Partial purification and characterization of β-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium,Pseudomonas 135

Summary An intracellular enzyme, d(—)--hydroxybutyric acid dehydrogenase involved in an intracellular poly-d(—)--hydroxybutyric acid degredation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of d(—)--hydroxybutyric acid (D-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5–6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at — 20° C. The K _m values for oxidation and reduction reactions were determined as 1.84 mm for D-HB, 0.244 mm for NAD⁺, 0.319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that d-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mm for d-lactate, 0.196 mm for NADH and 1.82 mm for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive. Correspondence to: J. M. Lebeault     

Biosynthesis of gallotannins. Enzymatic conversion of 1,6-digalloylglucose to 1,2,6-trigalloylglucose

Cell-free extracts from Rhus typhina L. (staghorn sumach) leaves were found to catalyze the transfer of the galloyl moiety of -glucogallin (1-O-galloyl--D-glucose) to 1,6-di-O-galloyl--D-glucose, resulting in the specific formation of 1,2,6-tri-O-galloyl--D-glucose, an intermediate of gallotannin biosynthesis. The reaction product was unequivocally identified by co-chromatography with authentic references using reversed-phase high-performance liquid chromatography and by ¹H-nuclear-magnetic-resonance spectroscopy.Abbreviations HPLC high-performance liquid chromatography - NMR nuclear magnetic resonance     

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.     

Exocellular pyruvate-containing galactoglucan ofAchromobacter spp.

Exopolysaccharides of a number of slime-producingAchromobacter strains, isolated from activated sludge, were prepared and analysed. They consist ofD-glucose,D-galactose, pyruvic acid and O-acetyl in the approximate molar ratio of 1:1:1:1/2. The polysaccharides were shown by methylation and partial hydrolysis to consist of unbranched chains of alternately arrangedD-glucose andD-galactose residues, exclusively linked by -1,3-linkages, and with pyruvate substituents linked by acetal bonds at positions 0–4 and 0–6 of theD-galactose residues. The significance of the exopolysaccharides in relation to some relevant properties of activated sludge organisms is discussed.     

Endo-β-mannanase,the leguminous aleurone layer and the storage galactomannan in germinating seeds ofTrigonella foenum-graecum L.

The activity ofendo--mannanase in the endosperm of fenugreek seeds at different stages of germination varies pari passu with storage galactomannan breakdown.Endo--mannanase is similarly associated with the galactomannan breakdown which occurs when isolated fenugreek half-endosperms are incubated under germination conditions. Metabolic inhibitors,acting on the aleurone layer reduceendo--mannanase production in the half-endosperms in proportion to their inhibition of galactomannan breakdown.It is concluded (a) thatendo--mannanase activity, like galactomannan breakdown, is regulated by the fenugreek aleurone layer and (b) that theendo--mannanase is almost certainly instrumental in bringing about galactomannanan hydrolysis in vivo.This is part five in a series of papers dealing with galactomannan metabolism. Part four: Planta (Berl.)112, 301–308 (1973)     

Antioxidant and antiviral activities ofEuphorbia thymifolia L.

The antioxidant and antiviral activities ofEuphorbia thymifolia L. (Euphorbiaceae) were investigated in this study. The results showed that all of the fractions (MeOH, CHCl₃, EtOAc, n-butanol and water) and pure compounds (3-O-galloyl-4,6-(S)-HHDP-D-glucose, rugosin B and 1,3,4,6-tetra-O-galloyl-K--D-glucose)tested possessed antioxidant activities, with the exception of the organic aqueous fraction in the anti-lipid and anti-super-oxide formation assays. The range of IC₅₀ of anti-lipid formation, anti-superoxide formation and free radical scavenging assays for all fractions and pure compounds were 2.81–7.63, 0.03–2.18 and 0.013–2.878 mg/ml, respectively. Electron spin resonance studies showed that water extract and pure compounds ofE. thymifolia exhibited superoxide radical and hydroxyl radical scavenging activities. Besides antioxidant activities, 3-O-galloyl-4,6-(S)-HHDP-D-glucose and EtOAc fraction also showed anti-HSV-2 activity. Thus,E. thymifolia was concluded to possess antioxidant and anti-HSV-2 activities.     

Pyranose oxidase and pyranosone dehydratase: enzymes responsible for conversion of d-glucose to cortalcerone by the basidiomycete Phanerochaete chrysosporium

Pyranose oxidase (glucose 2-oxidase) and pyranosone dehydratase were purified 27.6- and 43.9-fold respectively from mycelial extracts of the fungus Phanerochaete chrysosporium using hydrophobic interaction, anion exchange and gel filtration chromatography. The enzymes appeared substantially homogeneous on SDS-PAGE and were comprised of identical subuntis with apparent M_r values of 69 000 and 99 000 for pyranose oxidase and pyranosone dehydratase, respectively. The apparent M_r's of the native enzymes, based on equilibrium ultracentrifugation, were 308 000 and 221 000. In coupled reactions, the enzymes catalyzed conversion of d-glucose via d-glucosone (d-arabino-2-hexosulose) to the antibiotic -pyrone, cortalcerone. The latter compound was isolated as a diphenylhydrazone derivative and spectroscopically identified.Abbreviations DMAB 3-dimethylaminobenzoic acid - FPLC fast protein liquid chromatography - MBTH 3-methyl-2-benzothiazolinone hydrazone hydrochloride - PD pyranosone dehydratase - PMSF phenylmethylsulfonyl fluoride - POD pyranose oxidase     

Indigogenic methods for glycosidases

Summary The indigogenic method for -D-galactosidase of Pearson et al. (1963) with 4-Cl-5-Br-3-indolyl--D-galactoside was tested and evaluated.The acid -D-galactosidase is not firmly associated with structures and escapes from cryostat sections prepared in the usual manner into incubation solutions. This leakage cannot be prevented by a short postfixation of these sections in cold acetone or Baker's formol-calcium chloride. The leakage is negligible from frozen sections prepared from tissue blocks fixed 12–24 h in cold Baker's solution or in 3% buffered glutaraldehyde (the latter fixation is preferred). Even if this fixation causes about 70–80% inactivation of acid -D-galactosidase it is a prerequisite for studies concerned with its localization. The brush border -D-galactosidase of enterocytes is more firmly structurally bound. Since its activity against 4-Cl-5-Br-3-indolyl--D-galactoside cannot be proved after overnight fixation in cold aldehyde fixatives its demonstration is to be performed in sections prepared from specimens fixed in cold Baker's solution for 2 h at the most, or in cold microtome sections.The localization obtained with the original method is not correct. The addition of horseradish peroxidase did not result in any improvement of the localization because the employed samples of this peroxidase contained a concomitant -D-galactosidase activity.A striking improvement of the localization was achieved by a mixture of ferri- and ferrocyanide which causes a 40–75% inhibition of acid -D-galactosidase when used in concentrations of 1 · 10^–3 M to 1 · 10^–2 M.A new medium was devised consisting of 0,1 M citrate phosphate buffer pH 3,5–5,5, 8 · 10^–4M 4-Cl-5-Br-3-indolyL--D-galactoside, and 3,1 · 10^–3M potassium ferri- and ferrocyanide. This medium enabled to achieve a very good correlation with biochemical studies and to localize acid and neutral -D-galactosidases in situ.The acid enzyme was demonstrated first of all in lysosomes of many cells. Its activity is inhibited by galactonolactone, lactose and p-chloromercuribenzoate. The nature of the diffuse extralysosomal staining cannot be decided at present. The distribution pattern of this enzyme in many animal organs is given.The neutral -D-galactosidase (lactase) was localized by the improved method in the brush border of differentiated rat, human and monkey enterocytes and is inhibited by galactonolactone, lactose, gluconolactone, and cellobiose. In patients with celiac sprue this activity is very much reduced or absent. It is restituted after a gluten-free diet.Our revised method proved also very useful in processing zymograms and immunoprecipitation lines of -D-galactosidase(s) with homologous antisera obtained by Ouchterlony's technic and by immunoelectrophoresis.     

The formation,vacuolar localization,and tonoplast transport of salicylic acid glucose conjugates in tobacco cell suspension cultures

The metabolism of salicylic acid (SA) in tobacco (Nicotiana tabacum L. cv. KY 14) cell suspension cultures was examined by adding [7–¹⁴C]SA to the cell cultures for 24 h and identifying the metabolites through high performance liquid chromatography analysis. The three major metabolites of SA were SA 2-O--D-glucose (SAG), methylsalicylate 2-O--D-glucose (MeSAG) and methylsalicylate. Studies on the intracellular localization of the metabolites revealed that all of the SAG associated with tobacco protoplasts was localized in the vacuole. However, the majority of the MeSAG was located outside the vacuole. The tobacco cells contained an SA inducible SA glucosyltransferase (SAGT) enzyme that formed SAG. The SAGT enzyme was not associated with the vacuole and appeared to be a cytoplasmic enzyme. The vacuolar transport of SAG was characterized by measuring the uptake of [¹⁴C]SAG into tonoplast vesicles isolated from tobacco cell cultures. SAG uptake was stimulated eightfold by the addition of MgATP. The ATP-dependent uptake of SAG was inhibited by bafilomycin A₁ (a specific inhibitor of the vacuolar H⁺-ATPase) and dissipation of the transtonoplast H⁺-electrochemical gradient. Vanadate was not an inhibitor of SAG uptake. Several -glucose conjugates were strong inhibitors of SAG uptake, whereas glutathione and glucuronide conjugates were only marginally inhibitory. The SAG uptake exhibited Michaelis–Menten type saturation kinetics with a K_m and V_max value of 11 M and 205 pmol min^–1 mg^–1, respectively, for SAG. Based on the transport characteristics it appears as if the vacuolar uptake of SAG in tobacco cells occurs through an H⁺-antiport-type mechanism.     

Composition and Structure of Galactomannan from the Seed of Gleditsia ferox Desf.

Galactomannan, a heteropolysaccharide with a molecular weight of 1660 kDa, was isolated from the seed of Gleditsia ferox Desf., introduced in Russia, with a yield of 18.9%. Its aqueous solutions were optically active ([]_D = +30.5°) and highly viscous ([] = 1430 ml/g). An analysis of the heteropolysaccharide using chemical, enzymatic, and chromatographic procedures showed that it consists of D-mannopyranose and D-galactopyranose residues (molar ratio, 2.54 : 1). The main chain of this galactomannan consists of 1,4--D-mannopyranose residues, 39.2% of which are substituted at C6 with single residues of -D-galactopyranose. The probability of occurrence of mannobiose units differentially substituted with galactose was determined by ¹³C-NMR data and equaled, respectively, 0.37, 0.47, and 0.16 for non-substituted Man–Man units, monosubstituted Gal(Man–Man) and (Man–Man)Gal units taken together, and for the disubstituted Gal(Man–Man)Gal units.     

Synthesis and Antiaggregative Activity of a New RGDF Mimetic

m-[4-Oxo-4-(piperazin-1-yl)butyrylamino)benzoyl-D,L--(3,4-methylenedioxyphenyl)--alanine, a new mimetic of the peptide RGDF, was synthesized. This compound inhibited ADP-induced platelet aggregation in human blood plasma enriched with platelets with IC₅₀ = 3.5 × 10^–8 M.     

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     

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.     

Initial steps of αand β-d-glucose binding to intact red cell membrane

Summary The kinetics of the initial phases of d-glucose binding to the glucose transport protein (GLUT1) of the human red cell can be followed by stopped-flow measurements of the time course of tryptophan (trp) fluorescence enhancement. A number of control experiments have shown that the trp fluorescence kinetics are the result of conformational changes in GLUT1. One shows that nontransportable l-glucose has no kinetic response, in contrast to d-glucose kinetics. Other controls show that d-glucose binding is inhibited by cytochalasin B and by extracellular d-maltose. A typical time course for a transportable sugar, such as d-glucose, consists of a zero-time displacement, too fast for us to measure, followed by three rapid reactions whose exponential time courses have rate constants of0.5–100 sec^+–1 at 20°C. It is suggested that the zero-time displacement represents the initial bimolecular ligand/GLUT1 association. Exponential 1 appears to be located at, or near, the external membrane face where it is involved in discriminating among the sugars. Exponential 3 is apparently controlled by events at the cytosolic face. Trp kinetics distinguish the K _d of the epimer, d-galactose, from the K _dfor d-glucose, with results in agreement with determinations by other methods. Trp kinetics distinguish between the binding of the - and -d-glucose anomers. The exponential 1 activation energy of the -anomer, 13.6 ± 1.4 kcal mol^+–1, is less than that of -d-glucose, 18.4 ± 0.8 kcal mol^+–1, and the two Arrhenius lines cross at 23.5°C. The temperature dependence of the kinetic response following -d-glucose binding illustrates the interplay among the exponentials and the increasing dominance of exponential 2 as the temperature increases from 22.3 to 36.6°C. The existence of these interrelations means that previously acceptable approximations in simplified reaction schemes for sugar transport will now have to be justified on a point-to-point basis.We should like to express our thanks to Michael R. Toon for his important contributions. This work was supported in part by a grant-in-aid from the American Heart Association, by the Squibb Institute for Medical Research and by The Council for Tobacco Research.     

Production of steroid-ring-B-seco acid from β-sitosterol and influence of surfactants on sterol uptake in Nocardia sp. M 29–40

Summary The effect of several parameters (temperature; pH; main carbon source; time and amount of -sitosterol addition; Tween 20, 40, 60, 80; Span 20; pluronic F 68, L 64) on the conversion of -sitosterol to 3-(5-hydroxy-7a-methyl-1-oxo-3a-H-hexahydroindan-4-yl) propionic acid (I) by Nocardia sp. M. 29–40 was investigated. A maximal theoretical yield of 65 mol% I (with respect to substrate added) could be achieved during cultivation at pH 8.0 in presence of 6 g/l Tween 40 or Tween 60. Tween 40 and Tween 60 stimulate -sitosterol cooxidation not by improving the substrate suspension but by providing a fatty acid component as precursor for biosynthesis of surface active cell wall lipids.In memory of Professor David Perlmann     

New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids

Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: 3)--L-QuipNAc-(13)--D-GlcpNAc-(16)--D-GlcpNAc-(1 (S)-Lac-(2–3) (1) 4)--D-GlcpA-(13)--D-GalpNAc-(14)--D-Glcp-(13)--D-Galp-(14)--D-GalpNAc-(1 L-Ala-(2–6) (2) 3)--D-GalpNAc-(16)--D-GalpNAc-(14)--D-GlcpA-(1 L-Lys-(2–6)--D-GalpA-(14) (3) 4)--D-GlcpA-(16)--D-GalpNAc-(16)--D-GlcpNAc-(13)--D-GlcpNAc-(1 (R)-aLys-(2–6) (4) where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and N-[(R)-1-carboxyethyl]-L-lysine (alaninolysine), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.     

Solution structure of a synthetic peptide corresponding to a receptor binding region of FSH (hFSH-beta 33-53).

The receptor binding surface of human follicle-stimulating hormone (hFSH) is mimicked by synthetic peptides corresponding to the hFSH- chain amino acid sequences 33–53 [Santa-Coloma, T. A., Dattatreyamurty, D., and Reichert, L. E., Jr. (1990),Biochemistry 29, 1194–1200], 81–95 [Santa-Coloma, T. A., and Reichert, L. E., Jr. (1990),J. Biol. Chem. 265, 5037–5042], and the combined sequence (33–53)–(81–95) [Santa-Coloma, T. A., Crabb, J. W., and Reichert, L. E., Jr. (1991),Mol. Cell. Endocrinol. 78, 197–204]. These peptides have been shown to inhibit binding of hFSH to its receptor. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were used to determine the structure of the first peptide in this series, the 21 amino acid peptide hFSH--(33–53), H₂N-YTRDLVYKDPARPKIQKTCTF-COOH. Analysis of CD data indicated the presence of approximately equal amounts of antiparallel -pleated sheet, turns including a -turn, other structures, and a small amount ofa-helix. The major characteristics of the structure were found to be relatively stable at acidicpH and the predominant effect of increased solvent polarity was a small increase ina-helical content. One- and two-dimensional NMR techniques were used to obtain full proton and carbon signal assignments in aqueous solution atpH 3.1. Analysis of NMR results confirmed the presence of the structural features revealed by CD analysis and provided a detailed picture of the secondary structural elements and global folding pattern in hFSH--(33–53). These features included an antiparallel -sheet (residues 38–51 and 46–48), turns within residues 41–46, and 50–52 (a -turn) and a small N-terminal helical region comprised of amino acids 34–36. One of the turns is facilitated by prolines 42 and 45. Proline-45 was constrained to thetrans conformation, whereas proline-42 favored thetrans conformer (70%) over thecis (30%). Two resonances were observed for the single alanine residue (A-43) sequentially proximal to P-42, but the rest of the structure was minimally affected by the isomerization at proline-42. The major population of molecules, containingtrans-42 andtrans-45 prolines, presented 120 NOEs. Distance geometry calculations with 140 distance constraints and energy minimization refinements were used to derive a moderately well-defined model of the peptide's structure. The hFSH--(33–53) structure has a highly polar surface composed of six cationic amino acid (arginie-35, lysine-40, arginine-44, lysine-46, glutamine-48, and lysine-49) and two anionic residues (aspartate-36 and aspartic acid-41). A hydrophobic region in the structure is composed of residues in the antiparallel -sheet and -turn which fold to produce a distorted hairpin. The structure of this domain, together with the protruding and positively charged region in the vicinity of residues 42–45, may mimic the surface of hFSH that binds to the receptor.Abreviations used: hFSH, human follicle-stimulating hormone; PB, 25 mM Na₂KPO₄, 25 mM KH₂PO₄, and 5 mM Mg Cl₂; CD, circular dichroism spectrapolarimetry; NMR, nuclear magnetic resonance spectrometry; COSY, homonuclear correlated spectroscopy; NOESY, 2D nuclear Overhauser effect spectroscopy; HOHAHA, homonuclear Hartman-Han coherence transfer; HMQCHY, reverse-detected heteronuclear multiple shift correlation, one bond; HMBC, reverse-detected heteronuclear multiple bond correlation; S/N, signal to noise ratio; TFE, trifluoroethanol.Dr. Santa-Coloma is on leave of absence from the National Research Council of Argentina (CONICET).     

Cellular localization of glycoside hydrolases inBacteroides fragilis

The localizations of six glycosidases produced byBacteroides fragilis—-glucosidase, -glucosidase, -galactosidase, -galactosidase, -N-acetylglucosaminidase, and -l-fucosidase—were studied. Cell fractions and cell extracts were obtained by Triton X-100 release, by disruption by freeze-pressing and sonication, and by osmotic release. Isoelectric focusing of a cytoplasmic and of a Triton X-100 extract of the cell wall fraction was performed and revealed differences in the relative distribution of differently charged forms of -N-acetylglucosaminidase. -Galactosidase and alkaline phosphatase were used as cytoplasmic and periplasmic markers, respectively. It is concluded that inB. fragilis -glucosidase is periplasmic, -l-fucosidase and -galactosidase are cytoplasmic, and -n-acetylglucosaminidase is cell associated and bound to the cell envelope by hydrophobic interactions. -Glucosidase and -galactosidase are localized cytoplasmically and/or located in the cell envelope.     

Cellobiose phosphorylase (EC 2.4.1.20) of Cellulomonas: occurrence,induction, and its role in cellobiose metabolism

The occurrence of cellobiose cleavage by phosphorolysis and by hydrolysis was investigated in Cellulomonas spec., C. uda, C. flavigena, and C. cartalyticum. Cellobiose phosphorylase (EC 2.4.1.20) was shown to be produced by Cellulomonas spec. when cellobiose or cellulose was used as sole source of energy and carbon but not with glycerol or glucose. Using inhibitors of protein synthesis as well as double labelling techniques it was shown that cellobiose phosphorylase is synthesized de novo in Cellulomonas spec. Aryl--D-glucosidase which was shown to be present in crude extracts of this microorganism as well is not involved in cellobiose cleavage.Abbreviations oNPGluc ortho-nitrophenyl--D-glucopyranoside - oNPGlucase ortho-nitrophenyl--D-glucopyranoside hydrolase (aryl--D-glucosidase) - CMC carboxymethyl-cellulose - CMCase carboxymethyl-cellulase - PAGE polyacrylamde disc gel electrophoresis Parts of this work were presented on the Herbsttagung der Gesellschaft für Biologische Chemie (Schimz et al. 1979) and on the 14th FEBS Meeting (Schimz et al. 1981)     

Comparison of wild-type and UV-mutant β-glucanase-producing strains of Talaromyces emersonii with potential in brewing applications

A screen of 46 UV-mutant strains of the moderately thermophilic fungus Talaromyces emersonii yielded two mutants (TC2, TC5) that displayed gross morphological differences to the parent strain and enhanced activity against mixed linkage cereal -glucans. Activity against -(1, 3)(1, 4)-d-glucan from barley (BBGase) was measured during growth of the mutant and wild-type strains on a variety of carbon sources, ranging from solka floc to crude cereal fractions. In liquid culture, TC2 and TC5 secreted 1.2- to 8.6-fold more BBGase than the parent strain and markedly less -glucosidase (exo-activity); enzyme levels were dependent on the carbon source. Cellulose induced high BBGase. However, beet pulp, wheat bran, carob and tea-leaves were cheap and effective inducers. T. emersonii wild-type, TC2 and TC5 crude enzyme preparations achieved similar end-points during the hydrolysis of commercial barley -glucan (13.0–16.9%), but were more active against crude -glucan from barley (16.0–24.2% hydrolysis). The products of hydrolysis were quantified by high-performance anion-exchange chromatography. Mash trials indicated that enzyme preparations from all three organisms effected a significant reduction in wort viscosity and residual mash -glucan. Finally, TC2 and TC5 produce more efficient -glucan-depolymerizing enzymes; and wheat bran and solka floc can be used to provide inexpensive and potent enzyme cocktails with potential in brewing applications.