Utilization of Gluconate by Aspergillus niger. I. Enzymes of Phosphorylating and Nonphosphorylating Pathways

Zb l. Mikrobiol. 140 (J 985) , 475-484 [I nstitute of An imal Nutriti on, Un iversity of Hohenheim, St ut tgart , FRG]

Utilization of Gluconate by A spergillus niger. 1. Enz ymes of Phosphorylating and Nonphosphorylating Pathways R.-M . M ULLER W ith 6 Fi gures

Summary Gluco n a te which was produced in cult ures of Aspergillus nig er wit h glucose as the sole source of ca rbon, or which was added afte r exhaus t ion of glucose, wa s utilized by this mold. In cell-free ex t racts from gluconate degr adi ng my celia gluconokinase and f ive enzy me s of the pentose phosp hat e pat hway wer e iden tified . Enzy mes of the Entner-Doudor off pa thwa y a n d of a modified (no n -p hos phorylat ing) Entner-D oudor off system , as well as a gluconute deh ydrogenase could not bo de tected . I t is conc luded t hat gluco na te a fte r its pho sphoryl ation to 6-p hosp hog luc onate is metabo lized via th e pen to se phospha te pat hway by th e strain used ,

Zusammenfassung Gluco uat , das in Kulturen von A spergillus ni qer mi t Glucose a ls einz iger C-Que lle ge bi ldet od er das nach Ver bra uc h del' Glucos e zugeeetz t wurde, wu rde v on diese m Pilz v erwertet. In zellfre ien E xtrakten aus Gluconat abba ue nden l\ly celien wurden die Glu con ok ina se und fiinf Enzyme des P en t osephosphat- W eges nachgewiesen, Enzyme des Entner-Doudor off- W eges und eines rnodifizierten (nich t phosphoryl ier en den) E n t ne r-Do u do roff-Sys te me s, so wie eine Gluoonat -De hy drogen e se wa re n nich t zu fi nd en . E s wird gese hlossen, daB Glueo nat du rc h den beniitzten Stamm nach seiner Phosphoryli erung zu 6-Phosphogluconat iib er den Pentosep hos p hat-Weg metabolisiert wi rd .

A spergillus niger and some ot her molds have long been known for their ability to accumulate large amounts of gluconate from glucos e (FOSTER 1949, COCHRANE 1958, 1976). As yet, only few attempts have been made to explore th e control mechanism(s) by which this process is gov ern ed. :F UAK KE et al. (1963) and DREWS and SMALLA (1969) obser ved that during gluconate a ccumulation by A. niger th e activity of glucose oxidase in mycelial extracts increased whereas the activities of som e enzymes of the pentose ph osph ate and Embden-Meyerhof pa t hways and of th e tricarboxylic acid cycle d ecreased or did not increase with t he sa llie rate as th e act ivit y of glu cose oxidase. Th is suggests t hat t he gluconate accumulation by this mold is du e to a disequilibrium in t he conte nt of th e mycelium in enzymes inv olved in t he form ation and degradation of gluconate as well as in ot her pathways of glu cose metabolism. Th is hypothesis cannot be proved before the pathways of gluconate metabolization ha ve not been clarified. L AK SH MI N.A,RAYANA et aI. (1969) proposed t hat glu conate is ph osphorylated and t hen metaboliz ed via the pentose ph osph ate system. These a ut hors iden tifi ed in mycelial ex t rac ts of A. niger the enzy mes, gluconokina se, 6phosph oglu conate dehydrogena se a nd ribosephosphat e isomerase. C LE LAK D and J OH NSON (1956) following th eir st udies in pr efor med mycelia of A . niger incubated

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with HC-gluconolactone suggested that the Entner-Doudoroff pathway is involved in the utilization of 6-phosphogluconate. However, LAKSHMINARAYANA et al. (1969) and other authors could not detect the enzymes of this pathway in A. niger. ELZAINY et al. (1973) provided evidence for a modified Entner-Doudoroff pathway in A. niqer whose first step is the dehydration of gluconate to 2-keto-deoxygluconate which then is cleaved into pyruvate and glyceraldehyde. Another non-phosphorylative degradation of gluconate is its oxidation to 2-keto- or 5-keto-gluconate catalyzed by gluconate dehydrogenases. MARTIN and STEEL (1955) identified 5-ketogluconate in the culture fluid of A. niger, however, LA,KSHMINARAYANA et al. (1969) could not detect a gluconate dehydrogenase in mycelial extracts from this mold. In order to decide which of these enzymes are involved in the first steps of gluconate utilization under the conditions used by us, their activities were investigated in cellfree extracts.

Materials and methods I. Cultural conditions and determination of glucose and gluconate The strain used, the culture medium (containing 5 % glucose and 1.0 % KN0 3 as the sources of carbon and nitrogen), the inoculation with conidia and other experimental conditions were the same as described previously (MULLER 1966). The incubation temperature (43°C) allowed rapid growth but no formation of conidia. After the glucose of the medium had been consumed (78 h after inoculation) sodium gluconate (8.5 mmoles per flask, in 5 ml H 20) was added and the incubation continued for 48 h, if not otherwise stated (Fig. I). Glucose and glueonate were determinated as described previously (MULLER 1966).

II. Preparation of cell-free extracts Mycelial mats were removed from the culture fluid, washed twice with distilled water and gently pressed to remove as much water as possible. The whole mat was ground in a cooled mortar with a pestle together with quartz. After the mat was broken in small fragments extraction buffer was added in portions of 2-3 ml, and the homogenization was continued until a homogeneous paste was obtained. This paste was brought to 25 ml with extraction buffer, and an aliquot was centrifuged at 20.000 g for 10 min. All operations were carried out at I-3°C. The supernatant was kept in an ice bucket and was used for enzyme assays as soon as possible.

III. Enzyme assay procedures All enzyme assays were conducted in a spectrophotometer at 25°C if not otherwise stated. The following reaction mixtures were used: I. Gluconokinase (EC 2.7.1.12). 100 tJmoles Tris-HCl buffer (pH 8.3), 5 tJmoles MgSO j , 5ltmoles ATP, 0,7 ttmoles NADP, 150ttmoles sodium gluconate, IOttg yeast phosphogluconate dehydrogenase, cell-free extract (extraction with 0.5 M potassium phosphate buffer, pH 6.5), H 20 ad 1.5 rnI. The absorbance was measured at 334 nm against air. (Modified after LAKSHMI~ARAYANA et al. 1969).

2. Glucose-6-phosphate and 6-phosphogluconate dehydrogenase (EC 1.1.1.49, 1.1.1.44). 50 tJmoles Tris.HCI buffer (pH 7.7) 5ttrnoles MgS04 , 0.7 ttmoles NADP, 5ttmoles glucose-Bvphosphate or 6-phosphogluconate, cell.free extract (extraction of glncose.Bvphosphate dehydrogenase with 0.1 M Tris-HCI buffer, pH 7.5, extraction of 6-phosphogluconate rlehydrogenaRe with 0.4 M potassium phosphate buffer, pH 6.5), H 20 ad 1.5 m!. The absorbance was measured at 334 nrn against a blank without gluoose-B.phosphato or 6-phosphogluconate, respectively. 3. Ribosephosphate isomerase (EC 5.3.1.6). a. Enzymatic assay: A mixture of 5 ml 0.05 M Tris-HCI buffer (pH 7.I), 1.0 ml 0.25 M cysteine-H'Cl. H 20 (freshly neutralized), 1.0 ml 0.1 )1 D-ribose 5-phosphate (sodium salt, in H 20) or 1.0 ml H 20 was prewarmed at :37 °C for 5 min. then 1.0 ml cell.free extract (extraction with 0.1 M Tris.HCI buffer, pH 7.1) or 1.0 ml ribose-

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pnosphate isomerase solution (containing 25 fJ-g of the purified enzyme from spinach per ml of 0.05 M Tris·HCI buffer, pH 7.1) was added and the complete reaction mixture was incubated 10 min at 37°C in a water bath. PIodified after ASHWELL and HICKMAN 1957, KNOWLES et al. 1969, DOMAGK and DOEHRING 1975). b. Cysteine.carbazole test of DISCHE and BOREXFREUND (1951) for the detection of Dvribulose 5-phosphate: 0.5 ml of the reaction mixture from the enzymatic assay or 0.5 ml of 0.1 M D-ribulose 5.phosphate solution (sodium salt, in 0.05 M Tris-HCI buffer, pH 7.1) was added to 0.2 ml 1.2 % cysteine.HCI. H 20 solution, 5 ml 25 N H 2S04 , 0.2 ml 0.1 % carbazole in 95 % ethanol. The mixture was incubated for 30 min at 37°C. The absorption spectrum was recorded against a blank without Dvriboso 5.phosphate. 4. Ribulosephosphate 3-epimerase (EO 5.1.3.1). 50 "moles triethanolamine-HOI buffer (pH 7.2), 10 fJ-moles Dvribose 5.phosphate, 25 fJ-g ribosephosphate isomerase (from spinach), cell-free extract (extraction with 0.1 M Tris-HOI buffer, pH 7.1), H 20 ad 1.2 ml. The assay was performed at 37°C, the absorbance was measured at 290 nm against a blank without Dvriboso 5.phosphate (modified after WOOD 1970). 5. Transkotolaso (EO 2.2.1.1). 100 fJ-moles glycyl-glyeino buffer (pH 7.3), 7.5 fJ-moles MgS0 4 • 7H20, 1.2fJ-moles thiamine pyrophosphate, 200fJ-g NADH, 0.01 ml mixture of GDH/TIM (glycerol-3-phosphate dehydrogenase and triosephosphate isomerase, 5.ug/ml), 2 fJ-moles D-ribose 5-phosphate, cell-free extract (extraction with 0.4 M potassium phosphate buffer, pH 6.7), H 20 ad 1.2 ml. The absorbance was measured at 334 nm against air (modified after CASSELTON 1966). 6. Enzymes of Entner-Doudoroff pathway. 65 ,'moles triethanolarnine-Htjl buffer (pH 7.65), 0.5 fJ-moles MnCI 2 , 3 fJ-moles glutathione, 180 fJ-g NADH, 10 fJ-moles j\.phosphogluconate, 50 fJ-g lactate dehydrogenase, 50.ug GDH/TIM, cell-free extract (extraction with 0.4 M potassium phosphate buffer, pH 6.7 or 0.1 M Tris·HCI buffer, pH 7.1), H 20 ad 1.0 m!' The absorbance was measured at 334 nm against a blank without 6-phosphogluconate (modified after GOTTSCHALK et al. 1964). 7. Gluconate dehydratase (EC 4.2.1.39). a. Enzymatic assay: 50ltmoles potassium phosphate buffer (pH 7.5), 20 fJ-moles potassium glueonate, 5 fJ-moles MgCI2 , cell-free extract (extraction with 0.1 M or 0.4 M potassium phosphate buffer, pH 7.5), H 20 ad 3 ml. The reaction mixture was incubated with and without glueonate for 45 and 80 min (ELZAINY et al. 1973). b. Thiobarbituric acid test: 0.20 ml of the enzymatic assay was added to 0.25 ml of 0.025 N HI0 4 in 0.125 N H 2S0 4 , after 20 min at room temperature, 0.50 ml of 2 % sodium arsenite in 0.5 N HOI was added with shaking, and the solution permitted to stand two minutes. Two ml of 0.3 % thiobarbituric acid (pH 2) were added, and after stirring, the mixture was heated to 100°C for 10 min. 'When the mixture was cooled, the optical density was measured at 548 nm (WEISSBACH and HCRWITZ 1959). 8. Gluconate dehydrogenase (EC 1.1.99.3). a. Reduction of 2,6.dichlorophenol indophenol (DIP). 100 fJ-moles Tris·HCI buffer (pH 7.0), 70 fJ-g DIP, 100 fJ-g phenazine metosulphate, 10 fJ-moles MgS0 4, 200 fJ-g NADP, 10 fJ-moles potassium gluconate, cell-free extract, (extraction with 0.4 1\1 potassium phosphate buffer, pH 6.5 or 0.2:\1 potassium phosphate buffer pH 7.2 or with 0.1 M Tris·HOI buffer, pH 7.2), H 20 ad 3.0 ml. The absorbance was measured at 600 nm against a blank without gluconate. b. Reduction of NAD and NADP. 100 fJ-moles glycyl-glycine buffer (pH 7.6), 50 fJ-moles MgS04 , 300 fJ-g NAD or NADP, 50 fJ-moles potassium gluconate, cell-free extract, H 20 ad 3 ml. The absorption was measured at 334 nm against a blank without gluconate (LAKSHi\II. NARAYANA et al. 1969).

IV. Chemicals Sodium salts of Dvriboae 5.phosphate and Dvribulose 5-phosphate, and ribosephosphate isomerase (type I, from spinach) were purchased from Sigma Chemicals, St. Louis, 2.6.dichlorophenol indophenol from Fluka, Basel, thiamine pyrophosphate from Serva, Heidelberg. The other chemicals and the auxiliary enzymes used in tho enzyme assays were from Boehringer, :\Iannheim, all other chemicals from Merck, Darmstadt.

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Results A. D egrada tion of g l uconate b y th e in ta c t m y c elium I n batc h cult ures which were used in t he present st udy t he content of gluconate per flask increased to a ma ximum and decreased again. When gluconate was added after exhaust ion of glucose, it s degrad ation started immediat ely and it was used up a lmost complete ly within 100 h (Fig. 1). B. Id en tifi cation of e nzy mes in ce ll -f ree e xt racts 1. Gluconokinase (2.7.1.12) This enzyme was identified by coupling the phosphorylation of gluconate to t he oxidation of 6-phosphogluconate. Thus in the presence of yeast 6-phosphogluconate dehydrogenase the phosphorylation of gluconate resulted in the formation of reduc ed NADP. This reduction did not occur when gluconate but no ATP was added and also when t he test system contained all components except th e cell-free extract. Some increase in the absorbance at 334 nm was also observed when ATP but no gluconate was added, but it was mu ch slower than in the presence of all component s (Fig. 2). Thi s increase may have been du e to the presence of some gluconate in the cell-free extract, or NADP was redu ced by glucose-6-phosphate dehydrogenase. (The cell-free extract contained hexokin ase and glucose-6-phosphate dehydrogenase.)

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Fig. 1. Utilization of g lucose and g lu conate in batc h cu lt ures of A. niger with gluco se as the so le source of carbon in t he origin al cu lt u re medi um. No g luc onate added: (0) g luc ose , (0) gluconat e Glucona t e a dde d (8.4 pmoles/fl ask): (e) gluconate t : time of add it ion. F ig. 2. I dentifica tion of gluco n oki nase (Ee 2.7. 1.12) in a cell-free ex t ra ct from A . niger. (el Glucon a t e and AT P a d de d, (0) Gluc onate a d de d , AT P om itted, (0 ) Gluconate omitt ed, AT P

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Utilization of Gluconate by Aspergillu8 niger. 1.

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Fig. 6. Stimulation of NADP reduction by D-ribose 5-phosphate. The reaction mixture contained 100/lmoles glyeyl-glycine buffer (pH 7.3), 7.5/lmoles MgS04 , 1.2/lmoles thiamine pyrophosphate, 200/lg NADP, 2,umoles D-ribose 5-phosphate, cell-free extract (extraction with 0.4 M potassium phosphate buffer, pH 6.7), H 20 ad 1.30 ml. (e) Dvribose 5-phosphate added, (0) D-ribose 5phosphate omitted.

Utilization of GJuconatc by Aspergillus niger. 1.

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2. Enzymes of the pentose phosphate pathway a. Glucose-6-phosphate and 6-phosphogluconate dehydrogenase (EC 1.1.1.49, 1.1.1.44). The se enzymes were identified by th e reduction of NADP which was markedly stimulat ed by the addition of glucose-6-phosphate and 6-phosphogluconate, reo specti vely. The assay for glu cose-6-phosphate dehydrogenase was not disturbed by 6-phosphogluconate deh ydrogenase because the form er enzyme was extracted with Tri s-H CI buffer wich lowered the activity of 6-phosphogluconate dehydrogena se. b. Ribo sephosphate isomera se (EC 5.:U.6) Thi s enzy me was detected by incubating D-ribose 5-phosphate with cell-free ex t ract followed by the identification of the reaction produ ct D-ribulose 5-phosphate via the cyste ine-carbazole t est of DISClIE and BORENFREUND (1951). This test yielded a produ ct with a characteristic ab sorption spectrum (Fig. a), and the same spectrum was obtained when instead of th e cell-free extract purified ribos ephosphate isomerase from spinach was used, or when th e cysteine-carbazole rea ction was performed with D-ribulose 5 phosphate (Fig. ~). c. Ribulosephosphate 3 -epimerase (EC 5.1.3.1.) As described by WOOD (1970) a solution containing D-ribose 5-phosphate was pla ced in the spect rop hotomete r and purified ribo sephosphate isomerase from spinach was added . The rea ction was a llowed to come to an equilibrium, i.e. after some tim e the a bsorption remained constant except for a very slow increase du e to traces of ribulosephosphate 3-epimerase presentvBy the addition of cell-free ext rac t the increase in ab sorbance was accelerated t hus demonstrating t he presence of epimerase in the ex t ra ct (Fig. 4). d . Transketolase (EC 2.2.1.1 ) The presence of this enzyme in t he cell-free extract was indicated by the rapid oxidation of NADH in a reaction mixture containing D-ribose 5-phosphate as substrate, thiamine pyrophosphate as cofactor, and triosephosphate isomerase and glycerol-3-phospha te dehydrogenase as a uxiliary enzymes (Fig . 5). Ribosephosphate isomerase and ribulosephosphate 3-epimerase which are also needed for this oxidation are present in the extract (see above). When D-ribose 5-phosphate and the two auxiliary enzymes were omitted, the oxidation of NADH pro ceeded much slower (Fig. 5). e. Operation of the whole pentose phosphate pathway The operation of this pathway in a cyclic fashion leads to the production of fructose 6-phosphate. Thus it should be possible to increase the rate of reduction of NADP by an extract containing glu cose-6-phosphate dehydrogenase and glucosephosphate isomerase by supplying D-ribose 5-phosphate. This proved to be the case with a cell-free extract of A. niger (Fig. 6). The presence of glu cosepho sphate isomerase was demonstrated in an optical test by using the rea ction mixture for the assay of glucose6-phosphate dehydrogenase in which glucose-6-phosphate was replaced by fru ctose6-phosphate. 3. Enzymes of the Entner-Doudoroff (ED) pathway If the two enzymes of this pathway, 6-phosphogluconate deh ydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) ar e present, the formation of pyruvate ad gly ceraldehyde 3-phosphate can be stimulated in a suitable test sys te m by the addition of 6-pho sphogluconate. In the presence of the auxiliary

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enzymes, lactate deh yd rogenase, glycerol-3-phosp hate dehydrogenase and triosepho sphate isomera se, t his stimulation leads to an in crease in t he rate of oxidation of NADH 2 • With our cell-free extracts t he decrease in ab sorb an ce at 334 nm could not be accelerate d by addit ion of 6-phosphogluconate . Thi s indicat es that the t wo E D enzymes were not present. 4. Gluconate dehydratase (EC 4.2.1.39) This enzyme catalyzes t he dehydrati on of gluconate into 2-keto-3-deoxyglu conate (KDG) t hus initiating a nonpho sphorylative degrad ation ana logous to t he Entn erDou doroff reactions. As mentioned a bove it has been identified in A. niger by ELZAINY et al. (1973). With our cell-free ext racts there was no form ation of KDG in t he reacti on mixture described by these authors. Th erefore, it can be concluded th at t his enzyme was not present. 5. Gluconat e dehydrogenas e (EC 1.1.99.3) Thi s enzym e catalyzes th e oxidation of gluconate, using in vitro the hydrogen a cceptors, amongst others, 2.6-dichlorophenol indophenol and NAD(P). It has been described in ba cteria, e.g. for Alcaligenes (KERSTERS and DE LEY 1975). In our cellfr ee extrac ts it could no t be det ect ed because there was no stim ulation of th e redu ction of 2.6-dichlorop henol ind ophenol and NAD(P) by the addit ion of gluconate .

Discussion Th e identification of gluconokinase in our cell-free extract indicates that t he degrad at ion of glueonate by t he stra in of A . niger which was used in the pr esent study is initiated by t he formati on of 6-phosphoglucona te . Our results also provide evidence that t his intermediate is metabolized via t he pentose ph osph ate pathway. Almost all of t he enzymes of t his system could be identified in cell-free extracts and t he increase of t he redu ction of NADP in a su itabl e test system by sup plying ribo se 5-phosph a te suggests t hat t he cycle as a whole is operating in vivo. LAKSHMINARAYANA et al. (1969) ident ified gluconoki nase in a cell-free extrac t from A . niger. Accord ing to t hese authors the activity of t his enzyme increased considera bly during ad ap tation of their st rain to growth on glueonate as the sole carbon source. This strongly suggest s that glu conokin ase is induced by gluconate. In ou r case this inductive effect may have been elicit ed by th e accumulation of glucona te from glucose and/or by the addition of gluconate after exhaust ion of glucose (Fig. 1). The presence of glu conokinase in cell-free extracts from A. niger which decomposed gluconate in the cult ure fluid has been demonstrated also by BETRA,ND and DE WOLFE (1957a, b) and by 'l'ESSI et al. (1968). Of t he enzymes of t he pentose ph osphate pa thway glucose-6-phosphate dehydrogenase and 6-phosph oglu conate dehydro gena se ha ve been identified in cell-free extracts of A . niger by McD ONOUGH and MARTIN (1958), FRANKE et al. (1963), DREWS and SMA,LLA (1969) and LAKSHMINARA,YANA, et al. (1969). Th e lat ter aut hors also have det ected rib osephosph at e isomerase. In t he present study, also ribulose phosph ate 3-epimerase and t ra nsketolase hav e been demonstrated . On incu bating cell-free extracts fo A. niger with ribo se 5-ph osphate, McDONOUGH and :M.A,RTIN (1958) obser ved t hat t he conte nt in tot al pentose decreased, accompanied by a sequential appear an ce and disappearance of ketopentose, and in t he presence of hydrazin both heptulose and t riose were shown to increase. These observations too prov ide evidence for t he operation of t he pentose ph osph ate pathway .

Utilization of G1uconate by Aspergillus niger. 1.

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It seems that the production not only of gluconokinase but also of enzymes of the pentose phosphate pathways is favoured by the presence of glue onate in the culture fluid. According to LA,KSHMINARAYA,NA et al. (1969) the activities of 6-phosphogluconate dehydrogenase and of ribosephosphate isomerase were much higher in the cellfree extract from a gluconate adapted strain than in the extract from the parent strain. In contrast, the levels of hexokinase and fructose diphosphate aldolase were more or less the same in both strains. DREWS and SMALLA, (1969) observed that the activity of 6-phosphogluconate dehydrogenase in a cell-free extract from A. niger increased in a late phase of gluconate accumulation, whereas the activities of hexokinase and fructose diphosphate aldolase remained constant or decreased. These observations are of considerable interest from the point of view of inductive synthesis of enzymes of the pentose phosphate pathway in the presence of gluconate. The enzymes of the Entner-Doudoroff (ED) pathway and of nonphosphorylative degradation mechanisms of gluconate could not be detected by us. The failure to detect ED enzymes is in agreement with the results of McDONOUGH and MARTIN (1958), GESER (1962), TESSI et al. (1968) and LA,KSHMINARAYANA et al, (1969), which also could not identify these enzymes in cell-free extracts of A. niger. The first enzyme of a modified ED pathway which dehydrates gluconate into 2-keto-3-deoxygluconate has been found by ELZAINY et al. (1973) in a cell-free extract of a mycelium which had been grown from conidia on a medium containing at the time of inoculation gluconate as the sole carbon source. In our case the sole carbon source in the original medium was glucose and this may have been the reason why a gluconate dehydratase could not be detected. The same explanation may also be true for the failure to identify a gluconate dehydrogenase. Another reason may be that the occurrence of this enzyme or group of enzymes is restricted to bacteria.

References ASHWELL, G., and HICKMAN, J.: Enzymatic formation of xylulose 5.phosphate from ribose 5phosphate in spleen. J. BioI. Chern. 226 (1957),65-76. BERTRAND, D., and DE WOLFE, A.: L'acide gluconique, metabolite intermediaire normal chez Aspergillus niger. C. R. Acad. Sci. Paris 244 (1957a), 1558-1561. - - Voie de transformation de I'acide gluconique lors de sa fermentation par I'Aspergillus niger. C. R. Acad, Sci. Paris 244 (1957b), 2985-2987. CASSELTON, P. J.: Enzymes of the Ernbden-Meyerhof and pentose phosphate pathway in Polyporus brumalis extracts. J. Exp. Bot. 17 (1966), 579-589. CLELAND, W. W., and JOHNSON, M. J.: Studies on the formation of oxalic acid by Aspergillus niger. J. BioI. Chern. 220 (1956),595-606. COCHRANE, V. W.: Physiology of fungi, New York, 1958. - Glycolysis. In: The Filamentous Fungi, Vol. 2 (J. E. Smith, D. R. Berry, Eds.), London 1976, 65-91. DISCHE, Z., and BORENFREUND, E.: A new spectrophotometric method for the detection and determination of keto sugars and trioses. J. BioI. Chern. 192 (1951), 583-587. DOMAGK, G. F., and DOEHRING, K. M.: DvRibose-fi-phosphate isomerase from Candida utilis. In: Meth. Enzymol., Vol. XLI (W. A. Wood, Ed.), New York 1975, 427-429. DREWS, B., und SMALLA, H.: Beitrag zur Kenntnis der Glucoseoxydase und der Gluconsaurega, rung in Aspergillus niger. Branntweinwirtsch. 109 (1969), 21-27. ELZAINY, T. A. HASSAK, M. M., and ALLAM, A. M.: New pathway for nonphosphorylatod degradation of gluconate by Aspergillus niger. J. Bacteriol. 114 (1973), 457-459. FOSTER, J. W.: Chemical Activities of Fungi. New York 1949. FRANKE, W., EICHHORN, G., MOCHEL, L., und BERTRAM, L: Zur Kennt.nis der sogenannten Glucose-oxydase. V. :\fitt. Zur Physiologie und Enzymatik der Glnconsauregarung durch Asper. gillus niger. Arch. Mikrobiol. 46 (1963),96-116.

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