Pretreatment of beet molasses to increase pullulan production

Pretreatment of beet molasses with cation exchange resin, sulphuric acid, tricalcium phosphate, potassium ferrocyanide, and ethylenediaminetetraacetic acid and disodium salt (EDTA) to increase the production of pullulan was investigated. Among the above techniques used for the removal of heavy metals, sulphuric acid treatment gave better results regarding polysaccharide concentration, polysaccharide yield, and sugar utilization. Aureobasidium pullulans grown on beet molasses produced a mixture of pullulan and other polysaccharides. The pullulan content of the crude polysaccharide was 30–35%. The addition of nutrients improved the production of polysaccharide. A maximum polysaccharide concentration (32·0±1·0 g litre⁻¹) was achieved in molasses solution (70 g litre ¹ initial sugar concentration, pH 6·5–7·5) treated with sulphuric acid and supplemented with K₂HPO₄ 0·5%, -glutamic acid 1%, olive oil 2·5% and Tween 80 0·5%. In this case, the highest values of biomass dry weight (33·8±1·0 g litre⁻¹), polysaccharide yield (63·5±2·5%), and sugar utilization (97·5±1·5%) were obtained at pH 6·5, 3·5, and 4·5–7·5, respectively.     

Kerase Immobilization by Covalent Attachment to Porous Glass

Kerase, a serine protease from Streptomyces fradiae, was immobilized on porous glass (SIKUG®) by covalent attachment, through amino groups on the enzyme. Modifications of four lysine residues (44·4% of the accessible or superficial amino groups) results in a loss of 6·5% of the enzymic activity. After immobilization, the optimal reaction pH changed from a range of 7·5-8·5 to 9-10. The immobilized protease was stable in a broad pH range, 6-12, while the soluble protease was irreversibly denaturated at alkaline pHs (pH>8). The optimal reaction temperature was displaced from 55 to 65°C, showing a higher thermal stability of the immobilized enzyme. Kerase immobilized onto porous glass was stable for at least 28 days, working in a repeated-batch process of three cycles per day, with an activity loss of 22·1 ± 3·1%.     

Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid

Purified lipase from Mucor miehei (MML) has been covalently immobilized on different epoxy resins (standard hydrophobic epoxy resins, epoxy-ethylenediamine, epoxy-iminodiacetic acid, epoxy-copper chelates) and adsorbed via interfacial activation on octadecyl-Sepabeads support (fully coated with very hydrophobic octadecyl groups). These immobilized enzyme preparations were used under slightly different conditions (temperature ranging from 4 to 25 °C and pH values from 5 to 7) in the hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid.

Different catalytic properties (activity, specificity, enantioselectivity) were found depending on the particular support used. For example, the epoxy-iminodiacetic acid-Sepabeads gave the most active preparation at pH 7 while, at pH 5, the ethylenediamine-Sepabeads was superior.

More interestingly, the enantiomeric ratio (E) also depends strongly on the immobilized preparation and the conditions employed. Thus, the octadecyl-MML preparation was the only immobilized enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4 °C and pH 7 to 1.2 at pH 5 and 25 °C).

The other immobilized preparations, in contrast, were S selective. Immobilization on iminodiacetic acid-Sepabeads afforded the catalyst with the highest enantioselectivity (E=59 under optimum conditions).     

Optimization of culture conditions for production of cis-epoxysuccinic acid hydrolase using response surface methodology

Response surface methodology, which allows for rapid identification of important factors and optimization of them to enhance enzyme production, was employed here to optimize culture conditions for the production of cis-epoxysuccinic acid hydrolase from Bordetella sp. strain 1–3. In the first step, a Plackett–Burman design was used to evaluate the effects of nine variables (yeast extract, cis-epoxysuccinic acid, KH₂PO₄, K₂HPO₄ · 3H₂O, MgSO₄ · 7H₂O, trace minerals solution, culture volume, initial pH and incubation time) on the enzyme production. Yeast extract, cis-epoxysuccinic acid and KH₂PO₄ had significant influences on cis-epoxysuccinic acid hydrolase production and their concentrations were further optimized using central composite design and response surface analysis. A combination of adjusting the concentration of yeast extract to 7.8 g/l, cis-epoxysuccinic acid to 9.8 g/l, and KH₂PO₄ to 1.12 g/l would favor maximum cis-epoxysuccinic acid hydrolase production. An enhancement of cis-epoxysuccinic acid hydrolase production from 5.6 U/ml to 9.27 U/ml was gained after optimization.     

Rapid cultivation of stable aerobic phenol-degrading granules using acetate-fed granules as microbial seed

cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 °C in amount corresponding to 8% of the cell soluble protein (0.6 U mg⁻¹). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 °C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.     

The Sulfolobus tokodaii gene ST1704 codes highly thermostable glucose dehydrogenase

NAD(P)-dependent glucose-1-dehydrogenase (GDH) has been used for glucose determination and NAD(P)H production in bioreactors. Thermostable glucose dehydrogenase exhibits potential advantage for its application in biological processes. The function of the putative GDH gene (ST1704, 360-encoding amino acids) annotated from the total genome analysis of a thermoacidophilic archeaon Sulfolobus tokodaii strain 7 was investigated to develop more effective application of GDH. The gene encoding S. tokodaii GDH was cloned and the activity was expressed in Escherichia coli, which did not originally possess GDH. This shows that the gene (ST1704) codes the sequence of GDH. The enzyme was effectively purified from the recombinant E. coli with three steps containing a heat treatment and two successive chromatographies. The native enzyme (molecular mass: 160 kDa) is composed of a tetrameric structure with a type of subunit (41 kDa). The enzyme utilized both NAD and NADP as the coenzyme. The maximum activity for glucose oxidation in the presence of NAD was observed around pH 9 and 75 °C in the presence of 20 mM Mg²⁺. The enzyme showed broad substrate specificity: several monosaccarides such as 6-deoxy- -glucose, 2-amino-2-deoxy- -glucose and -xylose were oxidized as well as -glucose as the electron donor. -Mannose, -ribose and glucose-6-phosphate were inert as the donor. The enzyme showed high thermostability: remarkable loss of activity was not observed up to 80 °C by incubation for 15 min at pH 8.0. In addition, the enzyme was stable in a wide pH range of 5.0–10.5 by incubation at 37 °C. From the steady-state kinetic analysis, the enzyme reaction of -glucose oxidation proceeds via a sequential ordered Bi–Bi mechanism: NAD and -glucose bind to the enzyme in this order and then -glucono-1,5-lactone and NADH are released from the enzyme in this order. The amino acid sequence alignment showed that S. tokodaii GDH exhibited high homology with the Sulfolobus solfataricus hypothetical glucose dehydrogenase and a Thermoplasma acidophilum one.     

Regulation of colominic acid biosynthesis by temperature: role of cytidine 5''-monophosphate N-acetylneuraminic acid synthetase

Synthesis of colominic acid in Escherichia coli K-235 is strictly regulated by temperature. Evidence for the role of cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase in this regulation was obtained by measuring its level in E. coli grown at 20 and 37°C. No activity was found in E. coli grown at 20°C. CMP-Neu5Ac started to be quickly synthesized when bacteria grown at 20°C were transferred to 37°C and was halted when cells grown at 37°C were transferred to 20°C. These findings suggest that temperature regulates the synthesis of this enzyme and therefore the concentration of CMP-Neu5Ac necessary for the biosynthesis of colominic acid.     

The effect of phenolic acids and molasses spent wash concentration on distillery wastewater remediation by fungi

The effect of gallic acid, vanillic acid, and molasses spent wash (MSW) concentration on growth and decolourizing capability of four fungi (Geotrichum candidum, Coriolus versicolor, Phanerochaete chrysosporium and Mycelia sterilia) was studied. Fungal growth was inhibited to a varying extent in the presence of gallic and vanillic acid, except for G. candidum, which was unaffected by gallic acid. G. candidum and P. chrysosporium growth rates increased in the presence of increasing concentrations of MSW (up to 50% v/v), however, growth of M. sterilia and C. versicolor was inhibited at spent wash concentrations above 5% (v/v). Increasing the concentration of MSW from 6·25% (v/v) to 12·5% (v/v) increased the decolourizing ability of each fungus, except for M. sterilia. C. versicolor exhibited greatest colour removal with a reduction of 0·43 units at A₄₇₅ (equivalent to 53% colour reduction) after 10 days growth in 12·5% (v/v) MSW.     

The Production of Polyunsaturated Fatty Acids by Microalgae: from Strain Selection to Product Purification

A selection programme to increase the cellular eicosapentaenoic acid (EPA) content has been carried out with the microalga Isochrysis galbana. The selection process involved two stages of single selection. EPA content continuously increased from 2·4% dry weight (d.w.) of the ‘parent’ culture to an average value of 5·3% d.w. in the final stage. The proportion of total EPA variation attributable to the genetic variation (heritability in a broad sense) was 0·99 showing the importance of the genome in the determination of this fatty acid. The growth and fatty acid profile of an EPA-rich isolate grown as a chemostat in a cylindrical photobioreactor have been studied. A decrease in EPA content was observed (5·21% w/w to 2·8% w/w) at the lowest dilution rate D = 0·024 h⁻¹, up close to the maximum growth rate, D = 0·038 h⁻¹. At the same time, the biomass concentration also decreased from 1015 mg/litre to 202 mg/litre over the abovementioned range of dilution rate (D). Nonetheless, the EPA productivity increases with D, with a maximum of 15·26 mg/litre/day at D = 0·0208 h⁻¹. Furthermore, steady-state dilution rates may be related to average internal light intensity. Reverse-phase, high-pressure liquid chromatography (HPLC) on octadecylsilyl semi-preparative columns was used to separate stearidonic acid (SA), EPA and docosohexaenoic acid (DHA) in polyunsaturated fatty acid concentrate obtained by the urea complexation method from a fatty acid solution previously obtained by direct saponification of biomass. Isolate SA, EPA and DHA fraction purity was 94·8, 96·0 and 94·9%, respectively, with yields of 100·0, 99·6 and 94·0%.     

Structure and function of psychrophilic alanine racemase

We describe the structure and function of psychrophilic alanine racemases from Bacillus psychrosaccharolyticus and Pseudomonas fluorescens. These enzymes showed high catalytic activities even at 0°C and were extremely labile at temperatures over 35°C. The enzymes were also found to be less resistant to organic solvents than alanine racemases from thermophilic and mesophilic bacteria, both in vivo and in vitro. Both enzymes have a dimeric structure and contain 2 mol of pyridoxal 5′-phosphate (PLP) per mol as a coenzyme. The enzyme from B. psychrosaccharolyticus was found to have a markedly large K_m value (5.0 μM) for PLP in comparison with other reported alanine racemases, and was stable at temperatures up to 50°C in the presence of excess amounts of PLP. The dissociation of PLP from the P. fluorescens enzyme may trigger the unfolding of the secondary structure. The enzyme from B. psychrosaccharolyticus has a distinguishing hydrophilic region around residue no. 150 in its deduced amino acid sequence, whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of this region in the three dimensional structure of this enzyme was predicted to be in a surface loop surrounding the active site. This hydrophilic region may interact with solvent, reduce the compactness of the active site, and destabilize the enzyme.     

Effects of peptide YY and its analogues on chloride ion secretion in fed and fasted rat jejunum

The aim of our study was to determine whether a meal modifies the antisecretory response induced by PYY and the structural requirements to elicit antisecretory effects of analogue PYY(22–36) for potential antidiarrhea therapy. The variations in short-circuit current (Isc) due to the modification of ionic transport across the rat intestine were assessed in vitro, using Ussing chambers. In fasted rats, PYY induced a dose- and time-dependent reduction in Isc, with a sensitivity threshold at 5 × 10⁻¹¹ M (ΔIsc −2 ± 0.5 μA/cm²). The reduction was maximal at 10⁻⁷ M (Isc −23 ± 2 μA/cm²), and the concentration producing half-maximal inhibition was 10⁻⁹ M. At 10⁻⁷ M, reduction of Isc by PYY reached 90% of response to 5 × 10⁻⁵ M bumetanide. The PYY effect was partly reversed by 10⁻⁵ M forskolin (Isc +13.43 ± 2.91 μA/h·cm², p < 0.05) or 10⁻³ M dibutyryl adenosine 3′,5′ cyclic monophosphate (Isc +12 ± 1.69 μA/cm², p < 0.05). Naloxone and tetrodotoxin did not alter the effect of PYY. In addition, PYY and its analogue P915 reduced net chloride ion secretion to 2.85 and 2.29 μEq/cm² (p < 0.05), respectively. The antisecretory effect of PYY was accompanied by dose- and time-dependent desensitization when jejunum was prestimulated by a lower dose of peptide. The antisecretory potencies exhibited by PYY analogues required both a C-terminal fragment (22–36) and an aromatic amino acid residue (Trp or Phe) at position 27. At 10⁻⁷ M the biological activity of PYY was lower in fed than fasted rats (p < 0.001). Our results confirm the antisecretory effect of PYY, but show that the fed period is accompanied by desensitization, similar to the transient desensitization observed in the fasted period with cumulative doses. This suggests that PYY may act as a physiological mediator that reduces intestinal secretion.     

Gelling properties of water-soluble polysaccharides from proliferating marine green seaweeds (Ulva spp.)

Water-soluble polysaccharides (12·2% of the algal dry weight) were extracted from marine green seaweed (Ulva spp.) which proliferate along the Brittany shores of France. They were composed of 18·4% rhamnose, 4·4% glucose, 1·9% xylose, 0·9% mannose, 0·9% galactose, 15·2% uronic acid, 15·8% sulphate and 23·7% ash based on the extract dry weight. These polysaccharides, formed a weak gel (about 3 Pa) at a concentration of 1·6% (w/v) in deionized water. The elastic modulus increased to about 160 Pa when boric acid (15–33 m ) was added and reached 250 Pa when both boric acid (7 m ) and calcium chloride (7 m ) were present. Adjusting the pH to 7·5 or higher by sodium tetraborate, phosphate or Tris-HCl buffers was detrimental to the gel. These results demonstrate that the poorly exploited biomass of Ulva spp. is a source of gelling polysaccharides of potential economical value. Mechanisms for gel formation which unusually involve both boron and calcium ions are proposed and will be studied further.     

Cloning, expression and characterization of a novel thermal stable and short-chain alcohol tolerant lipase from Burkholderia cepacia strain G63

A lipase gene lipA and its chaperone gene lipB were cloned from Burkholderia cepacia strain G63. The lipA was composed of 1092 bp, encoding 363 amino acid residues, and the lipB composed of 1035 bp, corresponding to 344 amino acid residues. The significant amino acid similarity with Pseudomonas cepacia lipase revealed that this enzyme could be classified into the lipolytic subfamily I.2. The lipA and lipB genes were cloned into pBBR1Tp vector and conjugated into B. cepacia strains G63 with the help of pRK2013. The recombinant strain was fermented in 10 l bioreactor and the lipase was purified by a combination of ammonium sulfate fractionation, DEAE ion-exchange chromatography and gel filtration. The purified lipase kept stable at a temperature range of 40–70 °C. After incubated at 70 °C, the optimal temperature of this enzyme, for 10 h it remained 86.1% of its activity. The enzyme was also highly tolerant to a series of organic solution. Incubated in 50% methanol solution up to 48 h, the enzyme still kept 98.3% of its activity. The transesterification activity of soybean oil to fatty acid methyl esters (FAMEs) reached 87.8% after 72 h, indicating that it is a potential biocatalyzer for biodiesel production.     

Activity and stability of native and modified alanine aminotransferase in cosolvent systems and denaturants

Alanine aminotransferase (ALT) is used in clinical diagnostics, amino acid synthesis and in biosensors. Here we describe the stabilization of soluble porcine ALT by chemical modification with mono- and bis-imidates. The apparent transition temperatures (‘T_m’, the temperature where 50% of initial activity was lost in 10 min) for native and DMS-modified ALT were 46 and 56 °C respectively. The effects of water-miscible organic solvents (methanol, dimethylformamide, dimethylsulphoxide and 1,4-dioxane) on the activity/stability of native and modified forms were determined. In all systems studied, an abrupt decrease in ALT catalytic activity was observed on reaching a certain threshold concentration of the organic solvent. The modified derivatives were more organotolerant than native enzyme. Comparison of the apparent V_max and K_m for 2-oxoglutarate as substrate, determined in 10% (v/v) organic solvent, with the results of thermal inactivation studies showed that the solvents have different effects on ALT's catalytic parameters and on its conformational stability. At 35 °C with no organic solvent the dimethylsuberimidate (DMS)-modified derivative's half-life was 16 times greater than that for native enzyme; in 30% (v/v) solvent at 35 °C, the DMS-modified ALT's half-life was up to 4.6 times greater than native enzyme's. DMS-modified ALT was also more stable in urea and guanidine HCl, and its refolding was more noticeable, than that of native enzyme.     

Production d'NH4 par la crevette Crangon crangon L. dans deux écosystémes côtiers. approche expérimentale et étude de l'influence du sédiment sur le taux d'excrétion

Estimation of the ammonia production of the shrimp C. crangon in two littoral ecosystems (oligotrophic sand and eutrophic mud) was determined in winter and summer conditions from laboratory observations in experimental microcosms. The ammonia excretion rate of C. crangon was not influenced by either the sediment type or the ammonia concentration of the overlying water; on the other hand, the mean excretion rate and the response to initial handling stress increased markedly as shrimp were deprived of soft substratum.

The daily ammonia production of C. crangon was 16 μmol NH₃ · g ⁻¹ wet wt · day ⁻¹ in winter and 40 μmol in summer. A gross production of 12 μmol NH₃ · m⁻² · day ⁻¹ and 300–700 μmol μ m⁻² · day⁻¹, respectively, could be expected in the two ecosystems studied. This would account for 5% (winter) and 2–4% (summer) of the total NH⁺₄ flux at the sediment-water interface. The contribution of the excretion of all macrofauna to the NH⁺₄ flux from the sediment is discussed.     

The influence of salinity on certain aspects of the biology of Bulinus (Physopsis) africanus

The hatchability of eggs and the fecundity and survival of adult Bulinus (Physopsis) africanus was investigated in different salinities. Experimental results revealed egg masses and hatchlings to be considerably more sensitive to salinity than the adult snails. Egg-laying was recorded in salinities 4·5 ‰ and further increases in salinity resulted in a progressive reduction in the hatching success up to a lethal concentration of 5·25 ‰ Survival of these hatchings was adversely affected by salinities as low as 1·0 ‰ and a salinity of 4·5 ‰ was lethal within 6 days. In contrast, adult survival was unaffected in salinities < 3·5 ‰ while further increases in salinity resulted in significant reductions in survival up to a lethal salinity of 8·7 ‰, which caused 100% mortality within 24 h. The survival of B. africanus infected with Schistosoma haematobium and Schistosoma mattheei was lower in the different salinities and control than that of their uninfected counterparts.     

Characterization of Aspergillus niger catalase

Aspergillus niger catalase has been characterized by a variety of physical techniques including gel filtration, sedimentation rate and equilibrium methods and photon correlation spectroscopy. The catalase has a sedimentation coefficient (S₂₀⁰) of 14.2 ± 0.08 S and diffusion coefficient (D₂₀⁰) of 4.14 ± 0.35 × 10⁻⁷ cm² s⁻¹. The average molecular weight of the catalase from all available data including current sedimentation equilibrium measurements and two previous literature values is 345 000. The frictional ratio of the molecule assuming a hydration parameter similar to that of bovine liver catalase (.3 g H₂O g⁻¹) is 1.103, suggesting that Aspergillus niger catalase has an asymmetric structure with an axial ratio of approximately 3 (the Stokes radius is 5.83 ± 0.49 nm). The titration curve and amino acid analysis indicate that in the native conformation only 23% of the ionizable amino acid residues are titratable between pH 3 and 10.5. Denaturation with sodium n-dodecylsulphate increases the number of titratable groups to 46%. The ratio of anionic to cationic amino acid residues in Aspergillus niger catalase is 2.46 and the isoelectric point is 6.5. The optimum pH for catalytic activity is approximately 7.     

Redox potentials of the pyridine-haem c system

1. Haem c was synthesized and purified. It was shown unequivocally that the method gives a product with the cysteine residues on the -carbon atoms at the 2 and 4 positions of the haem.

2. Redox potentials of haem c in the presence of 2.5 M pyridine were determined in the pH range 1.5–13; it was found necessary to add cetyl trimethyl ammonium bromide (CTAB) to prevent precipitation in the acid range below about pH 4. The E_m vs pH curve shows three slopes (−dE/dpH) of value, 0.18, 0.01 and 0.06 with points of inflexion at pH 3.8 and 10.6. The potentials are intermediate between those of protohaem and mesohaem obtained under similar conditions.

3. With constant haem c concentration (a) 10⁻⁴ M and (b) 10⁻⁵ M and varying pyridine concentration (0.12–5 M) it was found at pH 9.0 that E_m values increased as the pyridine concentration was increased and there was a tendency to reach a plateau value. The explanation appears to be that pyridine binds more firmly to ferroporphyrin c than to ferriporhyrin c.

4. When the pyridine concentration was kept constant (2.5 M) and the haem c concentration was varied in the range 7 · 10⁻⁴–7 · 10⁻⁶ M, it was found that a decrease in haem c concentration brought about an increase in redox potential. The results are explained as being due to dimerization of the oxidized form.

5. The results are discussed in comparison with a number of related haem systems.     

Nucleotide sequence of the FokI restriction-modification system: separate strand-specificity domains in the methyltransferase

The genes for FokI, a type-IIS restriction-modification system from Flavobacterium okeanokoites (asymmetric recognition sequence: 5'-GGATG/3'-CCTAC), were cloned into Escherichia coli. Recombinants carrying the fokIR and fokIM genes were found to modify their DNA completely, and to restrict lambdoid phages weakly. The nt sequences of the genes were determined, and the probable start codons were confirmed by aa sequencing. The FokI endonuclease (R · FokI) and methyltransferase (M · FokI are encoded by single, adjacent genes, aligned in the same orientation, in the order M then R. The genes are large by the standards of type-II systems, 1.9 kb for the M gene, and 1.7 kb for the R gene. Preceding each gene is a pair of FokI recognition sites; it is conceivable that interactions between the sites and the FokI proteins could regulate expression of the genes. The aa sequences of the N- and C-terminal halves of M · FokI are similar to one another, and to certain other DNA-adenine methyltransferases, suggesting that the enzyme has a ‘tandem’ structure, such as could have arisen by the fusion of a pair of adjacent, ancestral M genes. Truncated derivatives of M · FokI were constructed by deleting the 5'- or 3' -ends of the fokIM gene. Deleting most of the C-terminus of M · FokI produced derivatives that methylated only the top (GGATG) strand of the recognition sequence. Conversely, deleting most of the N-terminus produced derivatives that methylated only the bottom (CATCC) strand of the recognition sequence. These results indicate that the domains in M · FokI for methylating the two strands of the recognition sequence are largely separate.     

Kinetic characterization of chiral biocatalysis of cycloarenes by the camphor 5-monooxygenase enzyme system

Redox enzyme mediated biocatalysis has the potential to regio- and stereo-specifically oxidize hydrocarbons producing valuable products with minimal by-product formation. In vitro reactions of the camphor (cytochrome P-450) 5-monooxygenase enzyme system with naphthalene-like substrates yield stereospecifically hydroxylated products from nonactivated hydrocarbons. Specifically, the enzyme system catalyzes the essentially stereospecific conversion of the cycloarene, tetralin (1,2,3,4-tetrahydronaphthalene) to (R)-1-tetralol ((R)-(−)-1,2,3,4-tetrahydro-1-naphthol). It is shown that this reaction obeys Michaelis–Menten kinetics and that interactions between the enzyme subunits are not affected by the identity of the substrate. This subunit independence extends to the efficiency of NADH usage by the enzyme system—subunit ratios do not effect efficiency, but substrate identity does. Tetralin is converted at an efficiency of 13±3%, whereas (R)-1-tetralol is converted at 7.8±0.7%. A model of this system based on Michaelis–Menten parameters for one subunit (Pdx: K_M=10.2±2 μM) and both substrates (tetralin: K_M=66±26 μM, ν_max=0.11±0.04 s⁻¹, and (R)-1-tetralol: K_M=2800±1300 μM, ν_max=0.83±0.22 s⁻¹) is presented and used to predict the consumption and production of all substrates, products and cofactors.