Immobilization of multienzymes and cofactors within lipid-polyamide membrane microcapsules for the multistep conversion of lipophilic and lipophobic substrates

Cofactors cannot be retained within polyamide membrane microcapsules unless the cofactors have been covalently linked to macromolecules. In this paper, a new approach using lipid-polyamide membrane microcapsules has resulted in the retention of unmodified cofactors. Lipid-polyamide microcapsules can be made to contain urease (urea amidohydrolase, EC 3.5.1.5), glutamate dehydrogenase (NAD(P)⁺) [l-glutamate: NAD(P)⁺ oxidoreductase (deaminating), EC 1.4.1.3], alcohol dehydrogenase (alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1), NAD⁺, NADH and α-ketoglutarate. Lipophilic substrates like ammonia can equilibrate rapidly into the microcapsules. The rate of conversion of ammonia into glutamate was studied. NAD⁺ retained in the microcapsules was effectively recycled into NADH and 0.25 μmol NAD⁺ converted 10 μmol ammonia into glutamate. Without cofactor recycling, 10 μmol NADH had to be microencapsulated to convert the same amount of ammonia into glutamate. By adjusting the ratio of cholesterol and lecithin in the lipid component of the membrane, it was also possible to achieve a good urea-permeable membrane without any leakage of cofactor or α-ketoglutarate. This way urea permeated through the lipid-polyamide membrane microcapsules was sequentially converted into ammonia and then glutamate.     

Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum.

Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways.     

Identification of two major ammonia-releasing reactions involved in secondary natto fermentation

Natto is a traditional Japanese food made from soybeans fermented by strains of Bacillus subtilis natto. It gives off a strong ammonia smell during secondary fermentation, and the biochemical basis for this ammonia production was investigated in this study. When natto was fermented by strain r22, ammonia production was shown to involve degradation of soybean proteins releasing amino acids, and only the glutamate contained in the natto obviously decreased, while the other amino acids increased during secondary fermentation. Strain r22 has two active glutamate dehydrogenase genes, rocG and gudB, and inactivating both genes reduced ammonia production by half, indicating that deamination of glutamate was one of the major ammonia-releasing reactions. In addition, urease encoded by ureABC was found to degrade urea during secondary fermentation. A triple mutant lacking rocG, gudB, and ureC exhibited minimal ammonia production, suggesting that the degradation of urea might be a further ammonia-releasing reaction.     

Hyperanodic forms of human glucose-6-phosphate dehydrogenase.

Pure glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) is transformed into 'hyperanodic forms' when incubated at acidic pH and in the presence of NADP+ with excess of glucose-6-phosphate or with some 'NADP+ modifying proteins' purified from the same cells. The enzyme hyperanodic forms exhibit low isoelectric point, altered kinetic properties and high lability to heat, urea, and proteolysis. Differences between hyperanodic and native forms of glucose-6-phosphate dehydrogenase are also noted by microcomplement fixation analysis, ultraviolet absorbance difference spectrum and fluorescence emission spectrum. Drastic denaturation of the enzyme by urea and acid treatment did not suppress the difference of isoelectric point between native and hyperanodic forms of glucose-6-phosphate dehydrogenase. From our data we suggest that the conversion into hyperanodic forms could be due to the covalent binding on the enzyme of a degradation product of the pyridine nucleotide coenzyme. This modification could constitute a physiological transient step toward the definitive degradation of the enzyme.     

Human erythrocyte glucose-6-phosphate dehydrogenase. Identification of a reactive lysyl residue labelled with pyridoxal 5'-phosphate

Human erythrocyte glucose-6-phosphate dehydrogenase contains a reactive lysyl residue, which can be labelled with pyridoxal 5'-phosphate. The binding of one mole of pyridoxal 5'-phosphate per mole of enzyme subunit produces substantial inactivation. The substrate glucose-6-phosphate prevents the loss of activity, suggesting that the reaction site is close to the substrate-binding site. A tryptic peptide containing the pyridoxal-5'-phosphate-binding lysyl residue has been isolated and characterised. The reactive lysyl residue has been identified in the glucose-6-phosphate dehydrogenase amino acid sequence. Comparison with glucose-6-phosphate dehydrogenase from other sources shows a high homology with a peptide containing a reactive lysyl residue, isolated from the enzyme from Saccharomyces cerevisiae; glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides also contains a region highly homologous with the sequence around the reactive lysyl residue in the human enzyme. The results of this communication provide the first direct evidence for the association of an essential catalytic function with a specific region of the molecule of human erythrocyte glucose-6-phosphate dehydrogenase.     

Interaction of an Antinucleating Chemical and an Ice Nucleation Active Bacterium: a Case Study with an n-Octylbenzyldimethyl-ammonium Salt and Erwinia ananas

Natto is a traditional Japanese food made from soybeans fermented by strains of Bacillus subtilis natto. It gives off a strong ammonia smell during secondary fermentation, and the biochemical basis for this ammonia production was investigated in this study. When natto was fermented by strain r22, ammonia production was shown to involve degradation of soybean proteins releasing amino acids, and only the glutamate contained in the natto obviously decreased, while the other amino acids increased during secondary fermentation. Strain r22 has two active glutamate dehydrogenase genes, rocG and gudB, and inactivating both genes reduced ammonia production by half, indicating that deamination of glutamate was one of the major ammonia-releasing reactions. In addition, urease encoded by ureABC was found to degrade urea during secondary fermentation. A triple mutant lacking rocG, gudB, and ureC exhibited minimal ammonia production, suggesting that the degradation of urea might be a further ammonia-releasing reaction.     

Conversion of ammonia or urea into essential amino acids, L-leucine, L-valine, and L-isoleucine, using artificial cells containing an immobilized multienzyme system and dextran-NAD+. 2. Yeast alcohol dehydrogenase for coenzyme recycling.

Semipermeable nylon-polyethylenimine artificial cells containing leucine dehydrogenase (EC 1.4.1.9), alcohol dehydrogenase (EC 1.1.1.1), urease (EC 3.5.1.5), and dextran-NAD+ were prepared. Artificial cells could convert ammonia or urea into L-leucine, L-valine, and L-isoleucine. For batch conversion in 20.0 mM of ammonium acetate substrate solutions, in 2 h 0.2 ml of artificial cells could produce 4.48 mumol of L-leucine, 9.98 mumol of L-valine, or 5.96 mumol of L-isoleucine. The corresponding conversion ratios were 22.4, 49.9, and 29.8%. In 20.0 mM of urea substrate solutions, 13.71 mumol of L-leucine, 16.12 mumol of L-valine, or 13.44 mumol of L-isoleucine was produced and the conversion ratios were 68.6, 80.6, and 67.2%. The substrate specificity of leucine dehydrogenase for the reductive amination was determined. Of the three branched-chain amino acids produced, the production rates of L-valine were the highest. The apparent Km values were as follows: 0.32 mM for alpha-ketoisocaproate, 1.63 mM for alpha-ketoisovalerate, and 0.73 mM for Dl-alpha-keto-beta-methyl-n-valerate. The leucine dehydrogenase multienzyme system had a good storage stability. It retained 72.0% of the original activity with artificial cells were stored at 4 degrees C for 6 weeks. The optimum conversion pH and temperature were 8.5-9.0 and 35-40 degrees C. The effects of urea and ammonium salts on conversion rate were also studied. The relative activities in ammonium salts solutions were 45.1-75.9% of those in urea solutions.     

Interaction between glutamate dehydrogenase (GDH) and L-leucine catabolic enzymes: intersecting metabolic pathways

Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5′-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5′-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations.     

Enantioselective synthesis of various D-amino acids by a multi-enzyme system

We have developed an effective method for the synthesis of various D-amino acids from the corresponding α-keto acids and ammonia by coupling four enzyme reactions catalyzed by D-amino acid aminotransferase, glutamate racemase, glutamate dehydrogenase, and formate dehydrogenase. In this system, D-glutamate is continuously regenerated from α-ketoglutarate, ammonia and NADH by the coupled reaction of glutamate dehydrogenase and glutamate racemase, and used as an amino donor for the enantioselective D-amino acid synthesis by the D-amino acid aminotransferase reaction. The unidirectional formate dehydrogenase reaction is also coupled to regenerate NADH consumed. Under the optimum conditions, D-enantiomers of valine, alanine, α-keto analogues with a molar yield higher than 80%.     

Nitrate assimilation in Crotalaria juncea (Linn.) pollen suspension culture

In vivo and in vitro activities of nitrate reductase were assayedin Crotalaria juncea pollen suspension cultures. This enzymewas found to be substrate-inducible and enhanced activity wasobserved when it was extracted with cysteine buffer or incubatedwith NADH (0.6 mM) at 25?C or when the germinated pollen grainswere treated with red light for 10 min. Enzymes of ammonia assimilation,glutamate dehydrogenase and glutamate synthetase, and also thepentose phosphate-shunt enzyme, glucose-6-phosphate dehydrogenase,which catalyzes the step that provides reducing power to thesystem, are described. (Received October 20, 1977; )     

13C and 31P NMR studies on sugar metabolism in Bombyx mori and Philosamia cynthia ricini larvae

Studies were made on ¹³C and ³¹P NMR in larvae of two species of silkworm, Bombyx mori and Philosamia cynthia ricini, in vivo as well as in vitro to determine the pathways of glucose utilization, especially those to amino acids as components of silk fibroin. Results showed that the ¹³C of [1-¹³C]glucose administered orally into 5th instar larvae of both species was incorporated into glucose-1-phosphate, glucose-6-phosphate and trehalose. Serine, glutamate, glutamine, citrate, malate, trehalose and sorbitol-6-phosphate were detected in the hemolymphs of these larvae as metabolites of [1-¹³C]glucose. Two days after [1-¹³C]glucose administration, labeled alanine, glycine, serine, urea, glycogen, trehalose and glycerol were clearly detected in Bombyx larvae. Starvation caused rapid consumption of administered [1-¹³C]glucose with very little accumulation of ¹³C in glycogen or trehalose. In the in vivo³¹P NMR spectra of Bombyx larvae, ATP, arginine phosphate, sorbitol-6-phosphate, uridine diphosphoglucose, phosphoenolpyruvate and inorganic phosphate were detected with some sugar phosphates, such as glucose-1-phosphate and glucose-6-phosphate. During starvation, the intensity of the signal of inorganic phosphate increased and those of sugar phosphate other than sorbitol-6-phosphate decreased, but these changes were reversed by oral administration of glucose.     

Production of aromatic d-amino acids from α-keto acids and ammonia by coupling of four enzyme reactions

A multi-enzyme system composed of glutamate racemase, thermostable d-amino acid aminotransferase, glutamate dehydrogenase and formate dehydrogenase was employed for the production of aromatic d-amino acids, d-phenylalanine and d-tyrosine, from the corresponding α-keto acids, phenylpyruvate and hydroxyphenylpyruvate, respectively. The optimal concentration of ammonium formate for the production of these d-amino acids was found in the range of 0.25–1.0 M. The optimal concentration of α-keto acid was determined to be 50 mM, above which the productivity greatly decreased. To keep the concentration of α-keto acid around this concentration, α-keto acid was intermittently fed into the multi-enzyme system during the production period. By running the multi-enzyme system for 35 h, 48 g l⁻¹ of d-phenylalanine and 60 g l⁻¹ of d-tyrosine were produced with 100% of optical purity from the equimolar amounts of phenylpyruvate and hydroxyphenylpyruvate, respectively. The production levels of both aromatic d-amino acids were demonstrated to be dependent on the stability of glutamate racemase.     

Carbon metabolism of chloroplasts in the dark: Oxidative pentose phosphate cycle versus glycolytic pathway

The conversion of U-labelled [¹⁴C]glucose-6-phosphate into other products by a soluble fraction of lysed spinach chloroplasts has been studied. It was found that both an oxidative pentose phosphate cycle and a glycolytic reaction sequence occur in this fraction. The formation of bisphosphates and of triose phosphates was ATP-dependent and occurred mainly via a glycolytic reaction sequence including a phosphofructokinase step. The conversion, of glucose-6-phosphate via the oxidative pentose phosphate cycle stopped with the formation of pentose monophosphates. This was found not to be because of a lack in transaldolase (or transketolase) activity, but because of the high concentration ratios of hexose monophosphate/pentose monophosphate used in our experiments for simulating the conditions in whole chloroplasts in the dark. Some regulatory properties of both the oxidative pentose phosphate cycle and of the glycolytic pathway were studied.Abbreviations DHAP dihydroxyacetone phosphate - GAP 3-phosphoglyceraldehyde - PGA 3-phosphoglycerate - HMP hexose monophosphates - including F6P fructose-6-phosphate - G6P glucose-6-phosphate - GIP glucose-1-phosphate - 6-PGL phosphogluconate - PMP pentose monophosphates - including R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate - X5P xylulose-5-phosphate - E4P erythrose-4-phosphate - S7P sedoheptulose-7-phosphate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

Kinetic properties of enzymes in particular of yeast alcohol dehydrogenase following their adsorption on polyaminomethylstyrene]

The adsorption of 8 enzymes to polyaminomethylstyrene was studied. While lactate dehydrogenase, alkaline phosphatase and glucose-6-phosphate dehydrogenase exhibit a relatively low affinity to the carrier, alcohol dehydrogenase, glutamate dehydrogenase and urease were found to form stabile complexes with the polymer that are enzymatically active. Adsorbed urease and beta-hydroxybutyrate dehydrogenase, are still active after several weeks; the other preparations lose their activity soon. It can be shown by the example of yeast alcohol dehydrogenase that the activity loss following adsorption is caused possibly by a process of reorientation of already bound enzyme molecules or by the increasing enzyme coverage of the carrier, with the active centres becoming more and more inaccessible for the substrate. During the substrate conversion catalysed by the alcohol dehydrogenase-polyaminomethylstyrene complex, a small amount of the enzyme is again detached from the carrier. The activity rises to a certain extent in the supernatant but drops to zero again. The stability of the adsorbed urease is distinctly increased compared with the dissolved enzyme. For the pH optimum and the KM value there are no differences between the two preparations. Continuous application of polyaminomethylstyrene-bound beta-hydroxybutyrate dehydrogenase and urease, respectively, in a column shows that both preparations have unchanged enzymatic activities even after running times of 5 and 24 days, respectively.     

Helicobacter pylori utilises urea for amino acid synthesis

Abstract Helicobacter pylori has one of the highest urease activities of all known bacteria. Its enzymatic production of ammonia protects the organism from acid damage by gastric juice. The possibility that the urease activity allows the bacterium to utilise urea as a nitrogen source for the synthesis of amino acids was investigated. H. pylori (NCTC 11638) was incubated with 50 mM urea, enriched to 5 atom% excess ¹⁵N, that is the excess enrichment of ¹⁵N above the normal background, in the presence of either NaCl pH 6.0, or 0.2M citrate pH 6.0. E. coli (NCTC 9001) was used as a urease-negative control. ¹⁵N enrichment was detected by isotope ratio mass spectrometry. H. pylori showed intracellular incorporation of ¹⁵N in the presence of citrate buffer pH 6.0 but there was no significant incorporation of ¹⁵N in unbuffered saline or by E. coli in either pH 6.0 citrate buffer or unbuffered saline. The intracellular fate of the urea-nitrogen was determined by means of gas chromatography/mass spectrometry following incubation with ¹⁵N enriched 5 mM urea in the presence of either 0.2 M citrate buffer pH 6.0 or 0.2 M acetate buffer pH 6.0. After 5 min incubation in either buffer the ¹⁵n label appeared in glutamate, glutamine, phenylalanine, aspartate and alanine. It appears, therefore, that at pH and urea concentrations typical of the gastric mucosal surface, H. pylori utilises exogenous urea as a nitrogen source for amino acid synthesis. The ammonia produced by H. pylori urease activity thus facilitates the organism's nitrogen metabolism at neutral pH as well as protecting it from acid damage at low pH.     

Les principales voies enzymatiques de l'assimilation de l'azote chez les microalgues eucaryotes

Nitrogen utilization by eukaryotic microalgae involves 1) the uptake of the nutrient nitrogen, 2) the intracellular processing of the nitrogen to convert it to ammonia if necessary, 3) the assimilation of the ammonia into small organic molecules (amino acids) and 4) the synthesis of larger organic macromolecules. The ammonia is assimilated into the intracellular amino acid pool of eukaryotic microalgae primarily by either the GS/GOGAT pathway or the reaction of GDH. Here we review our current knowledge on the characteristics of these enzymes and on their putative functions in ammonia assimilation. It seems to be generally accepted that the GS/GOGAT pathway is the main pathway for the ammonia assimilation in eukaryotic microalgae. There is, however, evidence for the role of GDH in ammonia assimilation in some species. In other species the role of this enzyme is thought to be in catalysing the conversion of glutamate to ammonia. The distribution of the isoforms of glutamate dehydrogenase that use NADH or 97 NADPH as reductant make any generalisation difficult.     

Methanol metabolism and ammonia assimilation in four methylophilns strains

Methanol assimilation and dissimilation pathways and ammonia assimilation pathway were investigated in four obligate methanol-utilizing bacteria through the detection of key enzymes. Both hexulose phosphate synthetase and hexulose phosphate isomerase, key enzymes of the ribulose monophosphate pathway (RMP) for methanol assimilation were detected whereas four key enzymes (hydroxy pyruvate reductase, isocitrate lyase, malyl-CoA-lyase and glyoxylate aminotransferase) that are characteristic of the serine assimilation pathway were absent. Key enzymes for the two methanol dissimilation pathways, the linear sequence enzymes formaldehyde and formate dehydrogenase and the RMP cyclic sequence enzymes glucose-6-phosphate dehydrogenase and 6-phosphate giuconate dehydrogenase were all detected. Ammonia was assimilated via the glutamate dehydrogenase pathway and not via the glutamine synthetase and glutamate synthase pathway.     

The Role of Glutamine Synthetase and Glutamate Dehydrogenase in Cerebral Ammonia Homeostasis

In the brain, glutamine synthetase (GS), which is located predominantly in astrocytes, is largely responsible for the removal of both blood-derived and metabolically generated ammonia. Thus, studies with [¹³N]ammonia have shown that about 25?% of blood-derived ammonia is removed in a single pass through the rat brain and that this ammonia is incorporated primarily into glutamine (amide) in astrocytes. Major pathways for cerebral ammonia generation include the glutaminase reaction and the glutamate dehydrogenase (GDH) reaction. The equilibrium position of the GDH-catalyzed reaction in vitro favors reductive amination of α-ketoglutarate at pH 7.4. Nevertheless, only a small amount of label derived from [¹³N]ammonia in rat brain is incorporated into glutamate and the α-amine of glutamine in vivo. Most likely the cerebral GDH reaction is drawn normally in the direction of glutamate oxidation (ammonia production) by rapid removal of ammonia as glutamine. Linkage of glutamate/α-ketoglutarate-utilizing aminotransferases with the GDH reaction channels excess amino acid nitrogen toward ammonia for glutamine synthesis. At high ammonia levels and/or when GS is inhibited the GDH reaction coupled with glutamate/α-ketoglutarate-linked aminotransferases may, however, promote the flow of ammonia nitrogen toward synthesis of amino acids. Preliminary evidence suggests an important role for the purine nucleotide cycle (PNC) as an additional source of ammonia in neurons (Net reaction: l-Aspartate?+?GTP?+?H₂O?→?Fumarate?+?GDP?+?P_i?+?NH₃) and in the beat cycle of ependyma cilia. The link of the PNC to aminotransferases and GDH/GS and its role in cerebral nitrogen metabolism under both normal and pathological (e.g. hyperammonemic encephalopathy) conditions should be a productive area for future research.     

Levels of glucose dehydrogenases with different substrate specificities in rat and beef livers

The relative substrate specificities of glucose dehydrogenases (E.C. 1.1.1.47) from beef liver and rat liver are very different. The beef enzyme oxidizes glucose more rapidly than either glucose-6-phosphate or galactose-6-phosphate. On the other hand, the dehydrogenase from rat liver prefers the hexose phosphates to glucose.A procedure for estimating the level of glucose dehydrogenase in rat and beef liver is described. The glucose-6-phosphate dehydrogenase activity attributed to glucose dehydrogenases is estimated to be about one-fifth and one-third that of cytoplasmic glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) in female and male rat liver respectively.A fluorometric adaptation of the less sensitive spectrophotometric assay for glucose dehydrogenase is described.