Suppression of transcription of the ribosomal RNA cistrons of Drosophila melanogaster in a structurally rearranged chromosome

Evidence has been sought on the possible existence of multiple forms of the enzyme controlled by the Li locus in white clover. During purification of enzyme from LiLi plants, there was no separation of activities against the -glucosides, p-nitrophenyl -d-glucoside, salicin, and linamarin-lotaustralin, and the -galactoside, p-nitrophenyl -d-galactoside. In addition, tests on mixtures of these four substrates provided no evidence for the existence of more than one enzyme. Immunological tests have shown that plants homozygous for the recessive li allele do not contain an enzymatically inactive protein, antigenically related to the normal enzyme. This suggests that li alleles either specify a low-activity immunologically altered protein or control the synthesis of very low levels of normal enzyme.     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 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 by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

Regulation of β-xylosidase formation by xylose in Trichoderma reesei

The soft-rot fungus Trichoderma reesei forms -xylosidase (EC 3.2.1.37) activity during cultivation on xylan and xylose, but not on glucose. When mycelia precultivated on glycerol were washed and transferred to fresh medium without a carbon and nitrogen source, -xylosidase formation was induced by xylan, xylobiose and xylose. A supply of 4 mm xylose and a pH of 2.5 provided optimal conditions for induction. -Xylosidase accounted for the major portion of total extracellular protein under these conditions, and could be purified to physical homogeneity by a single anion exchange chromatography step. A recombinant strain of T. reesei that carries multiple copies of the homologous xylanase II-encoding gene has a six-fold increased xylanase activity, but forms comparable -xylosidase activities. This shows that the rate of xylan hydrolysis has no effect on the induction of -xylosidase. Methyl--d-xyloside inhibited -xylosidase competitively and was a weak -xylosidase inducer. The induction by xylobiose and xylan was strongly enhanced by the simultaneous addition of methyl--d-xylosidese and xylan or xylobiose. The results suggest that a slow supply of xylose is a trigger for -xylosidase induction.     

Properties of β-glucosidase purified from Aspergillus niger mutants USDB 0827 and USDB 0828

Summary Two extracellular -glucosidases (EC 3.2.1.21) were isolated from Aspergillus niger USDB 0827 and A. niger USDB 0828, and their physical and kinetic properties studied. Both enzymes were very similar in terms of molecular size (230000 Da), pH optimum (pH 4.6), temperature optimum (65° C), stability at high temperatures and substrate preferences. They were capable of hydrolysing -linked disaccharides, phenyl -d-glucoside, p-nitrophenyl -d-glucoside (PNPG), o-nitrophenyl -d-glucoside, salicin and methyl -d-glucoside but lacked activity towards -linked disaccharides, a range of p-nitrophenyl monoglycosides and p-nitrophenyl diglycosides. Both -glucosidases were better at hydrolysing cellobiose than cellotriose, cellotetraose or cellopentaose. For both enzymes, glucose showed competitive inhibition with PNPG as substrate but had no effect with cellobiose. However, the two -glucosidases differed in inhibition by glucono-1,5-lactone and affinity for cellobiose. -Glucosidase from A. niger USDB 0827 also gave lower specific activity, and was more susceptible to metal ions (Ag⁺, Fe²⁺ and Fe³⁺) inhibition than that of A. niger USDB 0828. Correspondence to: Y. K. Hoh     

Purification and characterization of α(2-6)-sialyltransferase from human liver

A Gal1-4GlcNAc (2-6)-sialyltransferase from human liver was purified 34 340-fold with 18% yield by dye chromatography on Cibacron Blue F3GA and cation exchange FPLC. The enzyme preparation was free of other sialyltransferases. It did not contain CMP-NeuAc hydrolase, protease, or sialidase activity, and was stable at –20°C for at least eight months. The donor substrate specificity was examined with CMP-NeuAc analogues modified at C-5 or C-9 of theN-acetylneuraminic acid moiety. Affinity of the human enzyme for parent CMP-NeuAc and each CMP-NeuAc analogue was substantially higher than the corresponding Gal1-4GlcNAc (2-6)-sialyltransferase from rat liver.Abbreviations FPLC fast protein liquid chromatography - NeuAc 5-N-acetyl-d-neuraminic acid - 9-amino-NeuAc 5-acetamido-9-amino-3,5,9-trideoxy-d-glycero-2-nonulosonic acid - 9-acetamido-NeuAc 5,9-diacetamido-3,5,9-trideoxy-d-glycero--d-2-nonulosonic acid - 9-benzamido-NeuAc 5-acetamido-9-benzamido-3,5,9-trideoxy-d-glycero--d-galacto-2-nonulosonic acid - 9-fluoresceinyl-NeuAc 9-fluoresceinylthioureido-NeuAc - 5-formyl-Neu 5-formyl--d-neuraminic acid - 5-aminoacetyl-Neu 5-aminoacetyl--d-neuraminic acid - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - G_M1 Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-ceramide - ST sialyltransferase - DTE 1,4-dithioerythritol Enzyme: Gal1-4GlcNAc (2-6)-sialyltransferase, EC 2.4.99.1.     

Molecular cloning and expression of multiple cellulase genes of Ruminococcus flavefaciens strain 186 in Escherichia coli

Summary Cellulase genes of the ruminant micro-organism Ruminococcus flavefaciens strain 186 have been cloned and expressed in Escherichia coli using the bacteriophage vector NM1149. Twenty-six clones showed expression of endo--1,4-d-glucanases and were divided into four groups according to their insert sizes of approximately 2, 3, 4 or 9 kilobases (kb). Two of the clones with 4 kb inserts also showed exo--1,4-d glucanase activity while two clones with 9 kb inserts showed -glucosidase activity. One of the clones with 9 kb inserts (M903) showed the activities of all three cellulase activities. In addition, two of the 4 kb-insert clones and one 9 kb-insert clone degraded Avicel (PH101).     

Enzymatic β-galactosidation of β-xylopyranosides

Summary The disaccharides formed by enzymatic transfer of the -D-galactopyranosyl residue fromo-nitrophenyl -d-galactopyranoside to -d-xylopyranosides have been identified. The influence of different factors on the yields of the disaccharides obtained was evaluated. Significant changes in selectivity were observed when -galactosidase fromE. coli was used instead of -galactosidase fromA. oryzae.     

β-Galactosidase of Pediococcus species: induction,purification and partial characterization

Summary Six strains of Pediococcus pentosaceus and two of P. acidilactici had intracellular -galactosidase (-gal) activity when grown in the presence of lactose; all but two strains of P. pentosaceus and one of P. acidilactici had such activity when grown in the presence of glucose. Synthesis of -gal by P. pentosaceus ATCC 25745 was inducible with lactose, galactose, melibiose, lactobionic acid and possibly cellobiose but not with glucose, sucrose, maltose, glycerol, fructose or mannose. Lactose, galactose and possibly maltose, melibiose and lactobionic acid but not glucose, sucrose, glycerol, cellobiose, fructose or mannose induced -gal synthesis by P. acidilactici ATCC 25740. Synthesis of -gal was partially inhibited in P. pentosaceus ATCC 25745 and P. acidilactici ATCC 25740 by glucose added to the medium during growth in the presence of galactose or lactose. Isopropyl -d-thiogalactopyranoside failed to induce synthesis of -gal by either strain during growth on glucose. -Gal from P. pentosaceus ATCC 25745 had a molecular weight of 66,000 and activity optima of pH 6.5 and 45° C. Activity of the enzyme was stimulated by reducing agents, Mg²⁺, Mn²⁺, Zn²⁺ and Co²⁺ but not by Ca²⁺, and was markedly inhibited by ethylenediaminetetraacetate (EDTA), HgCl₂, 1,10-phenanthroline, and an oxidizing agent. The K _mvalues of the enzyme for o-nitrophenol--d-galactopyranoside and lactose were 3.07 and 7.0 mM, respectively, suggesting its low affinity for lactose. Offprint requests to: E. H. Marth     

Enzymic synthesis of lacto-N-triose II and its positional analogues

N-acetylhexosaminidase fromNocardia orientalis catalysed the synthesis of lacto-N-triose II glycoside (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OMe,3) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OMe (4) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OMe (5) throughN-acetylglucosaminyl transfer fromN,N-diacetylchitobiose (GlcNAc₂) to methyl -lactoside. The enzyme formed the mixture of trisac-charides3, 4 and5 in 17% overall yield based on GlcNAc₂, in a ratio of 20:21:59. Withp-nitrophenyl -lactoside as an acceptor, the enzyme also producedp-nitrophenyl -lacto-N-trioside II (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p,6) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p (7) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OC₆H₄NO₂-p (8). In this case, when an inclusion complex ofp-nitrophenyl lactoside acceptor with -cyclodextrin was used, the regioselectivity of glycosidase-catalysed formation of trisaccharide glycoside was substantially changed. It resulted not only in a significant increase of the overall yield of transfer products, but also in the proportion of the desired compound6.Abbreviations GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-d-glucose - NAHase N-acetylhexosaminidase - -CD -cyclodextrin     

Purification and characterization of an extracellular β-glucosidase from the anaerobic fungus Piromyces sp. strain E2

An extracellular -glucosidase (EC 3.2.2.21) from the anaerobic fungus Piromyces sp. strain E2 was purified. The enzyme is a monomer with a molecular mass of 45 kDa and a pI of 4.15. The enzyme readily hydrolyzes p-nitrophenyl--d-glycoside, p-nitrophenyl--d-fucoside, cellobiose, cellotriose, cellotetraose and cellopentaose but is not active towards Avicel, carboxymethylcellulose, xylan, p-nitrophenyl--d-galactoside and p-nitrophenyl--d-xyloside. To cleave p-nitrophenyl--d-glucoside the maximum activity is reached at pH 6.0 and 55°C, and the enzyme is stable up to 72 h at 40°C. Activity is inhibited by d-glucurono--lactone, cellobiose, sodium dodecyl sulfate, Hg²⁺ and Cu²⁺ cations. With p-nitrophenyl--d-glycoside, p-nitrophenyl--d-fucoside, and. cellobiose as enzyme substrates, the K _m and V _max balues are 1.5 mM and 25.5 IU·mg^-1, 1.1. mM and 133 IU·mg^-1, and 0.05 mM and 55.6 IU·mg^-1, respectively.     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

Purification and characterization of recombinant Escherichia coli-expressed Pichia etchellsii β-glucosidase II with high hydrolytic activity on sophorose

-Glucosidase II (Bgl II), encoded by the glu2 gene of the thermo-tolerant yeast Pichia etchellsii, was purified from recombinant Escherichia coli pBG22:JM109. The enzyme had a molecular mass of 176 kDa and was a dimer with an apparent subunit mass of 83 kDa. It exhibited broad substrate specificity and hydrolyzed -linked gluco-disaccharides and oligosaccharides, salicin, and cyanogenic glucoside amygladin. The unusually high hydrolytic activity of 7,680 units min^–1 g^–1 protein was obtained on sophorose. Competition experiments performed using differently linked -disaccharides indicated these to be hydrolyzed at the same active site. Transglycosylation activity leading to the biosynthesis of several disaccharides and oligosaccharides was observed. The enzyme was placed in glycosyl hydrolase family 3, based on a statistical approach using amino acid composition data. The involvement of His as a catalytically important residue was confirmed by diethylpyrocarbonate modification. Pre-incubation of the purified enzyme with 5 mM p-nitrophenyl--d-glucoside offered 2.5-fold higher residual activity compared with unbound enzyme, indicating protection at the active site. The feasibility of this enzyme as a biocatalyst of choice for the synthesis of glyco-conjugates is discussed.     

Properties of an intracellular β-glucosidase purified from the cellobiose-fermenting yeast Candida wickerhamii

An intracellular -glucosidase was isolated from the cellobiose-fermenting yeast, Candida wickerhamii. Production of the enzyme was stimulated under aerobic growth, with the highest level of production in a medium containing cellobiose as a carbohydrate source. The molecular mass of the purified protein was approximately 94 kDa. It appeared to exist as a dimeric structure with a native molecular mass of about 180 kDa. The optimal pH ranged from 6.0 to 6.5 with p-nitrophenyl -d-glucopyranoside (NpGlc) as a substrate. The optimal temperature for short-term (15-min) assays was 35°C, while temperature-stability analysis revealed that the enzyme was labile at temperatures of 28° C and above. Using NpGlc as a substrate, the enzyme was estimated to have a K _m of 0.28 mM and a V _max of 525 mol product min^–1 mg protein^–1. Similar to the extracellular -glucosidase produced by C. wickerhamii, this enzyme resisted end-product inhibition by glucose, retaining 58% of its activity at 100 mM glucose. The activity of the enzyme was highest against aryl -1,4-glucosides. However, p-nitrophenyl xylopyranoside, lactose, cellobiose, and trehalose also served as substrates for the purified protein. Activity of the enzyme was stimulated by long-chain n-alkanols and inhibited by ethanol, 2-propanol, and 2-butanol. The amino acid sequence, obtained by Edman degradation analysis, suggests that this -glucosidase is related to the family-3 glycosyl hydrolases.     

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.     

Glucosylation of cyclodextrin-complexed podophyllotoxin by cell cultures of Linum flavum L.

The glucosylation of the cytotoxic lignan podophyllotoxin by cell cultures derived from Linum flavum was investigated. Four cyclodextrins: -cyclodextrin, -cyclodextrin, dimethyl--cyclodextrin and hydroxypropyl--cyclodextrin were used to improve the solubility of podophyllotoxin by complexation. Dimethyl--cyclodextrin met our needs the best and the solubility of podophyllotoxin could be enhanced from 0.15 to 1.92 mM, using a podophyllotoxin/cyclodextrin ratio of 1:1. Growth parameters of the cell suspensions were not affected neither by the addition of cyclodextrins alone, nor when complexed podophyllotoxin was dissolved in the medium.The complexed lignan disappeared rapidly from the culture medium, within 24h, under all experimental conditions. Almost simultaneously, between 73 and 100% of detectable podophyllotoxin was bioconverted into podophyllotoxin--d-glucoside. A maximal bioconversion rate of 0.51 mmol l^-1 suspension day^-1 was calculated for the L. flavum cells growing in a medium which included the podophyllotoxin/dimethyl--cyclodextrin complex at a final concentration of 1.35 mM.     

Production,purification, and properties of β-glucosidase from Candida wickerhamii

Summary Candida wickerhamii growing on cellobiose produced -glucosidase with high activity against -nitrophenyl glucoside (PNPG) but low activity against cellobiose. -glucosidase production was constitutive, and was repressed by -glucosides and glucose. -glucosides containing an aromatic moiety in the aglycon were the best substrates for -glucosidase indicating that the enzyme is an aryl--glucosidase. A -glucosidase from C. wickerhamii cells was purified by (NH₄)₂SO₄ precipitation, dialysis, ion-exchange chromatography and gel filtration. The purified enzyme was homogeneous as shown by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme hydrolysed PNPG but not cellobiose. The K_m of the enzyme was 0.185 mM. Glucose inhibited the enzyme competitively and the K_i was 7.5 mM. The apparent molecular mass was 97,000. The optimum pH and temperature for enzyme activity were between pH 7 and 7.4 and 40°C respectively. At temperatures of 45°C and greater the enzyme was inactivated. The activation energy of the enzyme was 29.4 kJ · mol^-1.     

Purine glucosylating activity in cell suspension cultures ofSolanum tuberosum L.

Cell suspension cultures ofSolanum tuberosum L. cv. Adretta were established from leaf-derived calluses. In the search for purine glucosylating activity, the metabolism of 6-benzylaminopurine was studied. The main metabolite of BA was isolated and identified as 6-benzylaminopurine 7--d-glucopyranoside indicating the occurrence of purine glucosylating activity.Abbreviations BA 6-Benzylaminopurine - [3G]BA BA 3--d-glucopyranoside - [7G]BA BA 7--d-glucopyranoside - [9G]BA BA 9--d-glucopyranoside - RA Radioactivity - R _T Retention Time     

Purification and characterization of flavone-specific β-glucuronidase from callus cultures of Scutellaria baicalensis Georgi

-Glucuronidase from callus cultures of Scutellaria baicalensis Georgi was purified to apparent homogeneity by fractionated ammonium-sulfate precipitation and chromatography on diethylaminoethyl-cellulose, hydroxylapatite and baicalin-conjugated Sepharose 6B. A 650-fold purification was obtained by this purification system. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein migrated as a single band with a molecular mass of 55 kDa. We determined that the native enzyme has a molecular mass of 230 kDa using gel-filtration chromatography. These results suggested that the enzyme exists as a homotetramer composed of four identical 55-kDa subunits. The enzyme showed a broad pH optimum between 7.0 and 8.0. The K _m values were 9 M, 10 M, 30 M and 40 M for luteolin 3 -O--d-glucuronide, baicalin, wogonin 7-O--d-glucoronide and oroxlin 7-O--d-glucuronide, respectively. The enzyme was most active with flavone 7-O--d-glucuronides.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - pI isoelectric point - R _t retention time     

Isolation of a new cellulase gene from a thermophilic anaerobe and its expression inEscherichia coli

Summary A new cellulase gene was cloned and expressed inEscherichia coli from a thermophilic anaerobe, strain NA10. A 7.4 kbEcoRI fragment of NA10 DNA encoded the cellulase which hydrolyzed carboxymethyl cellulose, lichenan, andp-nitrophenyl--d-cellobioside, but could not digest laminarin andp-nitrophenyl--d-glucoside. The cloned enzyme could digest cellooligosaccharides and release cellobiose as a main product from cellotetraose but could not digest cellobiose. It was distinct from the endoglucanase which was cloned by us previously from NA10 strain in terms ofp-nitrophenyl--d-cellobioside degradation activity and the location of restriction enzyme sites. The enzyme produced byE. coli transformant was extremely heat-stable and the optimum temperature for the enzymatic reaction was 80°C. Fifty three percent of the cloned enzyme was detected in the periplasm and the remaining activity existed in the cellular fraction in theE. coli transformant.     

d-Xylose as inducer of the xylan-degrading enzyme system in the yeast Pullularia pullulans

Summary The specificity of induction of wooddegrading enzymes from Pullularia pullulans was investigated using series of mono-, di- and (14)--trisaccharides or glycanes. A strain of P. pullulans (1740), unable to grow on Avicel or carboxymethyl-cellulose (CMC), uses xylan and steamexploded wood as carbon sources. This strain, thus grown, was evaluated for various enzyme activities. d-Xylose was the nutritional inducer of -xylosidase and -xylanase. d-Glucuronic acid induced activity on CMC and -glucosidase activity was observed regardless of carbon source used. (14)--Xylobiose was not an inducer of -xylanase production, but high levels of this enzyme were obtained with either structural isomers (12) or (13)-. Since synthesis of this enzyme was stimulated by increasing xylose concentration yp to 40 g/l, it is suggested that xylose enters the cells by passive transport and is unable to induce a permease system.Affiliated to the Scientific, Technological and Medical University of Grenoble