Tolerance and metabolic response of acetogenic bacteria toward oxygen

The acetogens Sporomusa silvacetica, Moorella thermoacetica, Clostridium magnum, Acetobacterium woodii, and Thermoanaerobacter kivui (i) grew in both semisolid and liquid cultivation media containing O(2) and (ii) consumed small amounts of O(2). Low concentrations of O(2) caused a lag phase in growth but did not alter the ability of these acetogens to synthesize acetate via the acetyl coenzyme A pathway. Cell extracts of S. silvacetica, M. thermoacetica, and C. magnum contained peroxidase and NADH oxidase activities; catalase and superoxide dismutase activities were not detected.     

Electrosynthesis of organic compounds from carbon dioxide is catalyzed by a diversity of acetogenic microorganisms

Microbial electrosynthesis, a process in which microorganisms use electrons derived from electrodes to reduce carbon dioxide to multicarbon, extracellular organic compounds, is a potential strategy for capturing electrical energy in carbon-carbon bonds of readily stored and easily distributed products, such as transportation fuels. To date, only one organism, the acetogen Sporomusa ovata, has been shown to be capable of electrosynthesis. The purpose of this study was to determine if a wider range of microorganisms is capable of this process. Several other acetogenic bacteria, including two other Sporomusa species, Clostridium ljungdahlii, Clostridium aceticum, and Moorella thermoacetica, consumed current with the production of organic acids. In general acetate was the primary product, but 2-oxobutyrate and formate also were formed, with 2-oxobutyrate being the predominant identified product of electrosynthesis by C. aceticum. S. sphaeroides, C. ljungdahlii, and M. thermoacetica had high (>80%) efficiencies of electrons consumed and recovered in identified products. The acetogen Acetobacterium woodii was unable to consume current. These results expand the known range of microorganisms capable of electrosynthesis, providing multiple options for the further optimization of this process.     

Non-growth-associated demethylation of dimethylsulfoniopropionate by (homo)acetogenic bacteria

The demethylation of the algal osmolyte dimethylsulfoniopropionate (DMSP) to methylthiopropionate (MTPA) by (homo)acetogenic bacteria was studied. Five Eubacterium limosum strains (including the type strain), Sporomusa ovata DSM 2662(T), Sporomusa sphaeroides DSM 2875(T), and Acetobacterium woodii DSM 1030(T) were shown to demethylate DMSP stoichiometrically to MTPA. The (homo)acetogenic fermentation based on this demethylation did not result in any significant increase in biomass. The analogous demethylation of glycine betaine to dimethylglycine does support growth of acetogens. In batch cultures of E. limosum PM31 DMSP and glycine betaine were demethylated simultaneously. In mixed substrates experiments with fructose-DMSP or methanol-DMSP, DMSP was used rapidly but only after exhaustion of the fructose or the methanol. In steady-state fructose-limited chemostat cultures (at a dilution rate of 0.03 h(-1)) with DMSP as a second reservoir substrate, DMSP was biotransformed to MTPA but this did not result in higher biomass values than in cultures without DMSP; cells from such cultures demethylated DMSP at rates of approximately 50 nmol min(-1) mg of protein(-1), both after growth in the presence of DMSP and after growth in its absence. In cell extracts of glycine betaine-grown strain PM31, DMSP demethylation activities of 21 to 24 nmol min(-1) mg of protein(-1) were detected with tetrahydrofolate as a methyl acceptor; the activities seen with glycine betaine were approximately 10-fold lower. A speculative explanation for the demethylation of DMSP without an obvious benefit for the organism is that the DMSP-demethylating activity is catalyzed by the glycine betaine-demethylating enzyme and that a transport-related factor, in particular a higher energy demand for DMSP transport across the cytoplasmic membrane than for glycine betaine transport, may reduce the overall ATP yield of the fermentation to virtually zero.     

Transformation of tetrachloroethylene to trichloroethylene by homoacetogenic bacteria

Abstract Eight homoacetogenic strains of the genera Acetobacterium, Clostridium and Sporomusa were tested for their ability to dechlorinate tetrachloroethylene (perchloroethene, PCE). Of the organisms tested only Sporomusa ovata was able to reductively dechlorinate PCE with methanol as an electron donor. Resting cells of S. ovata reductively dechlorinated PCE at a rate of 9.8 nmol h⁻¹ (mg protein)⁻¹ to trichloroethylene (TCE) as the sole product. The dechlorination activity depended on concomitant acetogenesis from methanol and CO₂. Cell-free extracts of S. ovata, Clostridium formicoaceticum, Acetobacterium woodii , and the methanogenic bacterium Methanolobus tindarius transformed PCE to TCE with Ti(III) or carbon monoxide as electron donors. Corrinoids were shown in S. ovata to be involved in the dechlorination reaction of PCE to TCE as evident from the reversible inhibition with propyl iodide. Rates of dechlorination followed a pseudo-first-order kinetic.     

Hydrogen-dependent oxygen reduction by homoacetogenic bacteria isolated from termite guts

Although homoacetogenic bacteria are generally considered to be obligate anaerobes, they colonize the intestinal tracts of termites and other environments that are not entirely anoxic in space or time. In this study, we investigated how homoacetogenic bacteria isolated from the hindguts of various termites respond to the presence of molecular oxygen. All strains investigated formed growth bands in oxygen gradient agar tubes under a headspace of H(2)-CO(2). The position of the bands coincided with the oxic-anoxic interface and depended on the O(2) partial pressure in the headspace; the position of the bands relative to the meniscus remained stable for more than 1 month. Experiments with dense cell suspensions, performed with Clark-type O(2) and H(2) electrodes, revealed a large capacity for H(2)-dependent oxygen reduction in Sporomusa termitida and Sporomusa sp. strain TmAO3 (149 and 826 nmol min(-1) mg of protein(-1), respectively). Both strains also reduced O(2) with endogenous reductants, albeit at lower rates. Only in Acetonema longum did the basal rates exceed the H(2)-dependent rates considerably (181 versus 28 nmol min(-1) mg of protein)(-1)). Addition of organic substrates did not stimulate O(2) consumption in any of the strains. Nevertheless, reductive acetogenesis by cell suspensions of strain TmAO3 was inhibited even at the lowest O(2) fluxes, and growth in nonreduced medium occurred only after the bacteria had rendered the medium anoxic. Similar results were obtained with Acetobacterium woodii, suggesting that the results are not unique to the strains isolated from termites. We concluded that because of their tolerance to temporary exposure to O(2) at low partial pressures (up to 1.5 kPa in the case of strain TmAO3) and because of their large capacity for O(2) reduction, homoacetogens can reestablish conditions favorable for growth by actively removing oxygen from their environment.     

Interspecies hydrogen transfer in co-cultures of methanol-utilizing acidogens and sulfate-reducing or methanogenic bacteria

Abstract The metabolism of methanol by acidogenic bacteria ( Butyribacterium methylotrophicum, Sporomusa ovata and Acetobacterium woodii ) was studied in pure culture and in defined mixed cultures with sulfate-reducing bacteria ( Desulfovibrio vulgaris ) or methanogenic bacteria ( Methanobrevibacter arboriphilus strain AZ). In the mixed cultures, less acids (acetate and/or butyrate) were formed per unit methanol converted than in pure cultures. In these mixed cultures, a significant production of sulfide or methane was observed despite the inability of the sulfate reducer and the methanogen to use methanol as an energy substrate. These results are explained in terms of interspecies hydrogen transfer between the acidogens (converting part of the methanol to 1 CO₂ and 3 H₂) and the Desulfovibrio or Methanobrevibacter species. The bioenergetic aspects of this process and its ecological implications are discussed.     

Enhanced biotransformation of carbon tetrachloride by Acetobacterium woodii upon addition of hydroxocobalamin and fructose.

The objective of this study was to evaluate the effect of hydroxocobalamin (OH-Cbl) on transformation of high concentrations of carbon tetrachloride (CT) by Acetobacterium woodii (ATCC 29683). Complete transformation of 470 microM (72 mg/liter [aqueous]) CT was achieved by A. woodii within 2.5 days, when 10 microM OH-Cbl was added along with 25.2 mM fructose. This was approximately 30 times faster than A. woodii cultures (live or autoclaved) and medium that did not receive OH-Cbl and 5 times faster than those controls that did receive OH-Cbl, but either live A. woodii or fructose was missing. CT transformation in treatments with only OH-Cbl was indicative of the important contribution of nonenzymatic reactions. Besides increasing the rate of CT transformation, addition of fructose and OH-Cbl to live cultures increased the percentage of [(14)C]CT transformed to (14)CO(2) (up to 31%) and (14)C-labeled soluble materials (principally L-lactate and acetate), while decreasing the percentage of CT reduced to chloroform and abiotically transformed to carbon disulfide. (14)CS(2) represented more than 35% of the [(14)C]CT in the presence of reduced medium and OH-Cbl. Conversion of CT to CO was a predominant pathway in formation of CO(2) in the presence of live cells and added fructose and OH-Cbl. These results indicate that the rate and distribution of products during cometabolic transformation of CT by A. woodii can be improved by the addition of fructose and OH-Cbl.     

B cell growth modulating and differentiating activity of recombinant human 26-kd protein (BSF-2, HuIFN-beta 2, HPGF).

The human "26-kd protein' is a secreted glycoprotein expressed, for example, in (blood) leukocytes, in epithelial cells treated with various inducers, but most strongly in interleukin-1 (IL-1)-treated fibroblasts. After finding it has antiviral and 2-5A synthetase-inducing activity, one group of authors called this protein IFN-beta 2. However, recently the full-length 26-kd cDNA sequence was shown to be identical with that of a B-cell-differentiating lymphokine called BSF-2, and another report suggested that the 26-kd protein could support the growth of some transformed murine B cell lines. To define its biological activities, we expressed the recombinant 26-kd protein by translating in Xenopus laevis oocytes a pure, synthetic chimeric mRNA containing the 26-kd protein coding region surrounded by Xenopus laevis beta-globin untranslated regions. A similar construction, but containing the HuIFN-beta cDNA coding region, was used to produce HuIFN-beta by the same procedure. Both recombinant glycoproteins were secreted, glycosylated, and their amounts were measured by [35S]methionine incorporation by the oocyte. Here we show that the recombinant 26-kd protein exhibits a high growth factor activity when assayed on an IL-HP1-dependent murine B cell hybridoma (sp. act. approximately 2 X 10(8) U/mg) as well as a potent differentiating activity on human CESS cells (sp. act. approximately 5 X 10(7) U/mg). While rHuIFN-beta was inactive in the latter two assays, it had the expected antiviral activity of 1-5 X 10(8) U/mg. The parallel recombinant 26-kd protein preparations had no detectable antiviral activity (i.e. a maximal specific activity of 1-3 X 10(2) U/mg, if any). The 26-kd protein is thus clearly an interleukin, and considering the confusing nomenclature now in use, this factor may better be renamed "interleukin 6'.     

Degradation of glyoxylate and glycolate with ATP synthesis by a thermophilic anaerobic bacterium, Moorella sp. strain HUC22-1

The thermophilic homoacetogenic bacterium Moorella sp. strain HUC22-1 ferments glyoxylate to acetate roughly according to the reaction 2 glyoxylate --> acetate + 2 CO(2). A batch culture with glyoxylate and yeast extract yielded 11.7 g per mol of cells per substrate, which was much higher than that obtained with H(2) plus CO(2). Crude extracts of glyoxylate-grown cells catalyzed the ADP- and NADP-dependent condensation of glyoxylate and acetyl coenzyme A (acetyl-CoA) to pyruvate and CO(2) and converted pyruvate to acetyl-CoA and CO(2), which are the key reactions of the malyl-CoA pathway. ATP generation was also detected during the key enzyme reactions of this pathway. Furthermore, this bacterium consumed l-malate, an intermediate in the malyl-CoA pathway, and produced acetate. These findings suggest that Moorella sp. strain HUC22-1 can generate ATP by substrate-level phosphorylation during glyoxylate catabolism through the malyl-CoA pathway.     

Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli

A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.     

Two propanediol utilization-like proteins of <Emphasis Type="Italic">Moorella thermoacetica</Emphasis> with phosphotransacetylase activity

Moorella thermoacetica is one of the model acetogenic bacteria for the resolution of the Wood–Ljungdahl (acetyl-CoA) pathway in which CO₂ is autotrophically assimilated yielding acetyl-CoA as central intermediate. Its further conversion into acetate relies on subsequent phosphotransacetylase (PTA) and acetate kinase reactions. However, the genome of M. thermoacetica contains no pta homologous gene. It has been speculated that the moth_0864 and moth_1181 gene products sharing similarities with an evolutionarily distinct phosphotransacylase involved in 1,2-propanediol utilization (PDUL) of Salmonella enterica act as PTAs in M. thermoacetica. Here, we demonstrate specific PTA activities with acetyl-CoA as substrate of 9.05 and 2.03 U/mg for the recombinant enzymes PDUL1 (Moth_1181) and PDUL2 (Moth_0864), respectively. Both showed maximal activity at 65 °C and pH 7.6. Native proteins (90 kDa) are homotetramers composed of four subunits with apparent molecular masses of about 23 kDa. Thus, one or both PDULs of M. thermoacetica might act as PTAs in vivo catalyzing the penultimate step of the Wood–Ljungdahl pathway toward the formation of acetate. In silico analysis underlined that up to now beside of M. thermoacetica, only Sporomusa ovata contains only PDUL like class^III-PTAs but no other phosphotransacetylases or phosphotransbutyrylases (PTBs).     

Purification and characterization of the enantioselective esterase from Kluyveromyces marxianus CBS 1553

An intracellular esterase from the yeast Kluyveromyces marxianus CBS 1553 with interesting enantioselective hydrolytic activity towards racemic esters of 1,2-O-isopropylidene glycerol (IPG) was purified and characterized. Optimal culture conditions for the obtainment of the enantioselective esterase on a 5 l-fermentation scale were investigated. Two esterase activities (EST1 and EST2) in the crude cell extract were identified by native PAGE with specific activity staining and separated from each other by anion-exchange chromatography. EST1 showed higher activity and enantioselectivity than EST2 in the resolution of racemic IPG acetate and was further purified by hydrophobic interaction chromatography and preparative electrophoresis (final specific activity approximately = 300 U mg(-1), showing a main protein band with a molecular mass of 29 kDa. EST1 showed optimal activity between pH 8.0 and 10.0 and was stable in the pH range 7.0-10.0. Moreover, it was rather thermostable and active up to 80 degrees C, and retained most of its activity in the presence of 15% (v/v) of various organic solvents. The enzyme showed similar Vmax in the hydrolysis of the acetate esters of IPG, whereas the Km value towards (S)-IPG acetate was significantly lower than the one towards the (R)-enantiomer (5.3 and 70 microM, respectively). Finally, comparison of EST1 activity in the presence of different glycerol esters and synthetic substrates with different chain lengths showed a strong preference of this biocatalyst for short-chain substrates.     

Bacterial cyanide oxygenase is a suite of enzymes catalyzing the scavenging and adventitious utilization of cyanide as a nitrogenous growth substrate

Cyanide oxygenase (CNO) from Pseudomonas fluorescens NCIMB 11764 catalyzes the pterin-dependent oxygenolytic cleavage of cyanide (CN) to formic acid and ammonia. CNO was resolved into four protein components (P1 to P4), each of which along with a source of pterin cofactor was obligately required for CNO activity. Component P1 was characterized as a multimeric 230-kDa flavoprotein exhibiting the properties of a peroxide-forming NADH oxidase (oxidoreductase) (Nox). P2 consisted of a 49.7-kDa homodimer that showed 100% amino acid identity at its N terminus to NADH peroxidase (Npx) from Enterococcus faecalis. Enzyme assays further confirmed the identities of both Nox and Npx enzymes (specific activity, 1 U/mg). P3 was characterized as a large oligomeric protein (approximately 300 kDa) that exhibited cyanide dihydratase (CynD) activity (specific activity, 100 U/mg). Two polypeptides of 38 kDa and 43 kDa were each detected in the isolated enzyme, the former believed to confer catalytic activity based on its similar size to other CynD enzymes. The amino acid sequence of an internal peptide of the 43-kDa protein was 100% identical to bacterial elongation factor Tu, suggesting a role as a possible chaperone in the assembly of CynD or a multienzyme CNO complex. The remaining P4 component consisted of a 28.9-kDa homodimer and was identified as carbonic anhydrase (specific activity, 2,000 U/mg). While the function of participating pterin and the roles of Nox, Npx, CynD, and CA in the CNO-catalyzed scavenging of CN remain to be determined, this is the first report describing the collective involvement of these four enzymes in the metabolic detoxification and utilization of CN as a bacterial nitrogenous growth substrate.     

Proximal tubular metalloprotease activity is decreased in the senescent rat kidney.

Proximal tubule brush border membrane-enriched fractions (BBM) from young (4 months) and old (20-22 months) male Sprague-Dawley rats were prepared by differential centrifugation and metalloprotease activity was measured using radioiodinated insulin B chain as substrate. Proteolytic activity is expressed as Units (U), where 1 U = 1 microgram insulin B chain degraded per min and the specific activity is the U per mg BBM protein used in the assay. Total proteolytic activity (measured at pH 7) was decreased 2-fold in BBM from old rats (2.47 +/- 0.11 vs 4.71 +/- 0.35 U/mg BBM protein, p less than 0.01). The chelator, 1,10-phenanthroline, completely inhibited the proteolytic activity in both groups, suggesting that the BBM insulin B chain-degrading activity in both old and young rats was entirely due to metalloproteases. In the presence of thiorphan, a specific inhibitor of the metalloprotease endopeptidase 24.11, approximately 60% inhibition of proteolytic activity occurred in both groups. Thus, total metalloprotease and endopeptidase 24.11 activities are markedly diminished in the proximal tubule of the senescent rat kidney.     

Changes in carbonic anhydrase may be the initial step of altered metabolism in hypertension

Carbonic anhydrase (CA) is a well characterized pH regulatory enzyme in most of the tissues in the body. Changes in activities of CA have been associated with altered metabolism, especially in diabetes mellitus. Insulin resistance and hyperinsulinemia are common in hypertension. To investigate the possible role of CA, we measured the CA activity spectrophotometrically using p-nitrophenyl acetate as a substrate and acetazolamide, the specific inhibitor, in erythrocytes from normotensive and essential hypertensive subjects. Further, to evaluate the insulin action on CA, we used two different hemolysates; (i) insulin applied into hemolysate and (ii) hemolysate from insulin treated erythrocytes in vitro before the determination of CA activity. Two different levels of CA activities were obtained in these patients. CA activities were much lower (mean +/- SD, 0.88 +/- 0.19 U/min/mL) and higher (mean +/- SD, 1.77 +/- 0.23 U/min/mL) in patients than the normotensive controls (mean +/- 1 SD, 1.41 +/- 0.1 U/min/mL). These differences in both the groups were statistically significant (p less than 0.001). Similarly, total esterase activities in patients were (1.41 +/- 0.27 U/min/mL) that was 30% less in low activity group and (2.47 +/- 0.25 U/min/mL) that was 22% more in higher activity group in comparison with those from normotensives (2.02 +/- 0.17 U/min/mL). The relative percent of CA activities of insulin treated erythrocytes from normotensives and hypertensives were 11% and 18% higher than without insulin (p less than 0.05). No difference was observed when insulin was applied in the hemolysate. We conclude that essential hypertensive patients are associated with altered CA activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Na+-translocating pyrophosphatase in the acetogenic bacterium Acetobacterium woodii

The anaerobic acetogenic bacterium Acetobacterium woodii employs a novel type of Na(+)-motive anaerobic respiration, caffeate respiration. However, this respiration is at the thermodynamic limit of energy conservation, and even worse, in the first step, caffeate is activated by caffeyl-CoA synthetase, which hydrolyzes ATP to AMP and pyrophosphate. Here, we have addressed whether or not the energy stored in the anhydride bond of pyrophosphate is conserved by A. woodii. Inverted membrane vesicles of A. woodii have a membrane-bound pyrophosphatase that catalyzes pyrophosphate hydrolysis at a rate of 70-120 milliunits/mg of protein. Pyrophosphatase activity was dependent on the divalent cation Mg(2+). In addition, activity was strictly dependent on Na(+) with a K(m) of 1.1 mM. Hydrolysis of pyrophosphate was accompanied by (22)Na(+) transport into the lumen of the inverted membrane vesicles. Inhibitor studies revealed that (22)Na(+) transport was primary and electrogenic. Next to the Na(+)-motive ferredoxin:NAD(+) oxidoreductase (Fno or Rnf), the Na(+)-pyrophosphatase is the second primary Na(+)-translocating enzyme in A. woodii.     

Factorial effects of salinity, dietary carbohydrate and moult cycle on digestive carbohydrases and hexokinases in Litopenaeus vannamei (Boone, 1931)

Litopenaeus vannamei were reared in close cycle over seven generations and tested for their capacity to digest starch and to metabolise glucose at different stages of the moulting cycle. After acclimation with 42.3% of carbohydrates (HCBH) or 2.3% carbohydrates (LCBH) diets and at high salinity (40 g kg(-1)) or low salinity (15 g kg(-1)), shrimp were sampled and hepatopancreas (HP) were stored. Total soluble protein in HP was affected by the interaction between salinity and moult stages (p<0.05). Specific activity of alpha-amylase ranged from 44 to 241 U mg protein(-1) and a significant interaction between salinity and moult stages was observed (p<0.05), resulting in highest values at stage C for low salinity (mean value 196.4 U mg protein(-1)), and at D0 in high salinity (mean value 175.7 U mg protein(-1)). Specific activity of alpha-glucosidase ranged between 0.09 and 0.63 U mg protein(-1), an interaction between dietary CBH and salinity was observed for the alpha-glucosidase (p<0.05) and highest mean value was found in low salinity-LCBH diet treatment (0.329 U mg protein(-1)). Hexokinase specific activity (range 9-113 mU mg protein(-1)) showed no significant differences when measured at 5 mM glucose (p>0.05). Total hexokinase specific activity (range 17-215 mU mg protein(-1)) showed a significant interaction between dietary CBH and salinity (p<0.05) with highest value (mean value 78.5 mU mg protein(-1)) found in HCBH-high salinity treatment, whereas in the other treatments the activity was not significantly different (mean value 35.93 mU mg protein(-1)). A synergistic effect of dietary CBH, salinity and moult stages over hexokinase IV-like specific activity was also observed (p<0.05). As result of this interaction, the highest value (135.5+/-81 mU mg protein(-1)) was observed in HCBH, high salinity at D0 moult stage. Digestive enzymes activity is enhanced in the presence of high starch diet (HCBH) and hexokinase can be induced at certain moulting stages under the influence of blood glucose level. Perspectives are opened to add more carbohydrates in a growing diet, exemplifying the potential approach for less-polluting feed.     

Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers.

DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic O-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 +/- 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent Km values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60 degrees C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic O-demethylating enzyme system of A. woodii is oxygen sensitive. The THF- and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells.     

Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers.

DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic O-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 +/- 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent Km values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60 degrees C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic O-demethylating enzyme system of A. woodii is oxygen sensitive. The THF- and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells.     

12 alpha-hydroxysteroid dehydrogenase from Clostridium group P, strain C 48-50. Production, purification and characterization

NADP(H)-dependent 12 alpha-hydroxysteroid dehydrogenase (HSDH) from Clostridium group P, strain C 48-50, is still expressed at unusual high level (approximately 1% of total protein) under cultivation conditions where the usual expensive brain/heart infusion complex medium is replaced by inexpensive technical grade yeast autolysate. An inexpensive anaerobic bioprocess for the production of HSDH was developed provisionally up to 900-1 scale (9000 U/l, 7 g HSDH, specific activity 1.0 U/mg crude protein, 55 U/g wet cells). By a simple two-step affinity chromatography procedure, easily adaptable to a large-scale operation, using columns of small dimensions of Sephacryl-S-400-Procion-orange-P-2R (5 cm x 28 cm) and Sephacryl-S-400-Procion-red-HE-7B (2.6 cm x 14 cm) approximately 140 mg (1.8 x 10(4) U), HSDH was purified to apparent homogeneity and concentrated directly from a crude cell extract (overall yield 53%, specific activity 128 U/mg). As confirmed by fast native and SDS/PAGE, isoelectric focussing and electron microscopy, HSDH has a molecular mass of approximately 105 kDa and consists of four flattened tetrahedrically arranged identical subunits (26 kDa). The enzyme exhibits a rather low isoelectric point of 4.6, a pH optimum of 8.5-9.5 and a temperature optimum of approximately 55 C for the oxidation of cholic acid. Inhibition by SH reagents and pyridoxal 5'-phosphate has been observed. Chelating agents have no inhibitory effect. The presence of NADP increases considerably the thermostability (t 1/2 4-10 d, 25 C; 2-5 d, 37 C). Steady-state kinetic analysis for both reaction directions indicated that the reaction proceeds through an ordered bi bi mechanism with NADP(H) binding first to the free enzyme. Km, Vmax [forward (Vf) and reverse reactions (Vr)] and the dissociation constants Kd for the binary complexes with NADP and NADPH were as follows. NADP, Km = 35 microns, Kd = 35 microns; cholic acid, Km = 72 microns, deoxycholic acid, Km = 45 microns, Vf = 160 U mg; NAPDH, Kd = 16 microns; 12-oxochenodeoxylic acid, Km = 12 microns, 66 U/mg (conditions, 0.1 M potassium phosphate, pH 8.0, 25 degrees C). N6-functionalized NADP derivatives, e.g. N6-(2-aminoethyl)NADP (Km = 4.5 mM) are poorly accepted as coenzyme by HSDH.