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     

Influence of temperature on the properties of the xylanolytic enzymes of the thermotolerant fungus<Emphasis Type="Italic"> Aspergillus phoenicis</Emphasis>

This study reports on the effects of growth temperature on the secretion and some properties of the xylanase and -xylosidase activities produced by a thermotolerant Aspergillus phoenicis. Marked differences were observed when the organism was grown on xylan-supplemented medium at 25 °C or 42 °C. Production of xylanolytic enzymes reached maximum levels after 72 h of growth at 42 °C; and levels were three- to five-fold higher than at 25 °C. Secretion of xylanase and -xylosidase was also strongly stimulated at the higher temperature. The optimal temperature was 85 °C for extracellular and 90 °C for intracellular -xylosidase activity, independent of the growth temperature. The optimum temperature for extracellular xylanase increased from 50 °C to 55 °C when the fungus was cultivated at 42 °C. At the higher temperature, the xylanolytic enzymes produced by A. phoenicis showed increased thermostability, with changes in the profiles of pH optima. The chromatographic profiles were distinct when samples obtained from cultures grown at different temperatures were eluted from DEAE–cellulose and Biogel P-60 columns.     

Cloning of a gene encoding a thermo-stable endo-beta-1,4-glucanase from Thermoascus aurantiacus and its expression in yeast

A gene encoding a thermo-stable endo--1,4-glucanase was isolated from the thermophilic fungus, Thermoascus aurantiacusIFO9748, and designated as eg1. Induction of this gene expression at 50°C was stronger than at 30°C. The deduced amino acid sequence encoded by eg1 showed that it belongs to the glycoside hydrolase family 5. The cloned gene was expressed in Saccharomyces cerevisiae and the gene product was purified and characterized. No significant activity loss was detected over 2 h at 70°C and the product was stable from pH 3–10. The enzyme was optimally active at 70°C over 20 min and the optimal pH was 6.     

Hydrolysis of oligosaccharides of the β-(1→4)-linked d-xylose series by an endo(1→4)-β-d-xylanase from the anaerobic rumen fungus Neocallimastix frontalis

An endo-(14)--d-xylanase from Neocallimastix frontalis was purified by anion-exchange chromatography. The enzyme had an apparent molecular mass of 30 kDa on SDS-PAGE and exhibited maximum activity at 50°C and at pH values between 6.0 and 6.6. Kinetic studies on the hydrolysis of xylo-oligosaccharides, ranging from xylobiose to xylodecaose, showed that xylohexaose and xyloheptaose were the preferred substrates for the enzyme and that xylobiose, xylotriose and xylotetraose were not hydrolysed. Xylose was not a product of the hydrolysis of any of the xylo-oligosaccharide substrates tested. The enzyme appeared to have a strong preference for the hydrolysis of the internal glycosidic bonds of the oligosaccharides, which is typical of endo-(14)--d-xylanase activity, but it differed from other fungal endo-(14)--d-xylanases in that it had uniform action on the various internal linkages in the xylo-oligosaccharides.V. Garcia-Campayo, S.I. McCrae and T.M. Wood are with The Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB, UK     

Purification and biochemical properties of a thermostable xylose-tolerant β-<Emphasis Type="SmallCaps">D</Emphasis>-xylosidase from <Emphasis Type="Italic">Scytalidium thermophilum</Emphasis>

The thermophilic fungus Scytalidium thermophilum produced large amounts of periplasmic -D-xylosidase activity when grown on xylan as carbon source. The presence of glucose in the fresh culture medium drastically reduced the level of -D-xylosidase activity, while cycloheximide prevented induction of the enzyme by xylan. The mycelial -xylosidase induced by xylan was purified using a procedure that included heating at 50°C, ammonium sulfate fractioning (30–75%), and chromatography on Sephadex G-100 and DEAE-Sephadex A-50. The purified -D-xylosidase is a monomer with an estimated molecular mass of 45 kDa (SDS-PAGE) or 38 kDa (gel filtration). The enzyme is a neutral protein (pI 7.1), with a carbohydrate content of 12% and optima of temperature and pH of 60°C and 5.0, respectively. -D-Xylosidase activity is strongly stimulated and protected against heat inactivation by calcium ions. In the absence of substrate, the enzyme is stable for 1 h at 60°C and has half-lives of 11 and 30 min at 65°C in the absence or presence of calcium, respectively. The purified -D-xylosidase hydrolyzed p-nitrophenol--D-xylopyranoside and p-nitrophenol--D-glucopyranoside, exhibiting apparent K_m and V_max values of 1.3 mM, 88 mol min^–1 protein^–1 and 0.5 mM, 20 mol min^–1 protein^–1, respectively. The purified enzyme hydrolyzed xylobiose, xylotriose, and xylotetraose, and is therefore a true -D-xylosidase. Enzyme activity was completely insensitive to xylose, which inhibits most -xylosidases, at concentrations up to 200 mM. Its thermal stability and high xylose tolerance qualify this enzyme for industrial applications. The high tolerance of S. thermophilum -xylosidase to xylose inhibition is a positive characteristic that distinguishes this enzyme from all others described in the literature.     

Living cell reaction process forl-isoleucine andl-valine production

Summary A new process (Living Cell Reaction Process) forl-isoleucine production using viable, non-growing cells ofBrevibacterium flavum AB-07 was optimised using ethanol as the energy source and -ketobutyric acid (-KB) as precursor.l-valine also could be produced from glucose at high yield by this process. This process differs from the usual fermentation method in that non-growing cells are used, and the production ofl-isoleucine andl-valine were carried out under conditions of repressed cell division and growth. Minimal medium missing the essential growth factor, biotin was employed as the reaction mixture for the production ofl-isoleucine andl-valine. The productivity ofl-isoleucine andl-valine were 200 mmol·l^–1 · day^–1 (molecular yield to -KB: 95%) and 300 mmol · l^–1 · day^–1 (molecular yield to glucose: 80%) respectively. The content ofl-isoleucine andl-valine in total amino acids produced in the each mixture were 97% and 96% respectively.     

Uptake of acetyl-l-carnitine in the brain

Analysis in mouse brain slices of the uptake of acetyl-l-[N-methyl-¹⁴C]carnitine with time showed it to be concentrative, and kinetic analysis gave aK _m of 1.92 mM and aV _max of 1.96 mol/min per ml, indicating the presence of a low-affinity carrier system. The uptake was energy-requiring and sodium-dependent, being inhibited in the presence of nitrogen (absence of O₂), sodium cyanide, low temperature (4°C), and ouabain, and in the absence of Na⁺. The uptake of acetyl-l-carnitine was not strictly substrate-specific; -butyrobetaine,l-carnitine,l-DABA, and GABA were potent inhibitors, hypotaurine andl-glutamate were moderate inhibitors, and glycine and -alanine were only weakly inhibitory. In vivo, acetyl-l-carnitine transport across the blood-brain barrier had a brain uptake index of 2.4±0.2, which was similar to that of GABA. These results indicate an affinity of acetyl-l-carnitine to the GABA transport system.     

Extracellular mannanases and galactanases from selected fungi

Summary Eight thermophilic fungi were tested for production of mannanases and galactanases. Highest mannanase activities were produced byTalaromyces byssochlamydoides andTalaromyces emersonii. Mannanases from all strains tested were induced by locust bean gum except in the case ofThermoascus aurantiacus, where mannose had a greater inducing effect. Locust bean gum was also the best inducer of -mannosidase and galactanase except in the case ofT. emersonii where galactose was a better inducer of both these enzymes. Highest mannanase activity was produced byTalaromyces species when peptone was used as nitrogen source whereas sodium nitrate promoted maximum production of this enzyme byThielavia terrestris andT. aurantiacus. The pH optima of mannanases from the thermophilic fungi were in the range 5.0–6.6 and contrasted with the low pH optimum (3.2) of the enzyme fromAspergillus niger. Galactanases had pH optima in the range 4.3–5.8. The mannanase fromT. emersonii and the galactanase fromT. terrestris were most thermostable, each retaining 100% activity for 3 h at 60°C.     

An improved selection method for λlacZ − phages based on galactose sensitivity

The determination of thelacZ mutant frequency in gt10lacZ phage vectors isolated from the transgenic mouse strain 40.6 (MutaMouse), requires the screening of large numbers of phages on -galactosidase activity. Existing methods rely on distinguishing a few white plaques on X-gal containing plates amongst a multide of blue ones which is both time-consuming and expensive. The new screening method described here employs the galactose sensitiveEscherichia coli C lacZ recA galE strain into which a multicopy plasmid has been introduced, which results in over-expression of thegalK andgalT genes. In the presence of phenyl--d-galactopyranoside, a substrate for -galactosidase, this leads to the suppression of lacZ ⁺ phage propagation without affecting the ability of lacZ ^– phages to form plaques. With this method it is possible to screen 1.5×10⁶ phages on a single 9-cm Petri dish. Furthermore, the need for blue/white screening has been eliminated.     

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.     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Fungal thermostable α-dialkylamino acid aminotransferase: occurrence,purification and characterization

-Dialkylamino acid aminotransferase was found in various fungi; this is the first evidence for the occurrence of the enzyme in eukaryotes. The enzyme was purified from Fusarium solani and shown to be composed of four subunits with an identical molecular weight of 42,000. -Aminoisobutyrate and cycloleucine served as amino donors, and pyruvate, -ketobutyrate, -ketovalerate, -ketoisovalerate, and glyoxylate as amino acceptors. The K _m values for -aminoisobutyrate and -ketobutyrate were 28 and 0.3 mM, respectively. -Ketobutyrate inhibited the enzyme noncompetitively with -aminoisobutyrate, and showed K _i value of 8 mM. The significant inhibitory effect of l-cycloserine was observed, but d-cycloserine did not inhibit the enzyme. The pH and temperature optima for transamination of -aminoisobutyrate with pyruvate were about 8.0 and 60°C, respectively. Despite the production of this enzyme by the mesophile, the enzyme was thermostable; it retained its full activity upon heating at 60°C for 30 min.Abbreviations ACPC 1-aminocyclopropane-1-carboxylic acid - AIB -aminoisobutyrate - PLP pyridoxal 5-phosphate     

Induction and partial characterization of intracellular <Emphasis Type="Italic">β</Emphasis> from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> and its application in the synthesis of 6-kestose

Production of -fructofuranosidase from the thermophilic fungus Thermoascus aurantiacuswas enhanced by optimization of the type of nitrogen source as well as the type and concentration of carbon source. Submerged batch cultivation in a laboratory bioreactor (7 l) using the optimized medium allowed the production of 85 mU/ml of -fructofuranosidase. The enzyme showed both transfructosylating and hydrolytic activities and was optimally active at 60 °C and pH 5.0. The enzyme showed the ability to catalyse the synthesis of 1-kestose and the reaction was maximized at 30% (w/v) initial sucrose concentration.     

Cellodextrin utilization and β-glucosidase production by Bacteroides polypragmatus

Bacteroides polypragmatus, a mesophilic obligate anaerobe, was shown to simultaneously ferment glucose and cellobiose giving ethanol as a major metabolic end-product. A mixture of higher cellodextrins was also utilized. The bacterium produced a -glucosidase with a pI value of 4.2 and a molecular weight of approximately 100000 daltons. The enzyme was intracellular and functioned optimally at pH 7. The K _m values obtained with p-nitrophenyl--d-glucoside and cellobiose as substrates were 0.73 mM and 100 mM, respectively. The enzyme was quite stable at elevated temperatures; in the presence of 10% glycerol (v/v), it had a half-life of 4 h at 55°C. It was also stable during long-term storage at either 4°C or-20°C, provided that 10% (v/v) glycerol was added to preparations maintained at-20°C.Abbreviations HPLC high-performance liquid chromatography - IEF isoelectric focusing - pNPG p-nitrophenyl--d-glucoside NRCC No. 25676     

Abnormal expression ofα-L-fucosidase in lymphoid cell lines of fucosidosis patients

Fucosidosis is an autosomal recessive lysosomal storage disease due to a deficiency of-L-fucosidase activity in tissues and body fluids. Exponentially growing lymphoid cell cultures from four fucosidosis patients had 2.7-fold to 15.6-fold less extracellular-L-fucosidase protein and 28.8-fold to 144.0-fold less intracellular-L-fucosidase protein with negligible catalytic activity, compared to the mean of 19 control cultures. The percentage of total-L-fucosidase protein released extracellularly by cultures from the four patients was 64 to 85%, compared to 35±9% for control cultures. Intracellular and extracellular enzyme forms in fucosidosis and control cell lines were glycoproteins containing polypeptide chains ofM _r=52,000. During a 1.5-hr pulse-label with³⁵S-methionine,-L-fucosidase was synthesized by control cells and two fucosidosis cell lines as an intracellular form withM _r=58,000. During a subsequent 21-hr chase with unlabeled methionine, mutant enzyme was almost entirely processed to an extracellular form withM _r=62,000. In contrast, only 25–30% of control enzyme was processed to an extracellular form (M _r=62,000), with the remainder retained intracellularly (M _r=60,000). In the other two fucosidosis cell lines,-L-fucosidase was synthesized as an intracellular form withM _r=56,000 that was processed to an extracellular form withM _r=60,000. In summary, the fucosidosis mutation(s) affected the catalytic activity, quantity, and extracellular release of-L-fucosidase as expressed by lymphoid cells.This work was funded by NIH Grants DK 32161 to R. A. DiCioccio and GM 28428 to J. K. Darby.     

Short communication: Thermostability of α-amylase produced by Bacillus sp. E2—a thermophilic mutant

An -amylase from a hyper-producing strain of Bacillus (sp. E2) was stable at 70°C for 30 min but was quickly inactivated at higher temperatures. In the presence of 10mm Ca²⁺ and starch (20% w/v), however, the enzyme was stable at 90°C for 10 min and after 30 min at 100°C still retained 26% of its initial activity.     

Hyperthermophilic α-L-arabinofuranosidase from Thermotoga maritima MSB8: molecular cloning, gene expression, and characterization of the recombinant protein

A putative -L-arabinofuranosidase (AFase) gene belonging to family 51 of glycosyl hydrolases of a hyperthermophilic bacterium Thermotoga maritima MSB8 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein (Tm-AFase) was purified to apparent homogeneity by heat treatment (80°C, 30 min), followed by hydrophobic interaction, anion-exchange, and gel permeation column chromatography. Tm-AFase had a molecular mass of 55,284 Da on matrix assisted laser desorption ionization time-of-flight mass spectrometry and ~332 kDa on gel permeation column chromatography. Therefore, Tm-AFase comprised six identical subunits as in the case of homologous AFase from Geobacillus stearothermophilus. Regarding substrate specificity, Tm-AFase was active with p-nitrophenyl -L-arabinofuranoside but not with p-nitrophenyl -L-arabinopyranoside. Regarding polysaccharides, Tm-AFase hydrolyzed arabinan and debranched arabinan but not arabinoxylan, arabinogalactan, and carboxymethyl cellulose. Tm-AFase was extremely thermophilic, displaying an optimal reaction temperature of 90°C in a 10 min assay. When Tm-AFase was heated at 90°C, no loss of activity was observed for at least 24 h. At 100°C, the activity dropped to ~50% in 20 min; thereafter, inactivation occurred very slowly exhibiting a half-life of ~2.7 h, characterizing the enzyme to be the most thermophilic AFase reported thus far.     

Purification and some properties of β-mannanase from Bacillus sp.

-Mannanase produced by Bacillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NH₄)₂ SO₄ precipitation, adsorption to konjak gel, and column chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 70°C and was stable for at least 1 h between pH 5 and 10 and below 60°C. Its activity was completely inhibited by Hg²⁺. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose.The authors are with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University. Utsunomiya, Tochigi 321, Japan     

Properties of Aspergillus japonicus β-fructofuranosidase immobilized on porous silica

-Fructofuranosidase from Aspergillus japonicus MU-2, which produces fructo-oligosaccharides (1-kestose: O--D-fructofuranosyl-(2 1)--D-fructofuranosyl -D-glucopyranoside); and nystose: O--D-fructofuranosyl-(2 1)--D-fructofuranosyl-(2 1)--D-fructofuranosyl -D-glucopyranoside) from sucrose, was immobilized, covalently with glutaraldehyde onto alkylamine porous silica, at high efficiency (64%). Optimum pore diameter of porous silica for immobilization of the enzyme was 91.7 nm. After immobilization, the enzyme's stabilities to temperature, metal ions and proteolysis were improved, while its optimum pH and temperature were unchanged. The highest efficiency of continuous production of fructo-oligosaccharides (more than 60%), using a column packed with the immobilized enzyme, was obtained at 40% to 50% (w/v) sucrose. The half-life of the column during long-term continuous operation at 55°C was 29 days.