Properties of recombinant <Emphasis Type="Italic">Strep</Emphasis>-tagged and untagged hyperthermophilic D-arabitol dehydrogenase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

The first hyperthermophilic d-arabitol dehydrogenase from Thermotoga maritima was heterologously purified from Escherichia coli. The protein was purified with and without a Strep-tag. The enzyme exclusively catalyzed the NAD(H)-dependent oxidoreduction of d-arabitol, d-xylitol, d-ribulose, or d-xylulose. A twofold increase of catalytic rates was observed upon addition of Mg²⁺ or K⁺. Interestingly, only the tag-less protein was thermostable, retaining 90% of its activity after 90 min at 85 °C. However, the tag-less form of d-arabitol dehydrogenase had similar kinetic parameters compared to the tagged enzyme, demonstrating that the Strep-tag was not deleterious to protein function but decreased protein stability. A single band at 27.6 kDa was observed on SDS-PAGE and native PAGE revealed that the protein formed a homohexamer and a homododecamer. The enzyme catalyzed oxidation of d-arabitol to d-ribulose and therefore belongs to the class of d-arabitol 2-dehydrogenases, which are typically observed in yeast and not bacteria. The product d-ribulose is a rare ketopentose sugar that has numerous industrially applications. Given its thermostability and specificity, d-arabitol 2-dehydrogenase is a desirable biocatalyst for the production of rare sugar precursors.     

Enzyme activities associated with arabitol and mannitol biosynthesis and catabolism in Schizophyllum commune

Enzymes of polyol metabolism were studied in basidiospore germination of Schizophyllum commune during periods of in vivo arabitol and mannitol pool depletion (growth on glucose-asparagine) and during their subsequent synthesis (growth on acetate-NH ₄ ⁺ ). Optimal conditions for assays were established and specific activities of enzymes employing d-arabitol, d-mannitol, d-ribulose, d-fructose and d-xylulose as substrates were traced. Inquiries into the products formed during these reactions showed that d-ribulose generated arabitol while d-fructose produced mannitol with d-xylulose giving rise to xylitol. The dehydrogenase reactions were further investigated using polyacrylamide disc gel electrophoresis. Here was revealed the existence of at least two separate enzymatic activities pertaining to the catabolism of arabitol and mannitol. Also noted were the electrophoretic patterns when d-sorbitol, ribitol, xylitol and ethanol were used as substrates.     

Role of lactose on the production of d-arabitol by Kluyveromyces lactis grown on lactose

There are remarkably few reports on d-arabitol production from lactose. Previous studies in our laboratory have shown that the osmophilic yeast Kluyveromyces lactis NBRC 1903 convert lactose to extracellular d-arabitol. The present study was undertaken to determine the participation of osmotic stress caused by lactose on d-arabitol production by K. lactis NBRC 1903 and to provide the information on the kinetics of d-arabitol production from lactose by K. lactis NBRC 1903. It was confirmed that d-arabitol production was triggered when an initial lactose concentration was above 278 mmol L⁻¹. d-Arabitol yield increased with an increase in initial lactose concentration. The highest d-arabitol concentration of 79.5 mmol L⁻¹ was achieved in the cultivation of K. lactis NBRC 1903 in a medium containing 555 mmol L⁻¹ lactose and 40 g L⁻¹ yeast extract. Lactose was found to play two important roles in d-arabitol production by K. lactis NBRC 1903 grown on lactose. First, lactose was assimilated as the substrate both for cell growth and d-arabitol production. Second, a high lactose concentration induced cellular response to high osmotic stress and up-regulated the flow from d-glucose-6-phosphate to d-arabitol. The arrest of cell growth triggered d-arabitol production.     

Leclercia adecarboxylata Gen. Nov., Comb. Nov., formerly known asEscherichia adecarboxylata.

The nameLeclercia adecarboxylata is proposed for a group of the family Enterobacteriacae previously known asEscherichia adecarboxylata. Leclercia adecarboxylata can be phenotypically differentiated from all other species of Enterobacteriaceae. The members of this species are positive for motility, indole production, methyl red, growth in the presence of KCN, malonate, beta-galactosidase, beta-xylosidase, esculin hydrolysis, gas production fromd-glucose, and acid production fromd-cellobiose,d-lactose, melibiose,l-rhamnose, adonitol,d-arabitol, dulcitol, and salicin; the strains were negative for Voges-Proskauer, citrate (Simmons), H₂S (Kligler), lysine and ornithine decarboxylases, arginine dihydrolase, phenylalanine deaminase, gelatinase, DNase, Tween-80 hydrolysis, and acid production from myoinositol and alpha-methyl-d-glucoside. Fermentation ofd-raffinose,d-sucrose, andd-sorbitol is variable with strains. DNA relatedness of 11 strains ofL. adecarboxylata to three strains including the type strain of this species averaged 80% in reactions at 65°C. DNA relatedness to other species in Enterobacteriaceae was 2%–32%, indicating that this species was placed in a new genusLeclercia gen. nov. The type strain ofL. adecarboxylata is ATCC 23216.     

Effect of temperature on <Emphasis Type="SmallCaps">d</Emphasis>-arabitol production from lactose by <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

d-Arabitol production from lactose by Kluyveromyces lactis NBRC 1903 has been studied by following the time courses of concentrations of cell mass, lactose, d-arabitol, ethanol, and glycerol at different temperatures. It was found that temperature is a key factor in d-arabitol production. Within temperatures ranging from 25 to 39°C, the highest d-arabitol concentration of 99.2 mmol l⁻¹ was obtained from 555 mmol l⁻¹ of lactose after 120 h of batch cultivation at 37°C. The yield of d-arabitol production on cell mass growth increased drastically at temperatures higher than 35°C, and the yield reached 1.07 at 39°C. Increasing the cell mass concentration two-fold after 24 h of culture growth at 37°C, the d-arabitol concentration further increased to 168 mmol l⁻¹. According to the distribution of the metabolic products, metabolic changes related to growth phase were also discussed. The stationary-phase K. lactis cells in the batch culture that is started with exposing the precultured inoculum to high osmotic stress, high oxidative stress, and high heat stress are found to be preferable for d-arabitol production.     

Cloning,Purification and Characterization of an NAD-Dependent <Emphasis Type="SmallCaps">d</Emphasis>-Arabitol Dehydrogenase from Acetic Acid Bacterium, <Emphasis Type="Italic">Acetobacter suboxydans</Emphasis>

d-Xylulose-forming d-arabitol dehydrogenase (aArDH) is a key enzyme in the bio-conversion of d-arabitol to xylitol. In this study, we cloned the NAD-dependent d-xylulose-forming d-arabitol dehydrogenase gene from an acetic acid bacterium, Acetobacter suboxydans sp. The enzyme was purified from A. suboxydans sp. and was heterogeneously expressed in Escherichia coli. The native or recombinant enzyme was preferred NAD(H) to NADP(H) as coenzyme. The active recombinant aArDH expressed in E. coli is a homodimer, whereas the native aArDH in A. suboxydans is a homotetramer. On SDS–PAGE, the recombinant and native aArDH give one protein band at the position corresponding to 28 kDa. The optimum pH of polyol oxidation and ketone reduction is found to be pH 8.5 and 5.5 respectively. The highest reaction rate is observed when d-arabitol is used as the substrate (K _m = 4.5 mM) and the product is determined to be d-xylulose by HPLC analysis.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-arabitol by a newly isolated <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis>

A newly isolated Zygosaccharomyces rouxii NRRL 27,624 produced d-arabitol as the main metabolic product from glucose. In addition, it also produced ethanol and glycerol. The optimal conditions were temperature 30°C, pH 5.0, 350 rpm, and 5% inoculum. The yeast produced 83.4 ± 1.1 g d-arabitol from 175 ± 1.1 g glucose per liter at pH 5.0, 30°C, and 350 rpm in 240 h with a yield of 0.48 g/g glucose. It also produced d-arabitol from fructose, galactose, and mannose. The yeast produced d-arabitol and xylitol from xylose and also from a mixture of xylose and xylulose. Resting yeast cells produced 63.6 ± 1.9 g d-arabitol from 175 ± 1.8 g glucose per liter in 210 h at pH 5.0, 30°C and 350 rpm with a yield of 0.36 g/g glucose. The yeast has potential to be used for production of xylitol from glucose via d-arabitol route. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. department of Agriculture.     

Characterization of a xylitol dehydrogenase and a d-arabitol dehydrogenase from the thermo- and acidophilic red alga Galdieria sulphuraria

Galdieria sulphuraria (Galdieri) Merola can grow heterotrophically on at least ten different polyols. We investigated their metabolic path to glycolysis/gluconeogenesis and identified two NAD-dependent polyol dehydrogenases. Activity of other enzymes metabolizing mannitol or sorbitol could not be detected. The two dehydrogenases had a broad substrate specificity and were termed xylitol dehydrogenase (EC 1.1.1.14; substrate specificity: xylitol > d-sorbitol > d-mannitol > l-arabitol) and d-arabitol dehydrogenase (EC 1.1.1.11; substrate specificity: d-arabitol > l-fucitol > d-mannitol > d-threitol) according to the substrate with the lowest K _m value. The xylitol dehydrogenase was stable during purification. In contrast, the d-arabitol dehydrogenase was thermolabile and depended on divalent ions for stability and activity, preferentially Mn²⁺ and Ni²⁺. The molecular mass of the xylitol dehydrogenase was estimated to be 295 kDa by size-exclusion chromatography and 220 kDa by rate-sedimentation centrifugation. The d-arabitol dehydrogenase had a molecular mass of 105 kDa as determined by rate-sedimentation centrifugation. The specific activity of both enzymes increased about fourfold when cells were transferred from autotrophic to heterotrophic conditions regardless of whether sugars or polyols were supplied as substrates. The significance of polyol metabolism in Galdieria sulphuraria with regard to the natural habitat of the alga is discussed. Received: 15 January 1997 / Accepted: 12 February 1997     

Control of erythritol dehydrogenase in Schizophyllum commune

Summary An NAD-dependent erythritol dehydrogenase was detected in cell-extracts of basidiospore germinants of Schizophyllum commune following culture on either meso-erythritol or glycerol as sole carbon sources. Induction of erythritol dehydrogenase was also observed in purely vegetative mycelium (str. 845 or str. 699). Erythritol dehydrogenase was not observed in ungerminated basidiospores or germinants which arose on d-glucose, d-mannitol, sorbitol, ribitol, xylitol, d-arabitol or l-arabitol. NAD-coupled polyol dehydrogenases for all the latter sugar alcohols were observed in ungerminated basidiospores, germinants, and vegetative mycelium of S. commune cultured on d-glucose. Basidiospore germination on d-glucose plus meso-erythritol led to a 90% decrease in erythritol dehydrogenase and the specific activity of ribitol dehydrogenase was directly comparable to that seen in d-glucose germinants. Storage experiments of crude extracts of meso-erythritol germinants indicated differential enzyme decay of dehydrogenases for d-mannitol, sorbitol and erythritol while the respective enzymes could be further distinguished by heat-stability as well as preferential utilization of analogues of NAD. DEAE-cellulose column chromatography led to separation of sorbitol dehydrogenase which was also active with xylitol, erythritol dehydrogenase, and mannitol dehydrogenase which was also active with d-arabitol.     

Klebsiella ornithinolytica sp. nov., formerly known as ornithine-positiveKlebsiella oxytoca

The nameKlebsiella ornithinolytica sp. nov. is proposed for a group ofKlebsiella strains referred to previously as NIH Group 12 at the National Institute of Health, Tokyo. The members of this species are Gram-negative, encapsulated, nonmotile rods with the general characteristics of the familyEnterobacteriaceae and of the genusKlebsiella. They give positive results in tests for indole production, Voges-Proskauer, citrate utilization, lysine and ornithine decarboxylases, urease, -galactosidase, malonate utilization, growth in KCN, and esculin hydrolysis, and they produce acid and gas fromd-glucose, and acid froml-arabinose, cellobiose, lactose, melibiose, raffinose, rhamnose, sucrose, trehalose,d-xylose, adonitol,d-arabitol, myo-inositol, sorbitol, arbutin, salicin, -methyl-d-glucoside, and mucate. They give negative drolysis, DNase, pectinase, and acid production fromd-arabinose, melezitose, and dulcitol. They can grow at 4°C and 42°C, and do not produce any pigment. DNA relatedness of eight strains ofKlebsiella ornithinolytica to three strains including the type strain of this species averaged 88% in reaction at 75°C. DNA relatedness to the already recognizedKlebsiella species inEnterobacteriaceae was 1 to 20%. Phenotypic and DNA relatedness data also indicated that a group of organisms referred to as Enteric Group 47 orKlebsiella Group 47 at the Centers for Disease Control (Atlanta, Georgia) was identical withK. ornithinolytica. The type strain ofK. ornithinolytica is NIH 90-72 (JCM 6096).     

Purification and properties of D-gluconate dehydratase fromAchromobacter

d-Gluconate dehydratase fromAchromobacter, grown ond-gluconate, was purified 100-fold by a procedure involving ammonium sulfate fractionation and preparative acrylamide gel electrophoresis. The purified enzyme appeared to be homogeneous by disc gel electrophoresis. It is an inducible enzyme with an optimal activity in the pH region 8.4–8.8, a Km value of 2.08 × 10^–2 m ford-gluconate and a molecular weight of 270,000 ± 25,000. Only C₅ and C₆ aldonic acids possessing al-threo configuration at C₂ and C₃ are dehydrated. The dehydration products ofd-gluconate,d-xylonate,d-galactonate,d-fuconate andl-arabonate were identified as 2-keto-3-deoxy compounds by specific colour reactions and thin layer chromatography. Onemm Mg^{+ +} is a powerful activator, 0.1 mm Mn^{+ +} activates poorly and EDTA inhibits. Glutatione, dithiothreitol and mercaptoethanol had no effect, althoughp-chloromercuribenzoate (0.01 mm) decreased enzyme activity.We wish to thank Mr D. Dewettinck for skilful technical assistance. The senior author (J.D.L.) is indebted to the Fonds voor Kollektief en Fundamenteel Onderzoek (Belgium) for research and personnel grants. J.K.-M. is indebted to the Belgian government for a travel and study grant.     

Production of d-arabitol by a newly isolated Kodamaea ohmeri

A new yeast, isolated from natural osmophilic sources, produces d-arabitol as the main metabolic product from glucose. According to 18S rRNA analysis, the NH-9 strain belongs to the genus Kodamaea. The optimal culture conditions for inducing production of d-arabitol were 37 °C, neutral pH, 220 rpm shaking, and 5% inoculum. The yeast produced 81.2 ± 0.67 g L⁻¹ d-arabitol from 200 g L⁻¹ d-glucose in 72 h with a yield of 0.406 g g⁻¹ glucose and volumetric productivity Q_\textP Q_{\text{P}} of 1.128 g L⁻¹ h⁻¹. Semi-continuous repeated-batch fermentation was performed in shaker-flasks to enhance the process of d-arabitol production by Kodamaea ohmeri NH-9 from d-glucose. Under repeated-batch culture conditions, the highest volumetric productivity was 1.380 g L⁻¹ h⁻¹.     

Cellular compartmentation of photosynthetic and photorespiratory enzymes in the heterocystous cyanobacterium Anabaena cylindrica

Activities of enzymes of photosynthesis and photorespiration have been measured in extracts of vegetative cells and heterocysts from the filamentous cyanobacterium Anabaena cylindrica. Phosphoribulokinase, d-ribulose 1,5-bisphosphate carboxylase/oxygenase, phosphoglycollate phosphatase and glycollate dehydrogenase activities were readily measured in vegetative cell extracts, but were undetectable or negligible in heterocyst preparations. The data help to explain why heterocysts are unable to perform photosynthetic CO₂ fixation. They also exemplify the co-ordinate compartmentation of enzymes of photosynthesis and photorespiration which occur in a differentiated phototrophic prokaryote.Abbreviations Ru5P d-ribulose 5-phosphate - RuBP d-ribulose 1,5-bisphosphate - DCPIP 2,6-dichlorophenolindophenol - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulphonate     

Purification and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-tagatose

l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI activity was stimulated by Mn²⁺, Fe³⁺, Fe²⁺, Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Hg²⁺, Pb²⁺. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K _m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production corresponds to a 39% equilibrium.     

Catalytic properties and substrate specificity of 3-hexulose phosphate synthase fromMethylomonas M15

Summary 3-Hexulose phosphate synthase was purified in 94% yield from Methylomonas M15. The enzyme did not form a Schiff-base intermediate with d-ribulose 5-phosphate that could be reduced by NaBH₄. However, the enzyme required Mg²⁺ or Mn²⁺ ions for activity and was inactivated in the presence of EDTA. The latter is a property of class II aldolases. The enzyme accepted a wide range of other aldehydes in addition to its natural substrate formaldehyde, while d-ribulose 5-phosphate could not be replaced. This makes it an attractive tool for the synthesis of higher sugar phosphates. Offprint requests to: M.-R. Kula     

Structural analysis of a polysaccharide from<Emphasis Type="Italic">Chlorella kessleri</Emphasis> by means of gas chromatography—mass spectrometry of its saccharide alditols

Interpretation of gas chromatographic-mass spectrometric data of oligosaccharide alditols was used to determine their structures and to derive the structure of a water soluble polysaccharide isolated fromChlorella kessleri.¹H- and¹³C-NMR was employed to assess the configuration of glycosidic bonds and individual monosaccharides were assigned to thel ord series by means of gas chromatography of the acetylated (S)-2-butyl glycosides.     

Inhibition ofd-alanyl-d-alanine ligase in different bacterial species by amino phosphonic acids

A comparative study was performed on the kinetic properties and the specificity ofd-alanyl-d-alanine ligases fromPseudomonas aeruginosa, Streptococcus faecalis, andStaphylococcus aureus, using some aminophosphonic acids and related compounds.dl-I-Aminoethylphosphonic acid was shown to be a competitive inhibitor of theP. aeruginosa andS. faecalis ligases; assuming ad-form stereospecificity, its activity was nearly equal to that ofd-cycloserine. 2-Aminoethylphosphonate was found to be a weak inhibitor of the ligases, in contrast to the carboxylic analog, β-alanine. γ-Aminobutyric acid and phosphoethanolamine also exhibited some inhibitory properties.     

The α-d-mannan core of a complex cell-wall heteroglycan of Trichoderma reesei is responsible for β-glucosidase activation

A heteroglycan responsible for the binding of the enzyme β-1,4-d-glucosidase (EC 3.2.1.21) to fungal cell walls was isolated from cell walls of the filamentous fungusTrichoderma reesei. The heteroglycan, composed of mannose, galactose, glucose, and glucuronic acid, also activated β-1,4-d-glucosidase, β-1,4-d-xylosidase andN-acetyl-β-1,4-d-glucosaminidase activity in vitro. The structural backbone of this heteroglycan was prepared by acid hydrolysis and gel filtration. The molecular structure of the core of the heteroglycan was determined by NMR studies as a linear α-1,6-d-mannan. The mannan core obtained by acid degradation stimulated the β-glucosidase activity by 90%. Several glycosidases fromAspergillus niger were also activated by theT. reesei heteroglycan. The β-glucosidase ofTrichoderma was activated by mannan fromSaccharomyces cerevisiae to a comparable extent.     

Antheseabhängige UV-Muster in Blütenständen von Asteraceen

The pseudanthia ofHeliopsis scabra andRudbeckia vulgaris (Asteraceae) were examined during the anthesis for differences in their UV patterns. Distinct changes in the reflectance and absorbance properties could be observed. The results suggest a close correlation between different stages of floral development and pollinator attraction.

Herrn Prof. Dr.Lothar Geitler zum 90. Geburtstag gewidmet.     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.