Marczak Laboratoire de Biochimie des Bactéries Gram +, Domaine scientifique Victor Grignard, Université Henri Poincaré, Faculté des Sciences, BP 239, 54506 Vandoeuvre lès Nancy cédex, France (e-mail: email@example.com).
H. MALAOUI AND R. MARCZAK. 2001.
Aims:Clostridium butyricum E5 wild-type and mutant E5-MD were cultivated in chemostat culture on glycerol in order to compare the properties of two key enzymes of glycerol catabolism, i.e. propanediol and glycerol dehydrogenase.
Methods and Results: These two enzymes, which belong to the dha regulon, were separated by gel filtration. Both dehydrogenase activities displayed similar properties, such as pH optimum values, specificity towards physiological substrates and dependence on Mn2+. Both strains accumulate glycerol at high levels.
Conclusion: The mutant D strain contained a propanediol dehydrogenase activity which had a low affinity for its physiological substrate, leading to the conclusion that this strain would seem more resistant to the toxic effect of 3-hydroxypropionaldehyde than the wild-type.
Significance and Impacts of the study: These properties make Cl. butyricum mutant D strain the best candidate so far to be used as a biotechnological agent for the bioconversion of glycerol to 1,3-propanediol.
Clostridium butyricum, a strictly anaerobic spore-forming bacterium, usually metabolizes glycerol to 1,3-propanediol (1,3-PPD), acetate, butyrate, carbon dioxide (CO2) and molecular hydrogen (H2). Part of the reduced ferredoxin is reoxidized by the ferredoxin-NAD+ reductase, which explains why glycerol fermentation yields considerably less H2 than CO2 (Biebl et al. 1992). The enzymes glycerol dehydrogenase, diol dehydratase and 1,3-PPD dehydrogenase constitute the branch point that partitions the carbon flux between the competing pathways, i.e. formation of either 1,3-PPD or pyruvate. The increasing levels of these enzyme activities with increasing dilution rates explain the constant proportion of glycerol conversion into 1,3-PPD (Abbad-Andaloussi et al. 1996a). The same constancy in the conversion of glycerol into 1,3-PPD is also observed with hydrogenase-negative mutants obtained from Cl. butyricum DSM 5431 (Abbad-Andaloussi et al. 1995) and allyl alcohol-resistant mutants obtained from Cl. butyricum E5 (Abbad-Andaloussi et al. 1996b). Allyl alcohol resistance cannot be attributed to the loss of 1,3-PPD dehydrogenase. The apparent Km values of the various reactions catalysed by 1,3-PPD dehydrogenase in crude extracts of wild-type and mutant E5-MD do not differ greatly for NAD+ and NADH but show clearly in mutant D a decreasing Km value for glyceraldehyde, a non-physiological substrate, and an increasing Km value for 1,3-PPD and allyl alcohol (Abbad-Andaloussi et al. 1996b).
1,3-propanediol dehydrogenase (1,3-PPD dehydrogenase; EC 220.127.116.11) was detected originally in extracts of glycerol-grown cells as an enzyme that catalyses the oxidation of NADH at the expense of 3-hydroxypropionaldehyde (3-HPA) (Abeles et al. 1960). In contrast to the 1,3-PPD-forming enteric bacteria, little is known about the enzyme responsible for glycerol breakdown by clostridia. The activities of glycerol dehydrogenase, glycerol dehydratase and 1,3-PPD dehydrogenase have been determined in crude extracts of Cl. butyricum (Abbad-Andaloussi et al. 1996a) and the latter activity in Cl. pasteurianum (Heyndrickx et al. 1991). It is most likely that glycerol catabolism by Cl. butyricum requires 1,3-PPD dehydrogenase activity to avoid intracellular accumulation of 3-HPA, a very toxic compound (Axelsson et al. 1989). 1,3-PPD dehydrogenase has been purified from Klebsiella pneumoniae (Johnson and Lin 1987), Lactobacillus buchneri and Lact. brevis (Veiga-Da-Cunha and Foster 1992), Lact. reuteri (Talarico et al. 1990) and Citrobacter freundii (Daniel et al. 1995a). The entire dhaT regulon of C. freundii has been cloned and expressed in Escherichia coli (Daniel and Gottschalk 1992). The gene encoding glycerol dehydratase and 1,3-PPD dehydrogenase of Cl. pasteurianum has been cloned and expressed in E. coli and the sequence of the dha gene has been determined (Luers et al. 1997). Recently, 1,3-PPD dehydrogenase from Cl. butyricum E5 wild-type has been purified. Similarity was found between the N-terminal amino acid sequence of the 1,3-PPD dehydrogenase of Cl. butyricum and that of K. pneumoniae, C. freundii and Cl. pasteurianum (Malaoui and Marczak 2000).
The purpose of this study is the characterization of the 1,3-PPD and glycerol dehydrogenase of Cl. butyricum wild-type E5 and mutant E5-MD cultivated in chemostat culture on glycerol. These two enzymes are separated by Fast Protein Liquid Chromatography (FPLC) before characterization, which allows the properties of each enzyme to be studied without any cross-interference. The comparison of these properties should explain why these strains can be considered as competitive candidates for the bioconversion of glycerol to 1,3-PPD.
MATERIALS AND METHODS
Organism and medium
Clostridium butyricum E5 and E5-MD, resistant to allyl alcohol, were used. The spores of the strains were stored at 4°C in Hungate tubes in Reinforced Clostridial Medium (RCM; Oxoid). For inoculum preparation, spores were transferred to RCM medium, heat-shocked at 80°C for 10 min, and incubated at 34°C under anaerobic conditions in Hungate tubes. The pre-culture medium contained the following components (l–1 dionized water): glycerol 20 g; KH2PO4, 1·0 g; K2HPO4, 0·5 g; (NH4)2SO4, 2·0 g; MgSO4.7H2O, 0·2 g; CaCl2.2H2O, 15 mg; FeSO4.7H2O, 5 mg; CaCO3, 2·0 g; yeast extract, 1·0 g; trace element solution SL7 (Biebl and Pfenning 1982) 2 ml.
The growth temperature was 34°C. This medium, without CaCO3, and with 60 g l–1 glycerol, was used as the culture medium in the bioreactor.
Clostridium butyricum E5 and E5-MD were grown in glycerol-limited continuous culture. The continuous culture was carried out aseptically in 2 litre bioreactor (LSL-Biolafite (Saint Germain en Laye, France), 2 litre growth vessel with a 1 litre working volume). The temperature was controlled at 34°C and the pH at 6·8 with 2 mmol l–1 KOH. Anaerobic conditions were maintained by sparging with nitrogen. Agitation was kept constant at 100 rev min–1. The culture volume was kept constant at 1 litre by automatic regulation of the culture level. The bioreactor was inoculated (10%, v/v) with exponential pre-culture. The culture was grown in batch for 6–12 h, and then the continuous culture was started with a glycerol input concentration of 10 g l–1.
Preparation of cell-free extracts
Cells grown on glycerol were resuspended in Tris buffer (50 mmol l–1 Tris-HCl; 2·0 mmol l–1DL-dithiothreitol; 1 mmol l–1 MnCl2; pH 7·4), sparged with nitrogen and centrifuged at 12 000 g for 15 min. The cells were sonicated at 2°C for 20 s at a frequency of 20 kHz, followed by a 60 s pause (150 W ultrasonic disintegrator; MSE, Crawley, Sussex, UK); this cycle was repeated four times. The supernatant fluid was collected from the cell lysate by centrifugation at 12 000 g for 20 min at 4°C. At each step, extracts were maintained under a nitrogen atmosphere. The protein concentration of cell extracts was determined according to the method of Lowry et al. (1951), using crystalline bovine serum albumin as the standard. One unit of enzyme activity was the amount catalysing the formation of one micromole of product per minute under the specified conditions.
Part of the supernatant fraction of the cell extract from Cl. butyricum E5 was applied, through a 0·2 ml injection loop, to a high resolution (HR 10/30) superose 12 column of the Fast Protein Liquid Chromatography (FPLC) Pharmacia system (Pharmacia Biotech AB, Uppsala, Sweden). The column was equilibrated with 50 mmol l–1 potassium phosphate buffer (KPB, pH 7·4) containing 100 mmol l–1 KCl and 2 mmol l–1 DTT (flow rate, 0·5 ml min–1). The active fractions (1 ml volumes) were eluted with the same buffer and used for characterization of 1,3-PPD dehydrogenase and glycerol dehydrogenase activities.
The activities of 1,3-PPD dehydrogenase (EC 18.104.22.168) and glycerol dehydrogenase (EC 22.214.171.124) were assayed at 37°C in a SHIMADZU UV-160 A spectrophotometer (Shimadzu, Kyota, Japan) equipped with a Haake D1 circulating water bath (Thermo Haake, Karlsrule, Germany) to control the sample cell temperature. Activities were measured by the linear increase in absorbance at 340 nm (A340) produced by addition of the enzyme fraction. The assay mixture contained 100 mmol l–1 1,3-PPD or glycerol, 2 mmol l–1 NAD+, 30 mmol l–1 (NH4)2SO4 and 100 mmol l–1 potassium carbonate buffer (pH 9·7) in a 1 ml final volume.
Determination of the optimum pH
Assays to determine the optimum pH were performed with 0·2 mol l–1 KPB adjusted to the appropriate pH values with 3 mol l–1 KOH or 3 mol l–1 HCl. DL-glyceraldehyde (DL-Gld) 10 mmol l–1, dihydroxyacetone (DHA) 10 mmol l–1 and 3-HPA 3·5 mmol l–1 were reduced in the presence of 0·37 mmol l–1 NADH. 1,3-PPD 100 mmol l–1 and glycerol 100 mmol l–1 were oxidized in the presence of 2 mmol l–1 NAD+. The optimum pH values were calculated by non-linear regression to the Bell-Shaped Double pKa equation by use of the Curve Fit feature of the Grafit programme (Erithacus Software, London, UK).
Determination of kinetic parameters
The apparent Km values obtained with substrates and coenzymes were determined at 37°C with K2CO3 buffer (pH 9·7 for the oxidative reactions and pH 9·1 for the reductive reactions). They were determined from the results of experiments in which a fixed concentration of the other reactant was used.
The apparent Km values were expressed in mmol l–1 and calculated by non-linear regression to the Michaelis Menten equation by use of the Curve Fit feature of the Grafit programme.
Determination of substrate specificity
The activities of 1,3-PPD dehydrogenase and glycerol dehydrogenase in oxidation reactions were determined spectrophotometrically at 340 nm by use of the initial rate substrate-dependent NADH increase at 37°C. The assay mixture contained 100 mmol l–1 K2CO3 buffer (pH 9·7), 30 mmol l–1 (NH4)2SO4, 2 mmol l–1 NAD+ and 100 mmol l–1 substrate in a 1 ml final volume. The activities were expressed relative to those obtained with 1,3-PPD or glycerol, respectively.
The enzyme activities in reduction reactions were determined under the same conditions as those described for oxidation reactions, except that the assay mixture contained 100 mmol l–1 K2CO3 buffer (pH 9·1), 30 mmol l–1 (NH4)2 SO4, 0·37 mmol l–1 NADH and 100 mmol l–1 substrate in 1 ml final volume. Activities were expressed relative to those obtained with 3-HPA.
Effect of enzyme by mono and divalent cations
The chloride salts of ammonium, sodium, potassium, magnesium or lithium (10 mmol l–1), and iron, manganese or calcium (1 mmol l–1), were included with 100 mmol l–1 1,3-PPD or glycerol, 2 mmol l–1 NAD+ and 100 mmol l–1 carbonate buffer (pH 9·0) to determine the effect of these cations on the 1,3-PPD dehydrogenase and glycerol dehydrogenase activities.
Separation of 1,3-PPD dehydrogenase and glycerol dehydrogenase from Cl. butyricum by gel filtration
1,3-PPD dehydrogenase and glycerol dehydrogenase from Cl. butyricum E5 wild-type were separated by gel filtration as two enzymatic peaks (Fig. 1). The 1,3-PPD dehydrogenase was eluted as a single peak with an elution time of 17 min (fraction 8) and the glycerol dehydrogenase as a single peak with an elution time of 21 min (fraction 10).
According to the method used to determine the molecular mass of the native 1,3-PPD dehydrogenase (Malaoui and Marczak 2000), the molecular mass of the native glycerol dehydrogenase can be estimated to be 181 000 ± 15 000 Da.
A summary of the separation protocol of 1,3-PPD and glycerol dehydrogenase by gel filtration is presented in Table 1 and Table 2, respectively. 1,3-PPD dehydrogenase was purified fivefold with a 53% recovery, and glycerol dehydrogenase was also purified fivefold with a 35% recovery. Residual activities in fraction 10 for 1,3-PPD dehydrogenase and in fraction 8 for glycerol dehydrogenase were compared with those found in crude extract and can be attributed to non-specific substrate oxidation by the other enzyme.
Table 1. Recovery of 1,3-propanediol dehydrogenase after gel filtration from Clostridium butyricum E5 wild-type
Table 2. Recovery of glycerol dehydrogenase after gel filtration from Clostridium butyricum E5 wild-type
Similar results were obtained with Cl. butyricum E5-MD (data not shown).
Determination of the optimum pH of 1,3-PPD dehydrogenase from Cl. butyricum E5 wild-type and mutant D
The optimum pH values for the oxidation reaction of 1,3-PPD and for the reduction reaction of DL-Gld and 3-HPA catalysed by 1,3-PPD dehydrogenase are summarized in Table 3.
Table 3. Determination of the optimum pH of 1,3-propanediol dehydrogenase isolated from gel filtration for oxidation and reduction reactions
The enzyme of both strains exhibited Bell-Shaped-Double pKa kinetics. For the reduction of the 3-HPA and DL-Gld, Table 3 shows that the optimum pH was nearly the same whatever the strain under study. By contrast, the optimum pH differed by two units according to the substrate used. For the oxidation of 1,3-PPD, the optimum pH of the enzyme differed by one unit between the mutant D and the wild-type.
Determination of the optimum pH of glycerol dehydrogenase from Cl. butyricum E5 wild-type and mutant D
The optimum pH values for the oxidation reaction of glycerol and for the reduction reaction of DHA catalysed by glycerol dehydrogenase are summarized in Table 4.
Table 4. Determination of the optimum pH of glycerol dehydrogenase isolated from gel filtration for oxidation and reduction reactions
The glycerol dehydrogenase of both strains exhibited Bell-Shaped-Double pKa kinetics. Table 4 shows that there was no significant difference in the optimum pH value for glycerol dehydrogenase in DHA reduction, and in that of glycerol oxidation, between the two strains. On the other hand, the optimum pH value of the reaction of glycerol oxidation was one unit lower than that of the reaction of DHA reduction.
Determination of kinetic parameters of 1,3-PPD dehydrogenase from Cl. butyricum E5 wild-type and mutant D
The apparent Km values determined for various reactions catalysed by 1,3-PPD dehydrogenase are summarized in Table 5. The 1,3-PPD dehydrogenase of both strains exhibited Michaelis-Menten kinetics. Table 5 shows that the apparent Km of 1,3-PPD dehydrogenase of mutant D for the various substrates and coenzymes tested was two to five times higher than that of the enzyme of the wild-type. This implies that the enzyme of mutant D had less affinity than the enzyme of the wild-type for the respective substrates. Among the substrates studied, it appeared that the physiological substrate (3-HPA) had more affinity for the enzyme than the non-physiological substrate DL-Gld, nine times more in mutant D and seven times more in the wild-type.
Table 5. Determination of Km values for substrates and coenzymes of 1,3-propanediol dehydrogenase isolated from gel filtration
Determination of kinetic parameters of glycerol dehydrogenase from Cl. butyricum E5 wild-type and mutant D
The apparent Km values determined for various reactions catalysed by glycerol dehydrogenase are summarized in Table 6. The glycerol dehydrogenase of both strains exhibited Michaelis-Menten kinetics. Table 6 shows that contrary to 1,3-PPD dehydrogenase, the affinity of glycerol dehydrogenase of both mutant D and wild-type, with respect to the substrates and coenzymes tested, was relatively close. In both strains, the enzyme had much less affinity for its substrate the use of glycerol than in the opposite direction. This implies that the use of large amounts of glycerol was made possible in both strains.
Table 6. Determination of Km values for substrates and coenzymes of glycerol dehydrogenase isolated from gel filtration
Determination of substrate specificity of 1,3-PPD dehydrogenase from Cl. butyricum E5 wild-type and mutant D
Substrate specificity studies showed that 1,3-PPD dehydrogenase was capable of catalysing a number of oxidation and reduction reactions (Table 7).
Table 7. Substrate specifity of 1,3-propanediol dehydrogenase from Clostridium butyricum E5 wild type and mutant D
Table 7 shows that in the physiological direction, the enzyme of both strains was most active with the physiological substate (3-HPA). The enzyme of mutant D presented less specificity for other aldehydes tested because the relative activities were higher. Thus, reduction of propionaldehyde accounted for 40% of that of 3-HPA in mutant D and only 2·6% in the wild-type. In the non-physiological direction, it appeared that 1,3-PPD was the preferred substrate for the two strains. Glycerol was the second most active substrate in both wild-type and mutant D, with a relative activity of 28% and 11%, respectively. Other alcohols tested had little or no activity.
Determination of substrate specificity of glycerol dehydrogenase from Cl. butyricum E5 wild-type and mutant D
Substrate specificity studies showed that glycerol dehydrogenase was also able of catalysing a number of oxidation and reduction reactions (Table 8).
Table 8. Substrate specifity of glycerol dehydrogenase from Clostridium butyricum E5 wild type and mutant D
Table 8 shows that non-physiologically, glycerol dehydrogenase of both strains was most active with 3-HPA. The specificity of the enzyme for the other substrates studied was, in most cases, less extensive for the two strains than that of the 1,3-PPD dehydrogenase. As for the latter enzyme, the glycerol dehydrogenase of mutant D was relatively more active than in the wild-type with respect to the substrate. Physiologically glycerol was the preferred substrate for both strains. Other alcohols tested were oxidized with a much lower relative activity, displaying a maximum of 14% of that obtained with glycerol.
Effect of mono and divalent cations on 1,3-PPD dehydrogenase from Cl. butyricum E5 wild-type and mutant D
Table 9 shows that Mn2+ was the cation which most stimulated 1,3-PPD dehydrogenase activity in both strains. In mutant D, some other cations tested, in particular NH4+, Fe2+ and Li+, also stimulated enzymatic activity (in that order of decreasing relative activity). In the wild-type, the most effective cations after Mn2+ were Mg2+, Na+, Li+ and K+ (in that order of decreasing relative activity).
Table 9. Determination of the effect of mono and divalent cations on 1,3-propanediol dehydrogenase activity
Effect of mono and divalent cations on glycerol dehydrogenase from Cl. butyricum E5 wild-type and mutant D
Table 10 shows that Mn2+ was the most active cation on glycerol dehydrogenase activity in both strains, as for 1,3-PPD dehydrogenase. In the case of mutant D, no other cation allowed significant stimulation of activity compared with that of Mn2+. On the other hand, in the case of the wild-type, only Ca2+ allowed a significant stimulation of enzymatic activity.
Table 10. Determination of the effect of mono and divalent cations on glycerol dehydrogenase activity
Clostridium butyricum E5 wild-type and MD, a mutant resistant to allyl alcohol, have been shown to contain two key enzymes which take part in the catabolism of glycerol in the dha regulon (Forage and Foster 1982). The first enzyme, 1,3-PPD dehydrogenase, was purified and characterized in Cl. butyricum E5 wild-type (Malaoui and Marczak 2000).
The determination of the molar mass, the number of the subunits and the N-terminal sequence allows its properties to be compared to those reported in Kl. pneumoniae, C. freundii and Cl. pasteurianum. The separation of the two enzymes by gel filtration also allowed estimation of the molar mass of glycerol dehydrogenase of Cl. butyricum E5 wild-type.
The value of 181 000 ± 15 000 Da (supposed tetramer) approached that reported in C. freundii (hexamer of Mr=246 000 Da) more closely than those reported in S. pombe (octamer of Mr=400 000 Da) or E. coli K12-mutant (octamer of Mr=310 000 Da and dimer of Mr=81 000 Da).
The same technique enabled characterization of each enzyme by its specific properties. In both strains, the optimum pH of the two enzymes for the substrates studied did not differ to a significant degree and remained largely superior or equal to 7. In the case of 1,3-PPD dehydrogenase, a difference of one unit appeared according to the method employed. These values are close to that used or determined in Kl. pneumoniae (Johnson and Lin 1987).
On the contrary, the optimum pH of 1,3-PPD dehydrogenase for the reduction of the 3-HPA is lower in E. agglomerans (Barbirato et al. 1997), and even more so in Lact. reuteri (Talarico et al. 1990), with respective values of 7·8 and 6·2. The optimum pH of the glycerol dehydrogenase in DHA reduction is lower in E. coli K12 mutant (Tang et al. 1979) and S. pombe (Marshall et al. 1985) with a value of 6, while in the direction of glycerol oxidation it is higher with a value of around 10, contrary to the values observed in Cl. butyricum strains.
1,3-PPD dehydrogenase of the mutant exhibited an increase in apparent Km values for the respective substrates, in contrast to glycerol dehydrogenase. These results indicate that the mutation acted mostly on the 1,3-PPD formation pathway.
1,3-PPD dehydrogenase had a high affinity for its physiological substrate in the two strains. Thus, 3-HPA, which is an intermediary product, is quickly oxidized to 1,3-PPD, preventing its accumulation as a toxic product. The apparent Km of 3-HPA in the wild strain was close to that of C. freundii, which is 0·14 mmol l–1 (Daniel et al. 1995a). In mutant D it approached that of E. agglomerans (Barbirato et al. 1997), which is four times higher. Since mutant D had a lower affinity for 3-HPA, this strain would seem more resistant to the toxic effect of 3-HPA. The 1,3-PPD dehydrogenase of Lact. reuteri has one apparent Km distinctly higher (7·8 mmol l–1) than that of the two studied strains. This high Km value allows the bacterium to accumulate 3-HPA.
The glycerol dehydrogenase of both strains exhibited a low affinity for glycerol, which is the substrate of the metabolic pathway allowing the formation of the products. This low affinity allowed the bacterium to accumulate glycerol and to use it with a higher conversion rate to 1,3-PPD than in other strains, such as C. freundii, S. pombe and E. coli K12-mutant, for which the Km for glycerol is lower. On the other hand, the enzyme had an apparent Km value for DHA lower than that for glycerol. The reaction can therefore easily occur in the opposite direction. Consequently, both strains can accumulate glycerol and use it in different metabolic pathways, such as 1,3-PPD formation.
In both strains, there was a correlation between the affinity of 1,3-PPD dehydrogenase for 3-HPA and its specificity with respect to the substrates, as this enzyme is most effective with its physiological substrate. The enzyme of the wild strain retained 2–6% of the activity with other aldehydes tested. However, the enzyme of the mutant D is less specific as it retained 4–40% of the activity with other aldehydes. The low specificity of the enzyme of mutant D is due to the fact that it had a lower affinity for 3-HPA, which allowed the other substrates to replace it with a larger relative effectiveness. Like Cl. butyricum, 1,3-PPD dehydrogenase of C. freundii is also most active with 3-HPA and considerably less active with other aldehydes (Daniel et al. 1995a). On the contrary, the 1·3-PPD dehydrogenase of Lact. reuteri reduces at a similar rate the aldehydes 3-HPA, DHA and hydroxyacetone (Talarico et al. 1990).
Non-physiologically, the 1,3-PPD dehydrogenase of both strains was relatively specific for 1,3-PPD because, among other primary alcohols tested, only 1-butanol and glycerol were acted on significantly. The same specificity was found in the enzyme of Kl. pneumoniae (Johnson and Lin 1987). However, C. freundii, Lact. brevis and Lact. buchneri enzymes also oxidize n-propanol, n-butanol and 1·4-butanediol to their corresponding aldehydes, in addition to 1,3-PPD which remains the most active substrate. On the contrary, the 1,3-PPD dehydrogenase of Lact. reuteri is more active with the substrates which have adjacent hydroxyl functions, such as glycerol and 1,2-PPD. 1,3-PPD is oxidized at approximately 5% of the maximal rate observed for glycerol and 1,2-PPD (Talarico et al. 1990).
In both strains, glycerol dehydrogenase was most active physiologically with its natural substrate, glycerol. The same observation was made for the enzyme of C. freundii (Daniel et al. 1995b), whereas the enzymes of E. coli K12 mutant and S. pombe oxidized 1,2-PPD more quickly than glycerol. Non-physiologically, the glycerol dehydrogenase of both Cl. butyricum strains was more active with 3-HPA. However, the enzyme of mutant D also reduces DHA with significant activity. The glycerol dehydrogenase of mutant D appeared less specific with respect to the substrates than that of the wild strain. On the contrary, the enzyme of other strains, such as S. pombe, C. freundii and E. coli K12 mutant, is more active with DHA. However, the enzyme of E. coli K12 mutant reduces acetol with the same activity as that for DHA. The reducing pathway seemed more sensitive to mutation than the oxidative pathway because the two enzymes glycerol dehydrogenase and 1,3-PPD dehydrogenase of mutant D were less specific than those of the wild strain.
The specificity of the enzymes studied was also observed with respect to the added cations. In the absence of cations, 1,3-PPD dehydrogenase and glycerol dehydrogenase of the two strains can function, in contrast with 1,3-PPD dehydrogenase of Lact. buchneri and Lact. brevis whose activity is not detected (Veiga-Da-Cunha and Foster 1992). Like the 1,3-PPD dehydrogenase of C. freundii, both enzymes of the two studied strains are preferentially activated with Mn2+, whereas for the 1,3-PPD dehydrogenase of Kl. pneumoniae, Lact. brevis and Lact. buchneri, Mn2+ can be replaced by Fe2+. On the other hand, the 1,3-PPD dehydrogenase from C. freundii, as well as over-produced enzyme of the recombinant E. coli, are preferentially activated by Fe2+. Like glycerol dehydrogenase of S. pombe, 1,3-PPD dehydrogenase of Lact. reuteri requires for its optimal activity only one monovalent cation, i.e. K+. Contrary to the 1,3-PPD dehydrogenase of the other strains, the enzyme of E. agglomerans does not show obvious dependence for a cation, except that its activity is better preserved in the presence of Mn2+. Finally, NH4+ is the most effective activator for glycerol dehydrogenase of E. coli K12 mutant.
The two studied strains contain two enzymes, 1,3-PPD dehydrogenase and glycerol dehydrogenase, which share jointly several properties, such as pH optimum, specificity with respect to physiological substrates and dependence on Mn2+. These results suggest that the two proteins belong to the same dha regulon. The properties show that both strains are good candidates as biotechnological agents for the bioconversion of glycerol which they can accumulate. The mutant D strain has the advantage compared with the wild strain of being less sensitive to the toxic effect of 3-HPA, an intermediary product of this metabolic pathway. Therefore, it becomes the best candidate for use in a process of metabolic engineering leading to the production of 1,3-PPD starting from various carbon sources, such as glycerol.
This work was supported by the Délégation Régionale à la Recherche et à la Technologie pour la région Lorraine (Ministère de l’Enseignement Supérieur et de la Recherche, Paris, France). The authors thank Dr Thomas Haas (Degussa, Hanau, Germany) for giving 3-hydroxypropionaldehyde (3-HPA).