Role of NADP-dependent and quinoprotein glucose dehydrogenases in gluconic acid production by Gluconobacter oxydans

Role of NADP-dependent and

quinoprotein glucose dehydrogenases in

gluconic acid production by

G luconobacter oxydans

J. T. Pronk,*$ P. R. Levering,t W. Oiijvet and J. P. van Dijken*

*Laboratory o f Microbiology and Enzymology, Delft University o f Technology, Julianalaan 67a, 2628 B C Delft, The Netherlands

tMicrobiological Research and D e v e l o p m e n t Laboratories, Scientific D e v e l o p m e n t Group, Organon International, P.O. Box 20, 5340 B H Oss, The Netherlands (Received 22 October 1987; revised 3 May 1988)

Gluconobacter oxydans, an organism used for the industrial production of gluconic acid, contains two glucose dehydrogenases (GDHs) catalysing the direct oxidation of glucose to gluconic acid. In addition to a quinoprotein (PQQ-dependent), membrane-bound GDH (EC 1.1.99.17), an NADP-dependent, cytoplasmic GDH is present. From three types of experiments, evidence is presented that the quinoprotein GDH is the enzyme responsible.for gluconic acid production by G. oxydans. In cell-['tee extracts, the activity of quinoprotein GDH was 30-fold higher than the activity of NADP-dependent GDH. A kinetic analysis of glucose-dependent oxygen uptake showed that a system with an affinity constant similar to the Km of quinoprotein GDH is involved in the process of gluconic acid production. The conclusion that gluconic acid production is essentially an extracytoplasmic process catalysed by quinoprotein GDH was confirmed in uptake experiments, which demonstrated that inhibition of glucose transport does not result in inhibition of gluconic acid production.

Keywords: Gluconobacter oxydans; gluconic acid; glucose metabolism; quinoproteins

Introduction

Acetic acid bacteria are capable of carrying out a large number of incomplete oxidations. A number of these bioconversions are applied on a large scale in industry. Well-known examples are the production of acetic acid from ethanol by A c e t o b a c t e r spp. and the oxida- tion of glucose to gluconic acid by Gluconobacter oxydans.

The ability to produce gluconic acid from glucose is not restricted to G. oxydans but can be encountered in a wide variety of gram-negative bacteria. ~ In the majority of these bacteria, the conversion of glucose to gluconic acid constitutes the first step of the so-called direct oxidative pathway 2 of glucose catabolism. Glu- conic acid formed as a result of glucose oxidation can be further metabolized via either the Entner-Dou- doroff (KDPG) or pentose phosphate pathway. As an

~: To whom correspondence should be sent

alternative to the direct oxidative pathway, glucose may be phosphorylated prior to oxidation. 2

Glucose metabolism in G. oxydans proceeds exclu- sively via the pentose phosphate p a t h w a y ) 6-Phos- phogluconate, a key intermediate in this pathway, can be formed by phosphorylation of gluconic acid formed in the direct oxidation of glucose. 4 Alternatively, glucose may first be phosphorylated to glucose-6- phosphate by hexokinase and subsequently oxidized through the action of an NADP-dependent glucose-6- phosphate dehydrogenase. 5

The mechanism and physiological function of glu- conic acid production have been studied extensively in A c i n e t o b a c t e r calcoaceticus. In this bacterium, glu- cose metabolism is restricted to the formation of gluconic acid. 6 A membrane-bound quinoprotein (PQQ-containing) glucose dehydrogenase (EC 1.1.99.17) 7 is responsible for gluconic acid production by A . calcoaceticus. The enzyme is located at the outer side of the cytoplasmic membrane, facing the periplasm, a The same enzyme has been demonstrated

Gluconic acid production by G. oxydans: J. T. Pronk et al. to be responsible for gluconic acid production by

Pseudomonas aeruginosa, 9 Klebsiella aerogenes, l°

and Rhodopseudomonas sphaeroides 11 (now called

Rhodobacter sphaeroides).

G. oxydans is exceptional in its glucose metabolism since it contains two enzymes catalysing the direct oxidation of glucose to gluconic acid. In addition to the

membrane-bound quinoprotein GDH mentioned

above,~2 G. oxydans contains a soluble, NADP-depen- dent GDH (EC 1.1.1.47). 23 No conclusive evidence has been presented as to which,glucose oxidizing enzyme is responsible for the extensive product for- mation. Based on enzyme activities measured in cell- free extracts, it has been suggested 14'15 that NADP- dependent GDH provides a major contribution to gluconic acid production. In view of the significance of quinoprotein GDH in other bacteria, we decided to reinvestigate the role of both glucose dehydrogenases in the process of gluconic acid production by G.

oxydans.

Material and methods

Organism and growth conditions

G. oxydans ATCC 621H was obtained from the Ameri- can Type Culture Collection. The organism was grown under glucose limitation in a chemostat at a dilution rate of 0.10 h -~ at 28°C. A laboratory fermenter ~6 with a working volume of 1 1 was used. The dissolved oxygen concentration was recorded with a Clark-type oxygen electrode and maintained above 25% of air saturation. The pH was controlled at 5.5 by automatic addition of 2 M NaOH. The mineral salts medium contained, per litre: L-glutamine, 0.2 g; (NH4)2SO4, 2 g; MgSO4" 7H20, 0.2 g; KH2PO4, 2.2 g; Na2HPO4,

0.2 g; nitrilotriacetic acid, 5 mg; EDTA, 30 mg;

FeSO4.7H~O, 11 mg; ZnSO4"7H20, 9 mg; CoClz. 6H20, 0.6 mg; MnC1E-4H20, 2 mg; CuSO4.5H20, 0.6 mg; CaCl2 • 2H20, 9 mg; NaMoO4 • 2H20, 0.08 mg; H3BO3, 1 mg; KI, 0.2 mg; calcium panthotenate, 0.5 mg; nicotinic acid, 0.4 mg; p-aminobenzoic acid, 0.4 mg. The vitamins were filter-sterilized and added to the autoclaved medium. Glucose was autoclaved sepa- rately and added to a final concentration of 50 mM.

Preparation o f cell-free extracts

Cells were harvested by centrifugation (10,000g, 10 min, 4°C) and washed twice with 50 mM potassium phosphate buffer (pH 6.0) containing 5 mM MgC12. The final pellet was resuspended in the same buffer at a final concentration of approximately 20-30 mg dry weight ml -~. Portions of 2 ml were sonicated at 0°C in a Branson Sonifier B-12 Cell Disruptor (Branson Sonic Power Co., Danbury, Connecticut, USA) operating at 25 W output (15 × 15 s, with 30-s intermitting peri- ods). Whole cells were removed by centrifugation at 10,000g for 5 min at 4°C. The supernatant which contained 8-12 mg protein m1-1 was used as the cell- free extract.

Enzyme assays

Spectrophotometric assays were carried out at 280C with freshly prepared extracts. In all assays the reac- tion rate was linearly proportional to the amount of extract present. The assay mixtures used for the individual enzymes are described below.

Glucose dehydrogenase, PQQ-dependent (EC

1.1.99.17). Potassium phosphate buffer (pH 5.5), 75 mM; 2,6-dichlorophenol-indophenol (DCPIP), 0.15 mM; phenazine methosulfate (PMS), 2 mM; and cell- free extract. The reaction was started by the addition of glucose (20 mM). A molar extinction coefficient of DCPIP at 600 nm and pH 5.5 of 7.0 mM -~ cm -~ was used to calculate enzyme activity.

Glucose dehydrogenase, NADP-dependent (EC 1.1.1.47). Potassium phosphate buffer (pH 7.4), 75 mM; NADP, 0.5 mM; Triton X-100, 0.2% (in order to destroy NADPH oxidase activity~7); and cell-free ex- tract. The reaction was started by the addition of glucose (60 mM). A molar extinction coefficient of NADP at 340 nm of 6.22 mM -~ cm -~ was used to calculate enzyme activity.

Hexokinase (EC 2.7.1.1). Tris-HCI buffer (pH 7.4), 75 mM; MgCl2, 5 mM; NAD, 0.5 mM; glucose, 20 mM; Triton X-100, 0.05% (v/v); glucose-6-phosphate dehy- drogenase, 6 units m1-1. The reaction was started by the addition of ATP (2 mM).

Measurement o f glucose-dependent oxygen uptake

Cells were harvested by centrifugation (10,000g, 10 min). The pellet was resuspended in I00 mM potassium phosphate buffer (pH 5.5) containing 10 mM MgSO4, to a final concentration of 0.10-0.15 mg dry weight ml -~. Glucose-dependent oxygen uptake was mea- sured at 28°C using a Clark-type oxygen electrode. The solubility coefficient of oxygen at 28°C was taken to be 0.225 pmol m1-1.

Glucose transport by whole cells

Cells from a glucose-limited chemostat culture were harvested by centrifugation (10,000g, 10 min, 4°C), washed once with I00 mM potassium phosphate buffer (pH 5.5) containing 10 mM MgSO4, and resuspended in the same buffer. Uptake experiments were performed at 28°C as described previously. TM The experiments

were started by the addition of 14C-glucose (9.25 MBq mmol -~) to a final concentration of 5 mM. Cells were preincubated at 28°C for 5 min in the presence or absence of 2,4-dinitrophenol before the addition of 14C-glucose.

Gluconic acid production by suspensions o f chemostat-grown cells

Cells (0.8 g dry weight) were harvested by centri- fugation (10,000g, 10 min) and resuspended in mineral

P a p e r s

medium without glucose to a final concentration of 0.3 g dry weight 1-1. Gluconic acid production experi- ments were carried out at 28°C in a fermenter with a working volume of 1 1. The pH was maintained at 5.5 by automatic addition of 4 M NaOH. Experiments were started by the addition of glucose to a final concentration of 100 mM. At appropriate times, 5 ml samples were withdrawn and analyzed for glucose and gluconic acid. Growth was monitored by measuring the absorbance at 650 nm. CO2 production and Oz consumption were monitored continuously using an Uras 3E Infrared Gas Analyzer (Hartmann Braun AG, Frankfurt, FRG) and a Servomex 1100 A Model C Oxygen Analyser (Crowborough, Sussex, UK). Gas analysis data were acquired and processed with an Apple lie microcomputer.

i i m m

Analytical assays

Glucose was measured by the GOD-PAP method and gluconic acid with gluconate kinase/6-phosphoglu- conate dehydrogenase (testkits, Boehringer Mann- helm). Bacterial dry weight content of suspensions was calculated from the absorbance at 650 nm, using standard curves made with cells grown under identical conditions.

Chemicals and enzymes

(U-'4C)-o-Glucose was obtained from the Radio- chemical Centre (Amersham, Buckinghamshire, UK). 2,4-Dinitrophenol was obtained from Merck (Darmstadt, FRG). Glucose-6-phosphate dehydro- genase (from Leuconostoc) was purchased from Boeh- ringer (Mannheim, FRG). glucose J ~ gtuconate.~

t

t

Figure 1 Alternative routes of gluconate formation in G. oxy- dans. The numbered arrows represent the following enzymes or enzyme systems: (1) PQQ-dependent glucose dehydrogenase (EC 1.1.99.17); (2) glucose uptake system; (3) NADP-dependent glucose dehydrogenase (EC 1.1.1.47); (4) hexokinase (EC 2.7.1.1); (5) glucose-6-phosphate dehydrogenase (EC 1.1.1.49); (6) 6-phosphogluconate phosphatase (hypothetical); (7) gluco- nate transport system. The numbers in brackets indicate activi- ties [/~mol min -1 (mg protein) -1] measured in cell-free extracts prepared from cells grown in glucose-limited chemostat cul- tures (D = 0.10 h -1)

Kinetics o f glucose oxidation by intact cells

The kinetics of glucose oxidation by intact cells of G. oxydans were investigated by measuring the rate of Oz consumption at various glucose concentrations. A Lineweaver-Burk plot of the results (Figure 2) dem- onstrates the involvement of two kinetically different systems in the process of glucose-dependent oxygen consumption. The affinity constants and the maximal reaction rates of both systems differ by less than a factor of I0. This implies that it is not possible to

Results and discussion

Activities o f PQQ- and NADP-dependent glucose dehydrogenases in cell-free extracts G. oxydans cells can theoretically form gluconate from glucose in three ways (Figure 1). Gluconate may be formed by direct oxidation of glucose via either one or both of the glucose dehydrogenases. Furthermore, gluconate might be formed by dephosphorylation of 6-phosphogluconate. The latter route seems energeti- cally unfavorable, since it requires the consumption of I mol of ATP per mol of gluconate formed. Further- more, the presence of a 6-phosphogluconate phospha- tase has not been demonstrated in G. oxydans.

In cell-free extracts, PQQ-dependent GDH was by far the most active glucose oxidizing enzyme (Figure I). Its activity was some 27 times higher than the activity of NADP-dependent GDH. Hexokinase activ- ity could also be detected in cell-free extracts, al- though the activity was low when compared with either one of the GDHs. Since enzyme activities in cell-free extracts do not necessarily reflect in viva activities, the kinetics of glucose oxidation by intact cells were studied.

! o X i E i Z e I I I I I I 2 3 4 5 I/S ( mM -1 )

Figure 2 Kinetics of glucose-dependent oxygen uptake by intact cells of G. oxydans: double-reciprocal plot of glucose concentration versus rate of oxygen uptake. Cells were grown in a glucose-limited chemostat culture (D = 0.10 h -~)

Gluconic acid production by G. o x y d a n s : J. T. Pronk e t al. accurately calculate these parameters from a kinetic

plot. Nevertheless, it is clear that the Km of the low-affinity system (approximately 2 mM) is compa- rable with the affinity constants of quinoprotein GDH from Acinetobacter calcoaceticus, 19 Pseudomonas fluorescens, 2° and G. oxydans. 13 It therefore seems likely that the low-affinity system corresponds with glucose oxidation via quinoprotein GDH.

The Km of NADP-dependent GDH from G. oxydans was determined in cell-free extracts. The observed value (13 mM) corresponds neither with the low-affin- ity system nor with the high-affinity system. Since the Km values of bacterial and yeast hexokinases are usually around 0.1 mM, z='z2 the high-affinity glucose- oxidizing system may reflect glucose oxidation via the phosphorylative route (Figure I). Alternatively, the high-affinity system may represent a glucose uptake system, coupled to the cytoplasmic oxidation of glu- cose via either hexokinase or NADP-dependent GDH. The presence of a glucose uptake system with a Km of 0.05 mM in whole cells of G. oxydans has been demonstrated previously. 14

In order to study the contribution of PQQ-depen- dent GDH and the high-affinity system in gluconate production, we attempted to specifically inhibit the latter system.

Effect o f 2,4-dinitrophenol on glucose transport and gluconate production

Oxidation of glucose via quinoprotein GDH takes place in the periplasm. Both hexokinase and NADP- dependent G D H are located in the cytoplasm (Figure 1). Oxidation of glucose via either one or both of the latter enzymes requires uptake of glucose into the

200 150 u E 50 _-,,=, I 2 3 a 5 Time(min)

Figure 3 Uptake of 14C-glucose by intact cells of G. oxydans in the absence (0) and presence (©) of 1 m . DNP. Cells were preineubated for 5 rain in the absence or presence of DNP before the addition of ~4C-glucose. Cells were grown in a glucose-limited chemostat culture (D = 0.10 h -1)

Table 1 Initial rates of glucose consumption, gluconate pro- duction, 02 consumption, and COz production by G. oxydans in the absence and presence of 1 mM DNP

Initial rates [/~mol min -1 (mg dry weight) -11 -DNP +DNP Glucose consumption 2.0 2.1 Gluconate production 1.9 2.0 02 consumption 1.3 1.1 CO2 production 0.26 0.04

The organism was pregrown in a glucose-limited chemostat culture (D = 0.10 h -1) and resuspended to a final concentration of 0.3 g dry weight 1-1. At zero time glucose was added to a final concentration of 100 m i

cells. In the case that quinoprotein GDH is responsible for gluconate production, this process should be insen- sitive to inhibition of glucose transport.

2,4-Dinitrophenol (DNP) is a well-known uncoupler of membrane-associated energy-transducing pro- cesses. At a concentration of 1 mM, DNP almost completely inhibited uptake of glucose by intact cells of G. oxydans (Figure 3).

Parallel experiments were set up in which the .effect of 1 mM DNP on gluconate production was studied. Gluconate production rates were almost identical in the presence and absence of DNP (Figures 4A and 4B, Table 1). Growth was completely inhibited by 1 mM DNP, while CO2 production was greatly reduced. Furthermore, addition of DNP reduced the amount of oxygen consumed per mole of glucose. In the presence of DNP a I :2 stoichiometry of oxygen and glucose consumption was observed (Table 1). This observa- tion is consistent with the fact that complete oxidation of glucose to COz can only occur in the cytoplasm.

As mentioned above, DNP inhibits growth and CO2 production. However, from the data presented in Figure 4B, it is clear that only 90% of the glucose added can be recovered as gluconate. This discrep- ancy probably reflects the formation of small amounts of ketogluconates. 23

The observation that DNP, despite its effect on glucose transport, does not inhibit gluconate formation indicates that gluconate formation is an extracytoplas- mic process. This is consistent with the measured enzyme activities, which suggested a major invol- vement of PQQ-dependent GDH in the process of gluconate production.

Concluding remarks

The results clearly demonstrate that membrane- bound, quinoprotein glucose dehydrogenase is the enzyme responsible for the rapid oxidation of glucose to gluconate by G. oxydans. If the soluble, NADP- dependent GDH is involved in gluconate production, its contribution is quantitatively insignificant. This conclusion seems to be contradictory to results pres- ented previously, 14'15 where higher activities of

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F i g u r e 4 Effect of DNP on gluconate production by G. oxydans.

The organism was pregrown in a glucose-limited chemostat culture (D = 0.10 h -1) and resuspended in mineral medium to a final concentration of 0.3 g dry weight 1-1. At zero time, glucose w a s added to a final concentration of 100 mM. (A) Gluconate production, glucose consumption, and growth in the absence of DNP. (B) Gluconate production, glucose consumption, and growth in the presence of 1 mM DNP. Glucose (©), gluconic acid (O), and growth (r-I)

NADP-dependent GDH as compared to the mem- brane-bound enzyme were found in cell-free extracts of G. oxydans. However, the assay mixture for the measurement of quinoprotein GDH activity employed by these authors did not contain the mediator phen- azine methosulfate (PMS). Omission of PMS from the assay mixture used in our experiments resulted in a decrease of quinoprotein GDH activity of approxi- mately 75-80% (data not shown).

In many gram-negative bacteria, glucose oxidation via quinoprotein GDH leads to the generation of a proton motive force, which can be coupled effectively to the energization of various cellular processes. 19,24 It

has been shown that oxidation of glucose by mem- brane particles of G. oxydans can be coupled to the phosphorylation of ADP. 25 Furthermore, glucose oxi- dation can energize solute transport in cytoplasmic membrane vesicles of G. oxydans (J. T. Pronk, unpu- blished results). Therefore, it seems likely that gluco- nate formation in G. oxydans has a similar physiologi- cal function.

The major role of quinoprotein GDH in gluconate production by G. oxydans is in agreement with the theory of Hooper and Dispirito. 26 These authors stated that proton-yielding oxidation reactions preferably take place extracytoplasmically. It will be interesting to learn whether membrane-bound dehydrogenases also are primarily responsible for product formation in other bacteria that possess both cytoplasmic and peri- plasmic enzymes for the oxidation of a single sub- strate. Particularly interesting in this respect is the formation of acetate from ethanol by Acetobacter spp. 27

A c k n o w l e d g e m e n t s

The skillful technical assistance of Mrs. Anjo van Heijst is gratefully acknowledged. J. T. P. and J. P. v. D. are indebted to Wendy Levering for her kind hospitality during their stay at Organon.

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25 26 27