Pyruvate Decarboxylase Activity Research Articles

Physiological responses and quality attributes of Japanese pear ‘Kosui’ fruit kept in low oxygen atmospheres

SummaryJapanese pear ‘Kosui’ fruits were stored under a continuous flow of 0%, 1%, 3%, 5% and 10% O2 (balance N2) or air for 7 d at 20°C to study the effects of low O2 on their physiological responses and quality attributes. Low O2 treatments did not significantly influence changes in skin colour and soluble solids content. However, weak off-flavours were detected in the fruits stored at 0% O2 on day 3, and the intensity of these off-flavours increased as storage progressed. The concentrations of acetaldehyde in fruit increased throughout the storage period. The ethanol concentration was greatly increased in fruits stored at 0% O2. Moreover, ethanol concentrations were much higher than those of acetaldehyde and remained very low during storage in air, but their concentration were just slightly increased in fruits exposed to 1%, 3%, 5% and 10% O2. Pyruvate decarboxylase activity was greatly increased in fruits exposed to 1% and 3% O2, while its activity in fruits exposed to 5% and 10% O2 were only slightly higher than that of the control and at 0% O2 at the same level as the control. Alcohol dehydrogenase (ADH) activity greatly increased in fruit exposed to 0%, 1%, 3% and 5% O2, while at 10% O2, ADH was only slightly higher than the control. Changes in ADH isozymes correlated well with changes in ADH activity. The homogenate pH of fruits exposed to 1%, 3%, 5% and 10% O2 and air remained constant, while in fruit stored at 0% O2 their pH increased. The potential for using low O2 atmospheres to help in maintaining the quality of Japanese pear ‘Kosui’ is discussed.

Activity Profiles of Primary Metabolism Enzymes in Xanthomonas Campestris Strains During Xanthan Production

ABSTRACTThe activity of enzymes involved in the Entner-Doudor off pathway and citric acid cycle were confronted in high and low xanthan producing strains of Xanthomonas campestris. The effect of citric acid, added at different time intervals on the xanthan biosynthesis was also analyzed. It was shown that the activity of the enzyme pyruvate decarboxylase was 3 times high for the active variant strain ITS 342. Exist time dependence and quantity one for citric acid and intensity of xanthan production and enzyme activity.

Hypoxic induction of anoxia tolerance in Avena coleoptiles

Summary Seedlings of Avena sativa were subjected to 24 h anoxic stress after 24 h hypoxic-pretreatment (H-PT). The H-PT increased 4.5-fold the survivability of the anoxia in coleoptiles of the seedlings compared to that of non-pretreated (N-PT) seedlings. During pretreatment periods, alcohol dehydrogenase (ADH, EC 1.1.1.1) and pyruvate decarboxylase (PDC, EC 4.1.1.1) activities in the coleoptiles became 4.1- and 3.9-fold greater respectively than those of N-PT seedlings. After the onset of anoxic stress, ADH and PDC activities in all coleoptiles increased, but their activities in the H-PT coleoptiles remained much greater than those in the N-PT coleoptiles, and the average production rate of ethanol for the initial 6 h was 3.1 and 1.4 μmol g −1 fresh weight h −1 for the H-PT and N-PT coleoptiles, respectively. Coleoptiles of the seedlings lost ATP rapidly under anoxic stress; however, the decrease in ATP was much slower in the H-PT coleoptiles than in the N-PT coleoptiles. These results suggest that the H-PT may improve the subsequent tolerance of anoxia in Avena coleoptiles due to maintenance of ATP levels with rapid induction of ethanolic fermentation. The present results are consistent with the results that were found in roots of several plants, suggesting that the mechanism in hypoxic acclimation to anoxia implicated in their roots may act in Avena coleoptiles.

Hypoxic Induction of Anoxia Tolerance in Rice Coleoptiles

SummaryRice (Oryza sativa L.) seedlings were subjected to a 24-h anoxic stress after hypoxic pretreatment (H-PT) for evaluation of the hypoxic acclimation to anoxia tolerance in their coleoptiles. The elongation of the coleoptiles of the seedlings subjected to H-PT was greater than that of non-pretreated (N-PT) seedlings. During pretreatment periods, alcohol dehydrogenase (EC 1.1.1.1) and pyruvate decarboxylase (EC 4.1.1.1) activities in the H-PT coleoptiles became 5.3- and 3.6-fold greater than those in the N-PT coleoptiles, respectively. The average production rate of ethanol during the initial 6 h after the onset of the anoxia stress was 3.7 and 1.3 μmol g–1 fresh weight h–1 for the H-PT and N-PT coleoptiles, respectively. Coleoptiles of the seedlings lost ATP rapidly under the anoxic stress; however, the decrease in ATP was much slower in the H-PT coleoptiles than in the N-PT coleoptiles. These results are consistent with the results found in roots of several plants, suggesting that the mechanism in hypoxic acclimation to anoxia implicated in their roots may act in the rice coleoptiles.

Solvent kinetic isotope effects monitor changes in hydrogen bonding at the active center of yeast pyruvate decarboxylase concomitant with substrate activation: the substituent at position 221 can control the state of activation.

Substrate activation of yeast pyruvate decarboxylase has been studied extensively in the authors' laboratories providing strong evidence that interaction of substrate with residue C221 provides the trigger, and the information is then transmitted along the C221 to H92 to E91 to W412 to G413 pathway to the 4'-amino nitrogen of the thiamin diphosphate cofactor. Earlier, it was found that the C221S substitution reduced the Hill coefficient from 2.0 to 0.8-0.9, the C221A substitution to 1.0, even though C221 is located on the beta domain some 20 A from the active center thiamin diphosphate cofactor, which is at the interface of the alpha and gamma domains. Here are reported experiments on the C221D/C222A and C221E/C222A variants, in which a negative charge is built onto the C221 side chain, to better mimic the effect of a pyruvate molecule covalently bonded to C221 as a thiohemiketal. Both variants were purified to an optimal activity of 70% of the wild-type enzyme, higher activity than that with the earlier uncharged substitutions at this position. The Hill coefficient for both variants is exactly 1.0. The deuterium solvent kinetic isotope effects (SKIE) on k(cat) for these variants were similar to that for the wild-type enzyme and the C221A/C222A variant, suggesting that starting with the first irreversible step (decarboxylation) the rate-limiting transition states are very similar for all of these enzyme forms. In contrast, such SKIE on k(cat)/K(m) are quite different for the C221A/C222A variant (0.62) than for the C221E/C222A or C221D/C222A variants (0.80-0.82), clearly indicating the effect of the C221 substitutions on transition states starting with the binding of the first substrate to the enzyme and terminating with the decarboxylation step. The results provide strong additional evidence for the involvement of residue C221 in the substrate activation process and suggest that the C221D (C221E) substitution shifts the enzyme into a conformation that resembles the activated conformation. A comparison with SKIE for the wild-type enzyme provides insight to changes in hydrogen bonding at the active center as a result of substrate activation.

Efficient homolactic fermentation by Kluyveromyces lactis strains defective in pyruvate utilization and transformed with the heterologous LDH gene.

A high yield of lactic acid per gram of glucose consumed and the absence of additional metabolites in the fermentation broth are two important goals of lactic acid production by microrganisms. Both purposes have been previously approached by using a Kluyveromyces lactis yeast strain lacking the single pyruvate decarboxylase gene (KlPDC1) and transformed with the heterologous lactate dehydrogenase gene (LDH). The LDH gene was placed under the control the KlPDC1 promoter, which has allowed very high levels of lactate dehydrogenase (LDH) activity, due to the absence of autoregulation by KlPdc1p. The maximal yield obtained was 0.58 g g(-1), suggesting that a large fraction of the glucose consumed was not converted into pyruvate. In a different attempt to redirect pyruvate flux toward homolactic fermentation, we used K. lactis LDH transformant strains deleted of the pyruvate dehydrogenase (PDH) E1alpha subunit gene. A great process improvement was obtained by the use of producing strains lacking both PDH and pyruvate decarboxylase activities, which showed yield levels of as high as 0.85 g g(-1) (maximum theoretical yield, 1 g g(-1)), and with high LDH activity.

Enzymes of the Entner-Doudoroff and pyruvate decarboxylation pathways in Zymomonas mobilis wild-type CP4 and mutant strains grown in continuous culture.

The osmotolerant Zymomonas mobilis strain suc40, (containing plasmid pDS3154-inaZ), which is capable of producing simultaneously ethanol and ice nuclei protein, was cultivated in a chemically defined complete sucrose medium, as well as in a sugar beet molasses medium in continuous culture. The strain exhibited the normal Monod's relationship between biomass and dilution rate, and between growth substrate concentration and dilution rate. Specific activities of a number of enzymes that appear to control important steps in the metabolic flux of the Entner-Doudoroff and pyruvate decarboxylation pathways were investigated over a range of growth rates in steady state cultures. With the exception of glucose-6-phosphate dehydrogenase and gluconate kinase, all of the enzymes exhibited a very similar pattern for the wild type Z. mobilis CP4 and for the osmotolerant mutants, independent of the media used; the enzyme patterns remained relatively constant over the studied growth range. The specific activity of glucose-6-phosphate dehydrogenase was increased 2-fold by decreasing dilution rate in sugar beet molasses. The specific activity of gluconate kinase was 2-fold lower at medium growth rates compared with that at either low or high growth rates. Pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase activities were significantly higher compared with those of the enzymes governing the early steps of the Entner-Doudoroff pathway. The present study, which was designed to determine the behaviour of important enzymes in sucrose metabolism of Z. mobilis suc40/pDS3154-inaZ grown in continuous culture showed that the microorganism required regulation of specific enzyme activities at the transcriptional level when sugar beet molasses were used as the growth medium.

Regulation of ethanolic fermentation in bell pepper fruit under low oxygen stress

Abstract Bell pepper fruit were stored under a continuous flow of 0 and 1% O 2 (balance N 2 ) or air for 7 days at 20 °C to study the regulation of ethanolic fermentation. The concentrations of ethanol and acetaldehyde were very low during storage at 1% O 2 and air. At 0% O 2 , ethanol and to a lesser extent acetaldehyde, rapidly accumulated in the tissue. Pyruvate decarboxylase (PDC) activity greatly increased in fruit exposed to 1% O 2 , while activity in fruit exposed to 0% was only slightly higher than that of the control. Alcohol dehydrogenase (ADH) activity greatly increased in fruit exposed to 1% but at 0% O 2 was at the same level as the control. The activity of ADH was about 10 times that of PDC during storage. Changes in ADH isozymes correlated well with changes in ADH activity. Concentration of NADH did not change during storage in air, but in fruit in 0 and 1% O 2 showed a significant increase. Concentrations of pyruvate were very low and did not change during storage at 1% O 2 and air. At 0% O 2 , pyruvate rapidly accumulated in the tissue. The K m of ADH in bell pepper fruit was 0.74 mM for acetaldehyde, and 0.33 mM for NADH. The K m of PDC in bell pepper fruit was 0.92 mM for pyruvate. Possible regulation of ethanolic fermentation is briefly considered.

Carbohydrate–ethanol transition in cereal grains under anoxia

• Cereal grains differ greatly in their reponses to anaerobiosis. Here, the in vivo conversion of carbohydrates to ethanol and CO2 under anoxia is reported for three cereal grains. • The conversion of glucose, fructose or sucrose to ethanol under anaerobic conditions was investigated in rice (Oryza sativa), barley (Hordeum vulgare) and wheat (Triticum aestivum) grains; alcohol dehydrogenase (EC 1.1.1.1) and pyruvate decarboxylase (EC 4.1.1.1) activities were also analysed under aerobic and anaerobic incubation. • Our data suggest that rice grains are able to produce ethanol under anoxia for the whole period of anoxic treatment, whereas barley and wheat grains can produce this terminal product of fermentation only during the first days of anaerobiosis. The level of enzymes involved in the fermentation pathway increases strongly under anoxic conditions in all three cereals. • Conversion of hexose to CO2 is nearly unaffected by anoxia in wheat, barley and rice, whereas only rice grains are able to degrade and utilize sucrose efficiently under anoxia. By contrast, wheat and barley do not utilize sucrose efficiently under anaerobic conditions.

An Approach to the Mode of Action of a Novel Yeast Factor Increasing Yeast Brewing Performance

A yeast peptide complex (YPC) obtained by alcoholic extraction of yeast stimulates yeast metabolism. The addition of YPC during fermentation reduced the major negative effects of high-gravity brewing and considerably enhanced brewing performance and resistance to stress by increasing sugar metabolism and macromolecular biosynthesis. The presence of this yeast cell derivative influenced the activation of glycolysis at different crucial steps; it stimulated glucose uptake, increased fructose-2,6-biphosphate intracellular concentration, and increased pyruvate decarboxylase activity. Moreover, this stimulation of glucose metabolism had a positive effect on the synthesis of different cellular macromolecules such as lipids, proteins, and glycogen. In addition, results also demonstrated that YPC exhibits an insulin-mimetic activity not only on glycolysis but also on overall metabolism by stimulating mitochondrial enzymes, thereby confirming the crucial role of mitochondrial energy-generating systems in resistance to stress under fermentation conditions.

Physiological properties of Saccharomyces cerevisiae from which hexokinase II has been deleted.

Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. The hxk2 mutant had a higher mitochondrial H(+)-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2 mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.

Iron resupply-mediated deactivation of Fe-deficiency stress responses in roots of sugar beet

Different root zones with or without increased Fe-reducing activities have been studied in Fe-deficient sugar beet (Beta vulgaris L.) plants after Fe resupply to the nutrient solution. The subapical regions of roots from Fe-deficient plants decreased by 19 and 88% their capacity to reduce ferric chelates after 24 and 96 h of Fe resupply, respectively. Iron resupply caused 52 and 96% decreases in phosphoenolpyruvate carboxylase activity in root extracts after 24 and 96 h, respectively, and also caused general decreases in other enzyme activities involved in carboxylic acid metabolism. The large pools of carboxylic acids in Fe-deficient roots decreased by 9 and 48% after 24 and 96 h of Fe resupply, respectively. The activities of pyruvate decarboxylase and lactate dehydrogenase, enzymes related to anaerobic metabolism, decreased by 88% after 24 h of Fe resupply. The mitochondrial quinone and pyridine nucleotide pools became more oxidised in the Fe-deficient root tips after Fe resupply. Iron resupply caused a 70% decrease in root oxygen consumption rates 96 h after Fe resupply. Results indicate that deactivation of the Fe deficiency stress responses of sugar beet roots upon Fe resupply occurs in a progressive manner in a time scale of several days.

Role of fermentative and antioxidant metabolisms in the induction of core browning in controlled-atmosphere stored pears

Pears (Pyrus communis L cv Blanquilla) were stored at various CO2 concentrations to induce core browning. The severity of core browning and the products and enzymes associated with fermentative and antioxidant metabolisms were determined immediately after harvest and in healthy and damaged fruits after storage. The incidence and severity of brown core increased with CO2 concentration. Acetaldehyde and ethanol concentrations and alcohol dehydrogenase (ADH, EC 1.1.1.1) activity increased in all fruits during storage, but more in fruits with brown core than in healthy fruits. Pyruvate decarboxylase (PDC, EC 4.1.1.1) activity decreased slightly during this time. Ascorbate contents decreased whereas glutathione concentrations increased during storage in fruits with brown core compared to the same fruits at harvest. Superoxide dismutase (SOD, EC 1.15.11) activity increased during storage but was not affected by brown core. In contrast, a significant increase in both ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) activities and a significant decrease in catalase (CAT, EC 1.11.1.6) activity were found in damaged fruits. Malonaldehyde (MDA) and 4-hydroxyalkenal (4-HNE) contents increased significantly in the damaged fruits as a result of peroxidation. Collectively, our results suggest that brown core in pears is indirectly correlated with fermentation and involves oxidative damage which may be a causal factor in brown core development. © 2001 Society of Chemical Industry

Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae: role of the cytosolic Mg(2+) and mitochondrial K(+) acetaldehyde dehydrogenases Ald6p and Ald4p in acetate formation during alcoholic fermentation.

Acetic acid plays a crucial role in the organoleptic balance of many fermented products. We have investigated the factors controlling the production of acetate by Saccharomyces cerevisiae during alcoholic fermentation by metabolic engineering of the enzymatic steps involved in its formation and its utilization. The impact of reduced pyruvate decarboxylase (PDC), limited acetaldehyde dehydrogenase (ACDH), or increased acetoacetyl coenzyme A synthetase (ACS) levels in a strain derived from a wine yeast strain was studied during alcoholic fermentation. In the strain with the PDC1 gene deleted exhibiting 25% of the PDC activity of the wild type, no significant differences were observed in the acetate yield or in the amounts of secondary metabolites formed. A strain overexpressing ACS2 and displaying a four- to sevenfold increase in ACS activity did not produce reduced acetate levels. In contrast, strains with one or two disrupted copies of ALD6, encoding the cytosolic Mg(2+)-activated NADP-dependent ACDH and exhibiting 60 and 30% of wild-type ACDH activity, showed a substantial decrease in acetate yield (the acetate production was 75 and 40% of wild-type production, respectively). This decrease was associated with a rerouting of carbon flux towards the formation of glycerol, succinate, and butanediol. The deletion of ALD4, encoding the mitochondrial K(+)-activated NAD(P)-linked ACDH, had no effect on the amount of acetate formed. In contrast, a strain lacking both Ald6p and Ald4p exhibited a long delay in growth and acetate production, suggesting that Ald4p can partially replace the Ald6p isoform. Moreover, the ald6 ald4 double mutant was still able to ferment large amounts of sugar and to produce acetate, suggesting the contribution of another member(s) of the ALD family.

Fermentative metabolism in grape berries: isolation and characterization of pyruvate decarboxylase cDNA and analysis of its expression throughout berry development

The involvement of pyruvate decarboxylase (PDC) in the control of alcohol production during ripening of fruit tissues under aerobic conditions has been only partially studied. Enzymological studies showed a significant increase in PDC activity during the ripening of oranges and pears, concurrently with the induction of ethanol production. In tomato, on the other hand, the induction of ethanol production and ADH gene expression after the onset of ripening was not accompanied by induction of PDC activity. The isolation of PDC cDNA from fruits has not yet been reported, nor has its expression pattern during fruit development. We report here the isolation of a cDNA clone encoding for a grape PDC and the characterization of its expression throughout berry development. The pattern of PDC gene expression throughout berry development, combined with earlier findings on constitutive PDC activity in the berry, may suggest that PDC is not the limiting factor for the production of ethanol in the berry, which is induced only after the onset of berry ripening. Alternatively, the induction of ADH gene expression, which occurs only after the onset of ripening in both tomatoes and grape berries, may serve as a regulator of ethanol production in response to a ripening-related cue.

Fermentative capacity in high-cell-density fed-batch cultures of baker's yeast

High-cell-density fed-batch processes for bakers' yeast production will involve a low-average-specific growth rate due to the limited oxygen-transfer capacity of industrial bioreactors. The relationship between specific growth rate and fermentative capacity was investigated in aerobic, sucrose-limited fed-batch cultures of an industrial bakers' yeast strain. Using a defined mineral medium, biomass concentrations of 130 g dry weight/L were reproducibly attained. After an initial exponential-feed phase (μ = 0.18 h−1), oxygen-transfer limitation necessitated a gradual decrease of the specific growth rate to ca. 0.01 h−1. Throughout fed-batch cultivation, sugar metabolism was fully respiratory, with a biomass yield of 0.5 g biomass/g sucrose−1. Fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions with excess glucose) showed a strong positive correlation with specific growth rate. The fermentative capacity observed at the end of the process (μ = 0.01 h−1) was only half that observed during the exponential-feed phase (μ = 0.18 h−1). During fed-batch cultivation, activities of glycolytic enzymes, pyruvate decarboxylase and alcohol dehydrogenase in cell extracts did not exhibit marked changes. This suggests that changes of fermentative capacity during fed-batch cultivation were not primarily caused by regulation of the synthesis of glycolytic enzymes. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 517–523, 2000.

130 Response of Anaerobic Metabolism to Delaying Controlled Atmosphere Storage in Braeburn Apples

Apple fruits (Malus domestica Borkh. cv. Braeburn) harvested from two orchards (A and B) on the same day were stored in air or pretreated in air for 0, 2 (2dCA) or 4 weeks (4dCA) before moving into controlled atmosphere (CA) storage with 1.5% O2 + 5% CO2. During storage at 1 °C for 9 weeks in air and/or CA, changes of pyruvate decarboxylase (PDC) activity, alcohol dehydrogenase (ADH) activity, acetaldehyde (AA) and ethanol (EtOH) concentrations in flesh tissue were assayed in addition to the incidence of Braeburn browning disorder (BBD). Immediate introduction to CA conditions induced the development of BBD with the highest incidence 62.2%, however delaying application of CA for 2 and 4 weeks reduced the incidence of BBD to 38.5% and 27.0%. The development of disorder in grower B was less than in grower A. 2dCA and 4dCA treatments did not influence PDC activity compared with treatment of CA. However, ADH activity and the accumulation of AA and EtOH in treatments of 2dCA and 4dCA were markedly lower than those in CA. The accumulation of AA in grower B was lower than grower A. The results of this study suggest that the delayed application of CA reduced BBD and this may be due to reduced anaerobic metabolism of fruits in the delayed CA.

Evaluation of the importance of lactate for the activation of ethanolic fermentation in lettuce roots in anoxia

According to the Davies–Roberts hypothesis, plants primarily respond to oxygen limitation by a burst of lactate production and the resulting pH drop in the cytoplasm activates ethanolic fermentation. To evaluate this system in lettuce (Lactuca sativa L.), seedlings were subjected to anoxia and in vitro activities of alcohol dehydrogenase (ADH, EC 1.1.1.1), pyruvate decarboxylase (PDC, EC 4.1.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) and concentrations of ethanol, acetaldehyde and lactate were determined in roots of the seedlings. The in vitro activities of ADH and PDC in the roots increase in anoxia, whereas no significant increase was measured in LDH activity. At 6 h, the ADH and PDC activities in the roots kept in anoxia were 2.8‐ and 2.9‐fold greater than those in air, respectively. Ethanol and acetaldehyde in the roots accumulated rapidly in anoxia and increased 8‐ and 4‐fold compared with those in air by 6 h, respectively. However, lactate concentration did not increase and an initial burst of lactate production was not found. Thus, ethanol and acetaldehyde production occurred without an increase in lactate synthesis. Treatments with antimycin A and salicylhydroxamic acid, which are respiratory inhibitors, to the lettuce seedlings in the presence of oxygen increased the concentrations of ethanol and acetaldehyde but not of lactate. These results suggest that ethanolic fermentation may be activated without preceding activation of lactate fermentation and may be not regulated by oxygen concentration directly.

Influence of yeast immobilization on fermentation and aldehyde reduction during the production of alcohol-free beer.

Production of alcohol-free beer by limited fermentation is optimally performed in a packed-bed reactor. This highly controllable system combines short contact times between yeast and wort with the reduction of off-flavors to concentrations below threshold values. In the present study, the influence of immobilization of yeast to DEAE-cellulose on sugar fermentation and aldehyde reduction was monitored. Immobilized cells showed higher activities of hexokinase and pyruvate decarboxylase compared to cells grown in batch culture. In addition, a higher glucose flux was observed, with enhanced excretion of main fermentation products, indicating a reduction in the flux of sugar used for biomass production. ADH activity was higher in immobilized cells compared to that in suspended cells. However, during prolonged production a decrease was observed in NAD-specific ADH activity, whereas NADP-specific activity increased in the immobilized cells. The shifts in enzyme activities and glucose flux correlate with a higher in vivo reduction capacity of the immobilized cells.

Fermentative capacity in high-cell-density fed-batch cultures of baker's yeast

High-cell-density fed-batch processes for bakers' yeast production will involve a low-average-specific growth rate due to the limited oxygen-transfer capacity of industrial bioreactors. The relationship between specific growth rate and fermentative capacity was investigated in aerobic, sucrose-limited fed-batch cultures of an industrial bakers' yeast strain. Using a defined mineral medium, biomass concentrations of 130 g dry weight/L were reproducibly attained. After an initial exponential-feed phase (mu = 0.18 h(-1)), oxygen-transfer limitation necessitated a gradual decrease of the specific growth rate to ca. 0.01 h(-1). Throughout fed-batch cultivation, sugar metabolism was fully respiratory, with a biomass yield of 0.5 g biomass/g sucrose(-1). Fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions with excess glucose) showed a strong positive correlation with specific growth rate. The fermentative capacity observed at the end of the process (mu = 0.01 h(-1)) was only half that observed during the exponential-feed phase (mu = 0.18 h(-1)). During fed-batch cultivation, activities of glycolytic enzymes, pyruvate decarboxylase and alcohol dehydrogenase in cell extracts did not exhibit marked changes. This suggests that changes of fermentative capacity during fed-batch cultivation were not primarily caused by regulation of the synthesis of glycolytic enzymes.