A novel archaeal alanine dehydrogenase homologous to ornithine cyclodeaminase and mu-crystallin

A novel alanine dehydrogenase (AlaDH) showing no significant amino acid sequence homology with previously known bacterial AlaDHs was purified to homogeneity from the soluble fraction of the hyperthermophilic archaeon Archaeoglobus fulgidus. AlaDH catalyzed the reversible, NAD+-dependent deamination of L-alanine to pyruvate and NH4+. NADP(H) did not serve as a coenzyme. The enzyme is a homodimer of 35 kDa per subunit. The Km values for L-alanine, NAD+, pyruvate, NADH, and NH4+ were estimated at 0.71, 0.60, 0.16, 0.02, and 17.3 mM, respectively. The A. fulgidus enzyme exhibited its highest activity at about 82 degrees C (203 U/mg for reductive amination of pyruvate) yet still retained 30% of its maximum activity at 25 degrees C. The thermostability of A. fulgidus AlaDH was increased by more than 10-fold by 1.5 M KCl to a half-life of 55 h at 90 degrees C. At 25 degrees C in the presence of this salt solution, the enzyme was approximately 100% stable for more than 3 months. Closely related A. fulgidus AlaDH homologues were found in other archaea. On the basis of its amino acid sequence, A. fulgidus AlaDH is a member of the ornithine cyclodeaminase-mu-crystallin family of enzymes. Similar to the mu-crystallins, A. fulgidus AlaDH did not exhibit any ornithine cyclodeaminase activity. The recombinant human mu-crystallin was assayed for AlaDH activity, but no activity was detected. The novel A. fulgidus gene encoding AlaDH, AF1665, is designated ala.     

Alanine dehydrogenase of the N2-fixing blue-green alga, Anabaena cylindrica.

The L-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270,000, has 6 sub-units, each of molecular weight approximately 43,000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD+ specific and oxaloacetate can partially substitute for pyruvate. The Kampp for NAD+ is 14 muM and 60 muM at low and high NAD concentrations respectively.     

Dynamic affinity between dissociable coenzyme and immobilized enzyme in an affinity chromatographic reactor with single enzyme

The affinity chromatographic reactor (ACR) is a bioreactor which utilizes the dynamic interaction or the dynamic affinity between a free coenzyme and immobilized enzymes for the highly efficient regeneration of dissociable coenzymes. Dynamic affinity between free NAD and immobilized alcohol dehydrogenase (ADH) in ACR was investigated by three different methods. ADH catalyzed both oxidation and reduction of NAD, consuming propionaldehyde and ethanol. The theoretical model under consideration elucidated a criterion for the expression of the dynamic affinity as a relationship among the affinity constants and the concentrations of a coenzyme and immobilized enzyme. This criterion was confirmed experimentally by the measurements of the retention time of NAD and the half-life period of the reactor activity after one-shot pulse injection of NAD to ACR. In the stability measurement of the immobilized enzyme, it became clear that ADH was more stable at the higher concentration in immobilization. Although the present case of coenzyme cycling by a single enzyme is very special, with limited chance for the direct application, the results obtained here provide a theoretical basis for ACR with multienzymes-which is of more general use.     

Alanine dehydrogenase and its applications – A review

Alanine dehydrogenase (AlaDH) (E.C.1.4.1.1) is a microbial enzyme that catalyzes a reversible conversion of L-alanine to pyruvate. Inter-conversion of alanine and pyruvate by AlaDH is central to metabolism in microorganisms. Its oxidative deamination reaction produces pyruvate which plays a pivotal role in the generation of energy through the tricarboxylic acid cycle for sporulation in the microorganisms. Its reductive amination reaction provides a route for the incorporation of ammonia and produces L-alanine which is required for synthesis of the peptidoglycan layer, proteins, and other amino acids. Also, AlaDH helps in redox balancing as its deamination/amination reaction is linked to the reduction/oxidation of NAD⁺/NADH in microorganisms. AlaDH from a few microorganisms can also reduce glyoxylate into glycine (aminoacetate) in a nonreversible reaction. Both its oxidative and reductive reactions exhibit remarkable applications in the pharmaceutical, environmental, and food industries. The literature addressing the characteristics and applications of AlaDH from a wide range of microorganisms is summarized in the current review.     

Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering.

We report the engineering of Lactococcus lactis to produce the amino acid L-alanine. The primary end product of sugar metabolism in wild-type L. lactis is lactate (homolactic fermentation). The terminal enzymatic reaction (pyruvate + NADH-->L-lactate + NAD+) is performed by L-lactate dehydrogenase (L-LDH). We rerouted the carbon flux toward alanine by expressing the Bacillus sphaericus alanine dehydrogenase (L-AlaDH; pyruvate + NADH + NH4+ -->L-alanine + NAD+ + H2O). Expression of L-AlaDH in an L-LDH-deficient strain permitted production of alanine as the sole end product (homoalanine fermentation). Finally, stereospecific production (>99%) of L-alanine was achieved by disrupting the gene encoding alanine racemase, opening the door to the industrial production of this stereoisomer in food products or bioreactors.     

Purification and properties of L-alanine dehydrogenase of the phototrophic bacterium Rhodobacter capsulatus E1F1.

In the phototrophic nonsulfur bacterium Rhodobacter capsulatus E1F1, L-alanine dehydrogenase aminating activity functions as an alternative route for ammonia assimilation when glutamine synthetase is inactivated. L-Alanine dehydrogenase deaminating activity participates in the supply of organic carbon to cells growing on L-alanine as the sole carbon source. L-Alanine dehydrogenase is induced in cells growing on pyruvate plus nitrate, pyruvate plus ammonia, or L-alanine under both light-anaerobic and dark-heterotrophic conditions. The enzyme has been purified to electrophoretic and immunological homogeneity by using affinity chromatography with Red-120 agarose. The native enzyme was an oligomeric protein of 246 kilodaltons (kDa) which consisted of six identical subunits of 42 kDa each, had a Stokes' radius of 5.8 nm, an s20.w of 10.1 S, a D20,w of 4.25 x 10(-11) m2 s-1, and a frictional quotient of 1.35. The aminating activity was absolutely specific for NADPH, whereas deaminating activity was strictly NAD dependent, with apparent Kms of 0.25 (NADPH), 0.15 (NAD+), 1.25 (L-alanine), 0.13 (pyruvate), and 16 (ammonium) mM. The enzyme was inhibited in vitro by pyruvate or L-alanine and had two sulfhydryl groups per subunit which were essential for both aminating and deaminating activities.     

过量表达苹果酸脱氢酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌,厌氧条件下由于辅酶NAD(H) 的不平衡导致其丧失了代谢葡萄糖的能力。构建了苹果酸脱氢酶的重组菌大肠杆菌NZN111/pTrc99a-mdh,在厌氧摇瓶发酵过程中通过0.3 mmol/L的IPTG诱导后重组菌的苹果酸脱氢酶 (Malate dehydrogenase,MDH) 酶活较出发菌株提高了14.8倍,NADH/NAD+的比例从0.64下降到0.26,同时NAD+和NADH浓度分别     

Preparation and properties of a holo-lactate dehydrogenase enzyme reactor

NAD+ was the base material for syntheses of coenzyme analogs with reactive groups bound to N6 of the adenine moiety via spacers that are 3-17 A long. These analogs were used for the modification of dehydrogenases. Aromatic imidoesters and acyl azides are suitable reactive groups, which form covalent amidinium or amide bonds with amino acid residues such as the epsilon-amino groups of lysines. The catalytic function of the modified protein decreased only slightly. Coenzymes that are linked via a spacer to carboxyl and amino groups are fixed to the protein by means of carbodiimides and hydroxysuccinimide. Coenzyme-bound aromatic imidoesters with spacer lengths of more than 12 A were incorporated to the extent of 60% at the active site. Aliphatic imidoesters proved to be inefficient for protein modification because of fast hydrolysis. Fixing of coenzyme analogs containing appended carboxyl or amino groups to enzyme in the presence of carbodiimides resulted in a decrease of enzyme activity. Modified lactate dehydrogenase and L-alanine dehydrogenase formed an enzyme reactor for the production of L-alanine in the absence of free NAD+. Both enzymes were cross-linked by dimethyl suberimidate in the presence or absence of NAD+, bis-NAD+, pyruvate, and oxamate. Site-to-site directed cross-linking yielded a reaction mixture from which four protein fractions were obtained by isoelectric focusing; one of these showed a cycling rate of 600 h-1.     

淹水对两种甜樱桃砧木根系无氧呼吸酶及发酵产物的影响

以美早/东北山樱桃、美早/马哈利为试材,研究了淹水过程中两种甜樱桃砧木生长根、褐色木质根中无氧呼吸酶——丙酮酸脱羧酶(PDC)、乙醇脱氢酶(ADH)和乳酸脱氢酶(LDH)活性及褐色木质根的发酵产物——乙醛、乙醇和乳酸含量变化,结果表明:两类根系PDC、LDH活性均呈先升后降趋势,ADH活性变化在生长根中亦先升后降,而在褐色木质根中为上升趋势,三种酶活性变化幅度表现为生长根大于褐色木质根;美早/东北山樱桃两类根系中ADH和LDH活性增加幅度大于美早/马哈利,PDC则相反;两种砧木褐色木质根乙醛、乙醇含量呈升高趋势,乳酸含量先升后降;最终美早/东北山樱桃褐色木质根中乙醛含量低于美早/马哈利,乙醇含量则相反,而乳酸含量前者较早达峰值且高于后者峰值。     

低氧胁迫下24-表油菜素内酯对黄瓜幼苗根系生长及其无氧呼吸同工酶表达的影响

采用营养液水培,研究了低氧胁迫下24-表油菜素内酯(EBR)对黄瓜幼苗根系生长及其无氧呼吸同工酶表达的影响.结果表明:低氧胁迫增强了黄瓜幼苗根系丙酮酸脱羧酶(PDC)、乙醇脱氢酶(ADH)、乳酸脱氢酶(LDH)同工酶的表达,低氧胁迫下施用外源EBR的第3天PDC、ADH同工酶的表达量分别提高了18.8％、28.8％,而第6、第9天PDC、ADH、LDH同工酶的表达减弱,比单纯低氧处理分别降低19.5％、25.6％、53.4％及26.4％、26.0％、28.4％;低氧胁迫至第9天,黄瓜幼苗根系的生长受到了显著抑制(P＜0.05),而低氧胁迫下施用EBR,黄瓜幼苗根系的生长受抑制程度减轻,其根系总长、干重、根尖数较单纯低氧处理显著增加(P＜0.05),低氧抑制了黄瓜幼苗根系的生长,低氧胁迫下营养液添加EBR可调节黄瓜根系无氧呼吸同工酶的表达,缓解低氧胁迫对黄瓜幼苗根系的伤害.     

Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462. Purification, characterization and kinetic mechanism

Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM.     

Purification and properties of alanine dehydrogenase from Bacillus sphaericus.

1. The bacterial distribution of alanine dehydrogenase (L-alanine:NAD+ oxidoreductase, deaminating, EC 1.4.1.1) was investigated, and high activity was found in Bacillus species. The enzyme has been purified to homogeneity and crystallized from B. sphaericus (IFO 3525), in which the highest activity occurs. 2. The enzyme has a molecular weight of about 230 000, and is composed of six identical subunits (Mr 38 000). 3. The enzyme acts almost specifically on L-alanine, but shows low amino-acceptor specificity; pyruvate and 2-oxobutyrate are the most preferable substrates, and 2-oxovalerate is also animated. The enzyme requires NAD+ as a cofactor, which cannot be replaced by NADP+. 4. The enzyme is stable over a wide pH range (pH 6.0--10.0), and shows maximum reactivity at approximately pH 10.5 and 9.0 for the deamination and amination reactions, respectively. 5. Alanine dehydrogenase is inhibited significantly by HgCl2, p-chloromercuribenzoate and other metals, but none of purine and pyrimidine bases, nucleosides, nucleotides, flavine compounds and pyridoxal 5'-phosphate influence the activity. 6. The reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADH binds first to the enzyme followed by ammonia and pyruvate, and the products are released in the order of L-ALANINE AND NAD+. The Michaelis constants are as follows: NADH (10 microM), ammonia (28.2 mM), pyruvate (1.7 mM), L-alanine (18.9 mM) and NAD+ (0.23 mM). 7. The pro-R hydrogen at C-4 of the reduced nicotinamide ring of NADH is exclusively transferred to pyruvate; the enzyme is A-stereospecific.     

Overproduction of alanine by Arthrobacter strains with glucose-nonrepressible L-alanine dehydrogenase

Summary Two Arthrobacter strains were identified as having high alanine productivity and L-alanine dehydrogenase (ADH) activity upon growth on glucose. They excreted large amounts of DL-alanine (37 and 81 g/1), but Bacillus sphaericus with glucose-repressible ADH did not at all. These results suggest that the glucose-nonrepressible ADH might be involved in alanine overproduction in the Arthrobacter strains.     

Biotransformation of R-2-hydroxy-4-phenylbutyric acid by D-lactate dehydrogenase and Candida boidinii cells containing formate dehydrogenase coimmobilized in a fibrous bed bioreactor

R-2-hydroxy-4-phenylbutyric acid (R-HPBA) is an important intermediate in the manufacture of angiotensin converting enzyme inhibitors. In this work, a recombinant D-lactate dehydrogenase (LDH) was used to transform 2-oxo-4-phenylbutyric acid (OPBA) to R-HPBA, with concomitant oxidation of beta-nicotinamide adenine dinucleotide (NADH) to NAD(+). The cofactor NADH was regenerated by formate dehydrogenase (FDH) present in whole cells of Candida boidinii, which were pre-treated with toluene to make them permeable. The whole cells used in the process were more stable and easier to prepare as compared with the isolated FDH from the cells. Kinetic study showed that the reaction rate was dependent on the concentration of cofactor, NAD(+), and that both R-HPBA and OPBA inhibited the reaction. A novel method for co-immobilization of whole cells and LDH enzyme on cotton cloth was developed using polyethyleneimine (PEI), which induced the formation of PEI-enzyme-cell aggregates and their adsorption onto cotton cloth, leading to multilayer co-immobilization of cells and enzyme with high loading (0.5 g cell and 8 mg LDH per gram of cotton cloth) and activity yield ( > 95%). A fibrous bed bioreactor with co-immobilized cells and enzyme on the cotton cloth was then evaluated for R-HPBA production in fed-batch and repeated batch modes, which gave relatively stable reactor productivity of 9 g/L . h and product yield of 0.95 mol/mol OPBA when the concentrations of OPBA and R-HPBA were less than 10 g/L.     

Link between primary and secondary metabolism in the biotransformation of trimethylammonium compounds by escherichia coli

The aim of this work was to understand the steps controlling the process of biotransformation of trimethylamonium compounds into L(-)-carnitine by Escherichia coli and the link between the central carbon or primary and the secondary metabolism expressed. Thus, the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA (pyruvate dehydrogenase, acetyl-CoA synthetase, and ATP:acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (isocitrate dehydrogenase) and glyoxylate (isocitrate lyase) cycles, were followed in batch with both growing and resting cells and during continuous cell growth in stirred-tank and high-cell-density membrane reactors. In addition, the levels of carnitine, crotonobetaine, gamma-butyrobetaine, ATP, NADH/NAD(+), and acetyl-CoA/CoA ratios were measured to determine how metabolic fluxes were distributed in the catabolic system. The results provide the first experimental evidence demonstrating the important role of the glyoxylate shunt during biotransformation of resting cells and the need for high levels of ATP to maintain metabolite transport and biotransformation (2.1 to 16.0 mmol L cellular/mmol ATP L reactor h). Moreover, the results obtained for the pool of acetyl-CoA/CoA indicate that it also correlated with the biotransformation process. The main metabolic pathway operating during cell growth in the high cell-density membrane reactor was that related to isocitrate dehydrogenase (during start-up) and isocitrate lyase (during steady-state operation), together with phosphotransacetylase and acetyl-CoA synthetase. More importantly, the link between central carbon and L(-)-carnitine metabolism at the level of the ATP pool was also confirmed.     

Effect of ATP on purified L(+) lactate dehydrogenase from electric organ of Electrophorus electricus (L.)

L(+) lactate dehydrogenase (LDH) activity from the electric organ of Electrophorus electricus was measured in the presence of ATP in the forward (substrate lactate) and reverse (substrate pyruvate) enzymatic reactions. The I50 for ATP was first determined and then the kinetics of the reactions were investigated with either constant coenzyme (NAD or NADH) concentration and varying substrate (lactate or pyruvate) concentration, or, constant substrate and varying coenzyme concentration. The kinetic data showed that ATP inhibits LDH uncompetitively with respect to the reduced and the oxidized coenzyme. As for the substrates, ATP gives a mixed type inhibition for lactate and a noncompetitive inhibition for pyruvate.     

Purification, properties and primary structure of alanine dehydrogenase involved in taurine metabolism in the anaerobe Bilophila wadsworthia

Alanine dehydrogenase [L-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.4.] catalyses the reversible oxidative deamination of L-alanine to pyruvate and, in the anaerobic bacterium Bilophila wadsworthia RZATAU, it is involved in the degradation of taurine (2-aminoethanesulfonate). The enzyme regenerates the amino-group acceptor pyruvate, which is consumed during the transamination of taurine and liberates ammonia, which is one of the degradation end products. Alanine dehydrogenase seems to be induced during growth with taurine. The enzyme was purified about 24-fold to apparent homogeneity in a three-step purification. SDS-PAGE revealed a single protein band with a molecular mass of 42 kDa. The apparent molecular mass of the native enzyme was 273 kDa, as determined by gel filtration chromatography, suggesting a homo-hexameric structure. The N-terminal amino acid sequence was determined. The pH optimum was pH 9.0 for reductive amination of pyruvate and pH 9.0-11.5 for oxidative deamination of alanine. The apparent Km values for alanine, NAD+, pyruvate, ammonia and NADH were 1.6, 0.15, 1.1, 31 and 0.04 mM, respectively. The alanine dehydrogenase gene was sequenced. The deduced amino acid sequence corresponded to a size of 39.9 kDa and was very similar to that of the alanine dehydrogenase from Bacillus subtilis.     

亮氨酸脱氢酶偶联NADH再生体系合成L-2-氨基丁酸

文中以大肠杆菌BL21(DE3)为宿主,构建两株分别共表达亮氨酸脱氢酶(LDH,来源蜡样芽孢杆菌)/甲酸脱氢酶(FDH,来源水生弯杆菌)和亮氨酸脱氢酶(LDH,来源蜡样芽孢杆菌)/醇脱氢酶(ADH,来源红球菌)的重组大肠杆菌。通过偶联两种不同NADH再生体系,以L-苏氨酸为起始原料,利用苏氨酸脱氨酶(L-TD)与LDH-FDH或LDH-ADH一锅法合成L-2-氨基丁酸,并对LDH-FDH工艺和LDH-ADH工艺进行对比优化。LDH-FDH工艺的最适反应pH为7.5,最适反应温度为35℃,通过加入50 g/L甲酸铵、0.3 g/L NAD+、10%LDH-FDH粗酶液(V/V)和7 500 U/L的L-TD酶液,对L-苏氨酸进行分批补加,以便控制2-丁酮酸浓度小于15 g/L,反应28 h,实现了L-2-氨基丁酸的产量为161.8 g/L,产率97%。LDH-ADH工艺的最适pH为8.0,最适反应温度为35℃,通过加入0.3 g/L NAD+、10%LDH-ADH粗酶液(V/V)及7 500 U/L的L-TD酶液,分批补加L-苏氨酸及1.2倍摩尔量异丙醇,以便控制2-丁酮酸浓度小于15g/L,且每生成约40g/L的L-2-氨基丁酸,抽真空去除丙酮,反应24h,实现了L-2-氨基丁酸的产量为119.6 g/L,产率98%。文中所采用的工艺及结果可为L-2-氨基丁酸的工业化提供一定的参考依据。     

Continuous Production of l-Alanine with NADH Regeneration by a Nanofiltration Membrane Reactor

A conjugated enzyme system, alanine dehydrogenase (AIDH) for stereospecific reduction of pyruvate to l-alanine and glucose dehydrogenase (GDH) for regeneration of NADH, were coimmobilized in a nanofiltration membrane bioreactor (NFMBR) for the continuous production of l-alanine from pyruvate with NADH regeneration. Since pyruvate was proved to be unstable at neutral pH, it was kept under acidic conditions and supplied to NFMBR separately from the other substrates. As 0.2 m pyruvate in HCl solution (pH 4), 10 mm NAD, 0.2 m glucose, and 0.2 m NH₄Cl in 0.5 m Tris buffer (pH 8) were continuously supplied to NFMBR with immobilized AIDH (100 U/ml) and GDH (140 U/ml) at the retention time of 80 min, the maximum conversion, reactor productivity, and NAD regeneration number were 100%, 320 g/liter/d, and 20,000, respectively. To avoid the effect of pyruvate instability, a consecutive reaction system, lactate dehydrogenase (l-LDH) and AIDH, was also used. In this system, the l-LDH provides pyruvate, the substrate for the AIDH reaction, from l-lactate regenerating NADH simultaneously, so the pyruvate could be consumed as soon as it was produced. As 0.2 m l-lactate, 10 mm NAD, 0.2 m NH₄Cl in 0.5 m Tris buffer (pH 8) were continuously supplied to NFMBR with immobilized l-LDH (100 U/ml) and AIDH (100 U/ml) at the retention time of 160 min, the maximum conversion, reactor productivity, and the NAD regeneration number were 100%, 160 g/Iiter/d, and 20,000, respectively.     

Effect of oxygen transfer rate on levels of key enzymes of xylose metabolism in Debaryomyces hansenii

The titers of key enzymes of xylose metabolism were measured and correlated with the kinetics of xylitol production by Debaryomyces hansenii under different oxygen transfer rates (OTR) in a batch reactor. An OTR change from 2.72 to 4.22 mmol O₂ l⁻¹ min⁻¹ resulted in a decrease in NADPH-dependent xylose reductase (XR) and NAD ± -dependent xylitol dehydrogenase (XDH) activities. For higher values of OTR (12.93 mmol O₂ l⁻¹ min⁻¹, the XDH titer increased twofold whereas the XR titer did not show a significant change. At the lowest OTR (2.72 mmol O₂ l⁻¹ min⁻¹), xylitol (and ethanol) production rates showed the highest values. However, xylitol specific productivity was twice as high as ethanol specific productivity. The titer of the NADPH-forming enzyme, glucose-6-phosphate dehydrogenase (GPDH), increased from 333 to 412 mU mg⁻¹ when the OTR was increased. However, 6-phosphogluconate dehydrogenase (PGDH) activity remained unchanged and at a lower level, which indicates that this enzyme is responsible for the carbon flux control of the oxidative branch of the pentose phosphate pathway. The activity of the alcohol-forming enzyme was repressed at the higher amount of oxygen, decreasing its activity more than 50%. The changes in ADH suggested that two different metabolic regions under oxygen-limited conditions can be hypothesized for xylose metabolism by D. hansenii. For low OTR values (up to 4.22 mmol O₂ l⁻¹ min⁻¹), a fermentative-type activity is displayed. At higher OTR values (above 4.22 mmol O₂ l⁻¹ min⁻¹), no significant fermentative activity is reported.