characterized from E. coli KO11. The resulting plasmid was functionally expressed in SZ63 but was unstable in SZ85. Over 500 mM D(-)-lactate was produced from sucrose and from molasses by SZ63(pLOI3501).     

Purification and characterization of β‐methylaspartase from Fusobacterium varium

Methylaspartase (EC 4.3.1.2) was purified 20fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAESepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg²⁺ (K_m = 0.27 ± 0.01 mM) and K⁺ (K_m = 3.3 ± 0.8 mM) for maximum activity. Methylaspartasecatalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)3methylaspartate (major product) and (2S,3R)3methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)3methylaspartate (K_m = 0.51 ± 0.04 mM) was observed at pH 9.7. The Nterminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.     

Purification and Separation of A and B Forms of N-Acetyl-β-D-Hexosaminidase from Human Aorta

Human aorta has been shown to possess multiple forms of N-Acetyl-6-D-hexosaminidase (β-2-acetamido-2-deoxy-D-glucoside-acetamido-deoxyglucohydro-lase, EC 3.2, 1.30). The enzyme was separable, by gel electrophoresis, into 2 enzymatically active bands representing A and B forms. By gel electro-focussing, A and B forms were further subdivided into at least 5 and 8 bands, respectively. The B form consisted of 4 bands (B₁) and 4 bands (B₂), which were not inactivated at 50° for 3 hr. (at pH 4.4) in the presence of serum; whereas, the 5 bands found in A form were completely inactivated. All forms of the enzyme were active towards naphthol-AS-BI-N-acetyl-β-D-glucosaminide and the corresponding galactosaminide (about one-eighth of the hydrolysis rate of the former), suggesting each single enzyme acts on both substrates. The N-acetyl-hexosaminidases of bull epididymis, by comparison, were also found to be active towards both substrates and to possess 13 bands having pis more alkaline than those of the B form of the human enzyme, By heat inactivation we found that the aortic enzyme consisted of 51% of A and 49% of B (B₁ + B₂ .). Neuraminidase had no effect on either form of the aortic preparation. Both forms were partially purified and separated by conventional methods. They required BSA for their maximal activity; the A form being more dependent BSA than the B form, With PNP-N-acetyl-β-D-glucosaminide and the corresponding galactosaminide, Km of 1.04 mH and 0.54 mM, respectively, for A form and of 1.74 and 1.48 mM, respectively, for B form were obtained. While the purified B form was stable and did not transform into other species, the purified A form gradually transformed into B form as well as into other new forms during storage at -20°.     

Purification and characterization of α-glucosidase from Aspergillus carbonarious

Summary An -glucosidase was purified from Aspergillus carbonarious CCRC 30414 over 20 fold with 37 % recovery. Its molecular mass was estimated to be 328 kDa by gel filtration with an optimum pH from 4.2 to 5.0, and pI=5.0. The optimum temperature is at 60°C over 40 min. The enzyme was partially inhibited by 5 mM Ag⁺, Hg²⁺, Ba²⁺, Pb²⁺, and Aso₄ ⁺.     

Isolation and Identification of l-Menthyl-β-d-glucoside from Shubi

Quinoxalines derived from d-galactose with o-phenylenediamine (OPD) in acidic media under reflux were studied by using GLC and NMR measurements. Four quinoxaline derivatives were obtained from the reaction mixture, and were identical with those derived from d-glucose. The yields of 2-(D-lyxo-tetrahydroxybutyl)quinoxaline (GA-III), and the stereoisomeric derivative of GA-III, i.e., 2-(D-arabino-tetrahydroxybutyl)quinoxaline (ATBQ), were 13.2 and 5.3–, respectively. The ratio of GA-III to ATBQ derived from d-galactose was reciprocally coincident with that from d-glucose. Some proposals are made on the relationship between the isomerization of these sugars and the formation of quinoxaline derivatives.     

Purification and properties of α-d-mannosidase from jack-bean meal

1. α-Mannosidase from jack-bean meal was purified 150-fold. β-N-Acetyl-glucosaminidase and β-galactosidase were removed from the preparation by treatment with pyridine. Zn²⁺ was added during the purification to stabilize the α-mannosidase. 2. At pH values below neutrality, α-mannosidase undergoes reversible spontaneous inactivation at a rate dependent on the temperature, the degree of dilution and the extent of purification. The enzyme is also subject to irreversible inactivation, which is prevented by the addition of albumin. 3. Reversible inactivation of α-mannosidase is accelerated by EDTA and reversed or prevented by Zn²⁺. Other cations, such as Co²⁺, Cd²⁺ and Cu²⁺, accelerate inactivation; an excess of Zn²⁺ again exerts a protective action, and so does EDTA in suitable concentration. 4. Neither Zn²⁺ nor EDTA has any marked effect in the assay of untreated enzyme. In an EDTA-treated preparation, however, Zn²⁺ reactivates the enzyme during assay. 5. It is postulated that α-mannosidase is a dissociable Zn²⁺–protein complex in which Zn²⁺ is essential for enzyme activity.     

Partial purification and properties of γ-glutamyltranspeptidase from mycelia of Morchella esculenta

Three -glutamyltranspeptidase (enzymes I, II and III) were partially purified from the cell free extracts of the cultured mycelia of Morchella esculenta Fr. The molecular masses of enzymes were 155,000 (I), 219,000 (II) and 102,000 (III). All of them catalyzed both hydrolysis and transpeptidation of various -glutamyl compounds. -l-Glutamyl-cis-3-amino-l-proline occurring in the cultured mycelia of this fungus was a good substrate for both reactions. K _m values for hydrolysis were in the order of 10^-4 to 10^-5 M, and those for transpeptidation were in the order of 10^-2 to 10^-4 M. The enzymes were inhibited by a -glutamyltranspeptidase inhibitor, l-serine plus borate.Abbreviations -GTP -glutamyltranspeptidase - HPLC High-performance liquid chromatography