Dioxygenase activity and relative behaviour of Pseudomonas strains from soil in the presence of different aromatic compounds

Ten different Pseudomonas strains isolated from contaminated soils were tested for expression of active dioxygenases. Of these, two different clusters, related to strain origin were observed. The first included two P. fluorescens strains and two P. aeruginosa strains isolated from soils polluted with polyaromatic hydrocarbons and the second two P. cepacia strains and four P. chlororaphis strains from soils with polyphenols. All the isolates showed catechol 1,2-dioxygenase basal activity, while other dioxygenases (catechol 2,3-dioxygenase, protocatechuate 2,3-, 3,4- and 4,5-dioxygenases) were detected only after growth in the presence of suitable inducers (benzoate, catechol, salicylate, phenol). Significant induction of catechol 1,2-dioxygenase, the major activity of the tested strains, was also observed when combining starvation with the presence of high molecular weight aromatic hydrocarbons with recalcitrant structures (fluoranthene, chrysene, benzanthracene, pyrene).     

Aromatic metabolism in Rhizobium trifolii —protocatechuate 3,4-dioxygenase

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) has been purified 42-fold from 4-hydroxybenzoate-grown cells of Rhizobium trifolii TA1, where it constitutes about 2% of the cell protein. The dioxygenase has a molecular weight of 220,000, with two dissimilar sub-units of molecular weights 29,000 and 26,500, corresponding to an 44 composition. The enzyme is specific for protocatechuate, with a Km of 1.75×10^-5 M and maximum activity at pH 9.2. Metal removal and replacement studies indicate that the enzyme contains complexed Fe³⁺ which is required for activity. Direct atomic absorption analysis gave 1.3–1.5 g atoms Fe³⁺ per mole of isolated enzyme, but correction for metal-deficient proteins suggests that the value is close to 2.     

Purification and Properties of Catechol 1,2-Dioxygenase from Rhizobium leguminosarum biovar viceae USDA 2370

The catechol 1,2-dioxygenase of Rhizobium leguminosarum biovar viceae USDA 2370 was purified 296-fold, yielding a homogeneous preparation with a specific activity of 51.1 U mg of protein^-1. The molecular weight of the native protein was 70,000, with two identical subunits of 34,500 and 1 g-atom of iron per mol. The optimum pH for catalytic activity was 9.0 to 9.5.     

Aromatic metabolism in Rhizobium trifolii-catechol 1,2-dioxygenase

Catechol 1,2-dioxygenase has been purified 46-fold from cells of Rhizobium trifolii TA1 grown on benzoate plus glucose. The dioxygenase had a molecular weight of 107,000 and a sub-unit molecular weight of 59,000. The enzyme had a K _m of 2 M for catechol and also cleaved 4-methylcatechol. The dioxygenase contained 2 g atoms of Fe³⁺ per mole of enzyme which could be removed by treatment with 1,10-phenanthroline, resulting in a complete loss of activity; reactivation of the enzyme occurred specifically with Fe³⁺.     

Expression and homology modeling of 2′-aminobiphenyl-2,3-diol-1,2-dioxygenase from Pseudomonas stutzeri carbazole degradation pathway

The enzyme 2′-aminobiphenyl-2,3-diol-1,2-dioxygenase (CarB), encoded by two genes (carBa and carBb), is an α₂β₂ heterotetramer that presents meta-cleavage activity toward the hydroxylated aromatic ring in the carbazole degradation pathway from petroleum-degrader bacteria Pseudomonas spp. The 1082-base, pair polymerase chain reaction product corresponding to, carBaBb genes from Pseudomonas stutzeri ATCC 31258 was cloned by site-specific recombination and expressed in high levels in Escherichia coli BL21-SI with a histidine-tag and in native form. The CarB activity toward 2,3-dihydroxybiphenyl was similar for these two constructions. The α₂β₂ 3D model of CarB dioxygenase was proposed by homology modeling using the protocatechuate 4,5-dioxygenase (LigAB) structure as template. Accordingly, His12, His53, and Glu230 coordinate the Fe(II) in the catalytic site at the subunit CarBb. The model also indicates that His182 is the catalytic base responsible for deprotonating one of the hydroxyl group of the substrate by a hydrogen bond. The hydrophobic residues Trp257 and Phe258 in the CarB structure substituted the LigAB amino acid residues Ser269 and Asn270. These data could explain why the CarB was active for 2,3-dihydroxybiphenyl and not for protocatechuate.     

Influence of growth conditions on production of capsular and extracellular polysaccharides byRhizobium leguminosarum

The influence of growth rate and medium composition on exopolymer production byRhizobium leguminosarum was studied. When grown in medium containing 10g/l mannitol and 1g/l glutamic acid,Rhizobium leguminosarum biovartrifolii TA-1 synthesized up to 2.0g/l of extracellular polysaccharide (EPS), and up to 1.6g/l of capsular polysaccharide (CPS). Under non-growing cell conditions in medium without glutamic acid, CPS synthesis by strain TA-1 could proceed to 2.1g/l, while EPS-production remained relatively low (0.8g/l). Maximal CPS-yield was 2.9g CPS/l medium in a medium containing 20g/l mannitol and 2g/l glutamic acid. TheEPS-deficient strain R. leguminosarum RBL5515,exo4::Tn5 was able to produce CPS to similar levels as strain TA-1, but CPS-recovery was easier because of the low viscosity of the medium and growth of the cells in pellets. With strain TA-1 in nitrogen-limited continuous cultures with a constant biomass of 500mg cell protein/l, EPS was the most abundant polysaccharide present at every dilution rate D (between 0.12 and 0.02 h^–1). The production rates were 50–100mg/g protein/h for EPS and 15–20mg/g protein/h for CPS. Only low amounts of cyclic -(1,2)-glucans were excreted (10–30 mg/l) over the entire range of growth rates.Abbreviations bv biovar - CPS capsular polysaccharide - EPS extracellular polysaccharide - HM_r high molecular mass - LM_r low molecular mass - YEMCR Yeast Extract-Mannitol-Congo Red agar     

Catechol 1,2-dioxygenase from the new aromatic compounds - Degrading Pseudomonas putida strain N6

This study aimed to characterization of catechol 1,2-dioxygenase from a Gram-negative bacterium, being able to utilize a wide spectrum of aromatic substrates as a sole carbon and energy source. Strain designated as N6, was isolated from the activated sludge samples of a sewage treatment plant at Bentwood Furniture Factory Jasienica, Poland. Morphology, physio-biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicate that strain belongs to Pseudomonas putida. When cells of strain N6 grown on protocatechuate or 4-hydroxybenzoic acid mainly protocatechuate 3,4-dioxygenase was induced. The activity of catechol 1,2-dioxygenase was rather small. The cells grown on benzoic acid, catechol or phenol showed high activity of only catechol 1,2-dioxygenase. This enzyme was optimally active at 35 °C and pH 7.4. Kinetic studies showed that the value of K_m and V_max was 85.19 ??M and 14.54 ??M min⁻¹ respectively. Nucleotide sequence of gene encoding catechol 1,2-dioxygenase in strain N6 has 100% identity with catA genes from two P. putida strains. The deduced 301-residue sequence of enzyme corresponds to a protein of molecular mass 33.1 kDa. The deduced molecular structure of the catechol 1,2-dioxygenase from P. putida N6 was very similar and characteristic for the other intradiol dioxygenases.     

Osmotically-regulated trehalose accumulation and cyclic β-(1,2)-glucan excretion by Rhizobium leguminosarum biovar trifolii TA-1

Rhizobium leguminosarum biovar trifolii TA-1 produced high molecular weight extracellular (EPS) and capsular polysaccharides (CPS) as the main carbohydrate products in a medium (10 g of mannitol and 1 g of glutamic acid per liter) with low osmotic pressure of 0.20 MPa. By increasing the osmotic pressure of the medium with the addition of NaCl or other osmolytes up to 1.44 MPa, the synthesis of EPS and CPS was suppressed. Cyclic -(1,2)-glucans were excreted instead. Concentrations of over 1500 mg of glucans/l medium were produced by a biomass of 520 mg protein at 200 mM NaCl (1.20 MPa). Intracellular cyclic -(1,2)-glucan concentrations remained at 45 to 100 mg/g protein during the stationary phase, independent of the osmotic strength of the medium. Parallel to the increasing osmotic pressure of the medium, the disaccharide trehalose accumulated in the cells as osmo-protectant. Concentrations of up to 130 mg/g protein were reached. Strain TA-1 could tolerate 350 mM NaCl.Abbreviations CPS capsular polysaccharide - EPS extracellular polysaccharide - LM_r low molecular weight - HM_r high molecular weight     

Antioxidants from carbon dioxide fixing Chlorella sorokiniana

Chlorella sorokiniana H-84, which has toleranceto high temperatures and high concentrations ofCO₂, has been isolated from a hot spring inJapan. Large-scale culturing of C. sorokinianawas carried out in air containing 10% CO₂.Analysis of the biomass shows that protein, carbohydrate and lipids comprised 68.5, 11.9 and10.0% of dry matter, respectively. The totalcarotenoids comprised 0.69% dry matter. The luteinand -carotene contents were 4300 and 600 gg^-1 dry weight, respectively. The-tocopherol content was 112 g g^-1 dry weight. These carotenoids and -tocopherolare known to possess radical scavenging activity.Two fractions with radical scavenging activity wereisolated from the aqueous extract of C.sorokiniana H-84. The extract showed several singlepeaks by reversed phase HPLC analysis and two of themhad molecular weights of 710 and 1286, respectively.     

Purification and characterization of barley-aleurone xylanase

Xylanase (-1,4-D-xylan xylanohydrolase; EC 3.2.1.8) from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was purified and characterized. Purification was by preparative isoelectric focusing and a Sephadex G-200 column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight (Mr)=34000 daltons. The isoelectric point of the enzyme was 4.6. The enzyme had maximum activity on xylan at pH 5.5 and at 35° C. It was most stable between pH 5 and 6 and at temperatures between 0 and 4° C. The K_m was 0.86 mg xylan·ml^-1.Abbreviations GA₃ gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Utilization of aromatic compounds as carbon and energy sources during growth and N2-fixation by free-living nitrogen fixing bacteria

Six species of free-living nitrogen fixing bacteria, Azomonas agilis, Azospirillum brasilense, Azospirillum lipoferum, Azotobacter chroococcum, Azotobacter vinelandii, and Beijerinckia mobilis, were surveyed for their ability to grow and fix N₂ using aromatic compounds as sole carbon and energy source. All six species grew and expressed nitrogenase activity on benzoate, catechol, 4-hydroxybenzoate, naphthalene, protocatechuate, and 4-toluate. In many cases, growth rates on one or more aromatic compounds were comparable to or greater than those on the non-aromatic substrates routinely used for cultivation of the organisms. Specific activity of nitrogenase in extracts of aromatic-grown cells often exceeded that in cells grown on non-aromatic substrates. All six species growing on substrates typically converted to catechol expressed inducible catechol 1,2-dioxygenase and/or catechol 2,3-dioxygenase. When grown on substrates typically converted to protocatechuate, inducible protocatechuate 3,4-dioxygenase and/or protocatechuate 4,5-dioxygenase was expressed. A. chroococcum expressed only ortho cleavage dioxygenases during growth on naphthalene and 4-toluate and only meta cleavage dioxygenases on the other aromatics. B. mobilis expressed only ortho cleavage dioxygenases. The other four species examined expressed both ortho and meta cleavage enzymes.A preliminary account of this work was presented at the 91st General Meeting of the American Society for Microbiology, Dallas, TX, 1991     

A novel 2-aminophenol 1,6-dioxygenase involved in the degradation of p-chloronitrobenzene by Comamonas strain CNB-1: purification, properties, genetic cloning and expression in Escherichia coli

Comamonas strain CNB-1 was isolated from a biological reactor treating wastewater from a p-chloronitrobenzene production factory. Strain CNB-1 used p-chloronitrobenzene as sole source of carbon, nitrogen, and energy. A 2-aminophenol 1,6-dioxygenase was purified from cells of strain CNB-1. The purified 2-aminophenol 1,6-dioxygenase had a native molecular mass of 130 kDa and was composed of - and -subunits of 33 and 38 kDa, respectively. This enzyme is different from currently known 2-aminophenol 1,6-dioxygenases in that it: (a) has a higher affinity for 2-amino-5-chlorophenol (K_m=0.77 M) than for 2-aminophenol (K_m=0.89 M) and (b) utilized protocatechuate as a substrate. These results suggested that 2-amino-5-chlorophenol, an intermediate during p-chloronitrobenzene degradation, is the natural substrate for this enzyme. N-terminal amino acids of the - and -subunits were determined to be T-V-V-S-A-F-L-V and M-Q-G-E-I-I-A-E, respectively. A cosmid library was constructed from the total DNA of strain CNB-1 and three clones (BG-1, BG-2, and CG-13) with 2-aminophenol 1,6-dioxygenase activities were obtained. DNA sequencing of clone BG-2 revealed a 15-kb fragment that contained two ORFs, ORF9 and ORF10, with N-terminal amino acid sequences identical to those of the - and -subunits, respectively, from the purified 2-aminophenol 1,6-dioxygenase. The enzyme was actively synthesized when the genes coding for the ORF9 and ORF10 were cloned into Escherichia coli.     

Ammonium uptake by cowpea Rhizobium strain MNF 2030 and Rhizobium trifolii MNF 1001

Free-living Rhizobium trifolii MNF 1001 and cowpea Rhizobium MNF 2030 grown in chemostat culture under nitrogen limitation had high activities of an ammonium permease. In phosphate-limited, nitrogen-excess conditions, strains MNF 1001 and MNF 2030 retained 20% and 50%, respectively, of the ammonium uptake activity found in nitrogen-limited cells. Uptake in both strains was sensitive to azide, cyanide, carbonyl cyanide m-chlorophenyl hydrazone and 2,4-dinitrophenol. A gradient of ammonium concentration greater than 150-fold developed across the membrane within 20 min in cells of strain MNF 1001 grown under ammonia limitation. The pH optimum for ammonium uptake by N-limited cells of both MNF 1001 and MNF 2030 was around pH 7. The apparent K _m values for the ammonium permease in strains MNF 2030 and MNF 1001 were 3.9±1.6 M and 2.0±1.6 M respectively, and the V _max was 47±2.6 nmol min^-1 (mg protein)^-1 for MNF 2030 and 101±5.1 nmol min^-1 (mg protein)^-1 for MNF 1001. Isolated snake bean bacteroids of strain MNF 2030 capable of transporting succinate and l-glutamate had no detectable ammonium uptake activity. It therefore appears that the ammonium permeases in cells of these two strains are not as tightly regulated as in R. leguminosarum MNF 3841.Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrzone - HEPES N-Hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)^–1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52–55°C. The enzyme retained 97% activity after a 24-h incubation at 55°C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1–5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5–8 M), significantly affected phytase activity. The maximum hydrolysis rate (V_max) and apparent Michaelis-Menten constant (K_m) were 1,074 IU/mL and 606 M, respectively, with a catalytic turnover number of 3×10⁵ s^–1 and catalytic efficiency of 3.69×10⁸ M^–1 s^–1.     

Syntrophococcus sucromutans sp. nov. gen. nov. uses carbohydrates as electron donors and formate,methoxymonobenzenoids or Methanobrevibacter as electron acceptor systems

Most probable number (MPN) estimates indicated that a mean of 4.3×10⁷ and 5×10⁶ bacteria per ml of rumen fluid from a predominantly alfalfa hay-fed steer demethoxylated ferulate and syringate, respectively. After further enrichment from an MPN tube of the highest dilution showing demethoxylation of syringate, strain S195 was isolated using roll tubes with syringate as an added energy source. S195 was an anaerobic, Gram-negative, nonmotile coccus, 1 to 1.3 m in diameter, and was unique in using various carbohydrates as electron donor with acetate as the sole organic product. One of the following electron acceptor systems allowed growth (organic products in parentheses): Methanobrevibacter simithii (CH₄), formate (acetate), 3,4,5-trimethoxybenzoate and syringate (acetate and gallate), vanillate (acetate and protocatechuate), vanillin (acetate, protocatechuic aldehyde and protocatechuate), ferulate (acetate, caffeate and hydrocaffeate), caffeate (hydrocaffeate). Strain S195 required 30% (v/v) rumen fluid in the medium for good growth. S195 was placed in a new genus and species, Syntrophococcus sucromutans, of the family Veillonellaceae.Abbreviations G+C Guanine plus cytosine - MPN most probable number - OD optical density     

NADP+-dependent acetaldehyde dehydrogenase from Zymomonas mobilis

An NADP⁺-linked acetaldehyde dehydrogenase (EC 1.2.1.4) from the ethanol producing bacterium Zymomonas mobilis was purified 180-fold to homogeneity. The enzyme is a cytosolic protein with an isoelectric point of 8.0 and has an apparent molecular weight of 210000. It showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 55000, which indicates that it consists of four probably identical subunits. The apparent K _m values for the substrate acetaldehyde were 57 M and for the cosubstrate NADP⁺ 579 M. The enzyme was almost inactive with NAD⁺ as cofactor. Several other aldehydes besides acetaldehyde were accepted as a substrate but not formaldehyde or trichloroacetaldehyde. In anaerobically grown cells of Zymomonas mobilis the enzyme showed a specific activity of 0.035 U/mg protein but its specific activity could be increased up to 0.132 U/mg protein by adding acetaldehyde to the medium during the exponential growth phase or up to 0.284 U/mg protein when cells were grown under aeration. The physiological role of the enzyme is discussed.Abbreviations ALD-DH acetaldehyde dehydrogenases from Z. mobilis - DTT dithiothreitol - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - SDS sodium dodecylsulfate Dedicated to Prof. Dr. H.-G. Schlegel, Universität Göttingen, on the occasion of his 65th birthday     

Selection of Rhizobium leguminosarum strains for lentil (Lens culinaris) under growth room and field conditions

Most of the production of lentil (Lens culinaris) on the Great Plains occurs on soils that are free of indigenous Rhizobium leguminosarum. Inoculation is required to increase yields through N₂ fixation. A screening program to evaluate the effectiveness of R. leguminosarum strains for lentil was initially carried out under controlled environments followed by an evaluation under field conditions. In two separate growth room experiments, the effectiveness of 185 and 24 different strains of R. leguminosarum were tested for Laird and Eston lentil. Significant differences between strains in number of nodules, shoot weight and nitrogenase activity (acetylene reduction activity, ARA) were found for lentil grown for 5 weeks. When lentil were grown for 7 weeks, significant differences between strains in number of nodules, total plant weight, total N, and % N were observed.Fourteen strains plus Nitragin C inoculant were selected for further field testing on Eston and Laird lentil at two locations in 1986 and one site in 1987. Inoculation increased yield up to 135%. Percent Ndfa and total N₂ fixed ranged from 0 to 76 and 0 to 105 kg ha^-1, respectively. N₂-fixing activity was site specific and higher spring soil NO₃-levels resulted in lower N₂-fixing activity. Depending on site and growing conditions, strains 99A1 and I-ICAR-SYR-Le20 appeared to be superior to the other strains tested. A good agreement was found between the estimates for N₂ fixation based upon the ¹⁵N-isotope dilution and the classical N difference methods. Number of nodules, dry weight of nodules and ARA of Eston and Laird lentil grown under growth room conditions failed to show positive correlations with total dry matter production, total N or total N₂ fixed of field grown lentil. However, total plant weight and total N of lentil grown under growth room conditions were highly correlated with field parameters, and were the most reliable screening parameters for the selection of superior rhizobial strains.     

Purification and characterization of ferredoxin from Peptostreptococcus productus (strain Marburg)

Ferredoxin was purified to apparent homogeneity from cell extracts of the homoacetogen Peptostreptococcus productus (strain Marburg). The yield was 70 g ferredoxin per g wet cells of P. productus. The UV-vis spectrum exhibited characteristics of a typical clostridial ferredoxin spectrum with a molar extinction coefficient ₃₈₅ of 30000 M^-1 cm^-1 and an A₃₈₅/A₂₈₀ ratio of 0.76. The molecular weight M_r was near 5700 as calculated from the amino acid composition. The protein contained per mol 9.9 mol iron, 8.2 mol acid-labile sulfide, and near 7 mol cysteine indicating the presence of two 4 Fe/4 S clusters. The redox potential was determined to be-410 mV. The purified ferredoxin was reduced with carbon monoxide by the carbon monoxide dehydrogenase from crude extracts and by the partially enriched enzyme of P. productus.     

Phosphoenolpyruvate carboxylase from Acetobacter aceti

In Acetobacter aceti growing on pyruvate as the only source of carbon and energy, oxaloacetate (OAA) is produced by a phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31). The enzyme was purified 122-fold and a molecular weight of about 380,000 was estimated by gel filtration.The optimum pH was 7.5 and the K _m values for PEP and NaHCO₃ were 0.49 mM and about 3 mM, respectively. The enzyme needed a divalent cation; the K _m for Mn²⁺, Co²⁺ and Mg²⁺ were 0.12, 0.26 and 0.77 mM, respectively. Maximal activity was only obtained with Mg²⁺. Mn²⁺ and Co²⁺ became inhibitory at high concentrations.The activity was inhibited by succinate and, to a lesser extent, by fumarate, citrate, -ketoglutarate, aspartate and glutamate.As compared with the corresponding enzyme from A. xylinum, the PEP carboxylase of A. aceti showed the following differences: a) It had an absolute requirement for acetyl CoA (K _a 0.18 mM) or propionyl CoA (K _a 0.2 mM). b) It was not affected by ADP. c) It was sensitive to thiol blocking agents.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - MW molecular weight - TEMG buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM glutathione - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid     

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 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD⁺ specific and oxaloacetate can partially substitute for pyruvate. The K _m ^app for NAD⁺ is 14 M and 60 M at low and high NAD⁺ concentrations, respectively. The K _m ^app for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K _m ^app for NH ₄ ⁺ varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N₂-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N₂-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.Abbreviations ADH alanine dehydrogenase - DEAE diethylamino ethyl cellulose - EDTA ethylenediamine tetraacetic acid - GDH glutamic dehydrogenase - GS glutamine synthetase - GOT aspartate-glutamate aminotransferase - NAD⁺ nicotinamide adenine dinucleotide - NADH reduced nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH reduced nicotinamide adenine dinucleotide phosphate - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl) aminomethane