Zum Abbau derl-Äpfelsäure durch Milchsäurebakterien

Zusammenfassung Es wird die Aktivität von intaktenL. plantarum-Zellen verschiedener Arten gegenüberl-Äpfelsäure, Oxalessigsäure und Brenztraubensäure getestet. Bei der Dissimilation vonl-Äpfelsäure lassen sich zwei pH-Optima unterscheiden, 2,6–3,0 für eine MDH-Aktivität und 3,6–4,0 für eine Malic-Enzym-Aktivität. Stoffwechselprodukte der Brenztraubensäure-Decarboxylierung sind außer Kohlendioxid Äthylalkohol und Acetoin bzw. Diacetyl.L. plantarum ist außerdem zur Oxydation der Brenztraubensäure befähigt. Ausl-Äpfelsäure entsteht kein Acetoin (Stamm L).

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The dissimilation ofl-malic acid by lactic acid bacteriaIV. The activity of intact cells ofLactobacillus plantarum particularly considering the decarboxylation of pyruvic acid

Summary The decomposition ofl-malic, oxaloacetic and pyruvic acids by intact cells of three strains ofL. plantarum is investigated. The dissimilation ofl-malic acid shows two pH-optima, at pH2.6–3.0 for a malatedehy drogenase activity and at pH 3.6–4.0 for a malic enzyme activity. The decarboxylation of pyruvic acid yields CO₂, ethyl alcohol, acetoin and diacetyl.L. plantarum is also able to oxidize pyruvic acid. The acetoin produced byL. plantarum Strain L does not originate froml-malic acid.

     

The cytosolic pathway of l-malic acid synthesis in Saccharomyces cerevisiae: the role of fumarase

Saccharomyces cerevisiae accumulates l-malic acid but only minute amounts of fumaric acid. A ¹³C-nuclear magnetic resonance study following the label from glucose to l-malic acid indicates that the l-malic acid is synthesized from pyruvic acid via oxaloacetic acid. From this, and from previously published studies, we conclude that a cytosolic reductive pathway leading from pyruvic acid via oxaloacetic acid to l-malic acid is responsible for the l-malic acid production in yeast. The non-production of fumaric acid can be explained by the conclusion that, in the cell, cytosolic fumarase catalyzes the conversion of fumaric acid to l-malic acid but not the reverse. This conclusion is based on the following findings. (a) The cytosolic enzyme exhibits a 17-fold higher affinity towards fumaric acid than towards l-malic acid; the K _m for l-malic acid is very high indicating that l-malic acid is not an in vivo substrate of the enzyme. (b) Overexpression of cytosolic fumarase does not cause accumulation of fumaric acid (but rather more l-malic acid). (c) According to ¹³C NMR studies there is no interconversion of cytosolic l-malic and fumaric acids.     

Obtaining functional membrane vesicles from Leuconostoc oenos to study l-malate transport mechanisms

Membrane vesicles from the malolactic bacterium Leuconostoc oenos were obtained by a modified version of the procedure of Kaback [Methods Enzymol 22:99–120 (1971)]. Protoplasts were produced at frequencies greater than 95% by a method entailing mutanolysis digestion and osmotic shock. Glycerol or polyethyleneglycol 600 was required as an osmotic stabilizer while the use of sucrose prevented closed vesicle formation during osmotic shock. The membrane vesicles retained their functional properties and accumulated l-malic acid in response to an ATPase-induced proton gradient across the membrane of ATP-loaded vesicles. l-Malate uptake was strongly inhibited by dicyclohexylcarbodiimide, a specific inhibitor of membrane-bound ATPase. These data support the possibility of a pH-dependent transport of l-malate. Vesicles not loaded with ATP were slightly permeable to malic acid with an initial uptake rate (0.5 nmol·l^–1·s^–1) similar to the diffusion rate obtained previously in a L. oenos malate-transport-deficient strain. These results confirm two simultaneous uptake mechanisms in L. oenos, a permease-mediated transport and a passive diffusion for the anionic and the undissociated forms of l-malic acid respectively.     

Malic acid accumulation by Aspergillus flavus

Summary ¹³C Nuclear magnetic resonance and fumarase and NAD-malate dehydrogenase isoenzyme studies were carried out in a strain of A. flavus which produces relatively high levels of l-malic acid from glucose. The results of the ¹³C NMR showed that the ¹³C label from [1-¹³C] glucose was incorporated only to C-3 (-CH₂-) of l-malic acid and indicated that this acid must be synthesized from pyruvate mainly via oxaloacetate. Electrophoretic analysis has established the presence of unique mitochondrial and cytosolic isoenzymes for fumarase and malate dehydrogenase. Changes in the isoenzyme pattern were observed for malate dehydrogenase but not for fumarase during acid production. Cycloheximide inhibited profoundly both l-malic acid production and the increase in the major isoenzyme of malate dehydrogenase, without affecting either the total activity of fumarase or its isoenzyme pattern. The results suggested that de novo protein synthesis is involved in the increase in the activity of the major isoenzyme of malate dehydrogenase and that this isoenzyme is essential for l-malic acid production and accumulation.     

Living cell reaction process forl-isoleucine andl-valine production

Summary A new process (Living Cell Reaction Process) forl-isoleucine production using viable, non-growing cells ofBrevibacterium flavum AB-07 was optimised using ethanol as the energy source and -ketobutyric acid (-KB) as precursor.l-valine also could be produced from glucose at high yield by this process. This process differs from the usual fermentation method in that non-growing cells are used, and the production ofl-isoleucine andl-valine were carried out under conditions of repressed cell division and growth. Minimal medium missing the essential growth factor, biotin was employed as the reaction mixture for the production ofl-isoleucine andl-valine. The productivity ofl-isoleucine andl-valine were 200 mmol·l^–1 · day^–1 (molecular yield to -KB: 95%) and 300 mmol · l^–1 · day^–1 (molecular yield to glucose: 80%) respectively. The content ofl-isoleucine andl-valine in total amino acids produced in the each mixture were 97% and 96% respectively.     

Cultivation ofCandida blankii in simulated bagasse hemicellulose hydrolysate

Summary All fourCandida blankii isolates evaluated for growth in simulated bagasse hemicellulose hydrolysate utilized the sugars and acetic acid completely. The utilization ofd-xylose,l-arabinose and acetic acid were delayed by the presence ofd-glucose, but after glucose depletion the other carbon sources were utilized simultaneously. The maximum specific growth rate of 0.36 h^–1 and cell yield of 0.47 g cells/g carbon source assimilate compared with published results obtained withC. utilis. C. blankii appeared superior toC. utilis for biomass production from hemicellulose hydrolysate in that it utilizedl-arabinose and was capable of growth at higher temperatures.     

Functional characteristics ofl-glutamate,N-methyl-d-aspartate and kainate receptors in isolated brain synaptic membranes

l-Glutamic acid (l-Glu) and other excitatory amino acids and amino acid analogs enhanced [³⁵S]thiocyanate (SCN^–) uptake in isolated-resealed synaptic membrane vesicles. The SCN^– uptake was used as a measure of membrane depolarization to evaluate the characteristics of functional excitatory amino acid receptors in the synaptic membranes.N-Methyl-d-aspartate (NMDA) andl-Glu produced additive effects on SCN^– accumulation indicating the presence of distinctl-Glu and NMDA receptors. On the other hand, kainic acid (KA) andl-Glu shared either common receptor sites or ion channels. The effects of antagonists on NMDA,l-Glu, and KA stimulation of SCN^– influx were consistent with previously reported electrophysiologic observations in intact neurons.     

Multidimensional1H and15N NMR investigation of glutamine-binding protein ofEscherichia coli

Summary Specific and uniform¹⁵N labelings along with site-directed mutagenesis of glutamine-binding protein have been utilized to obtain assignments of the His¹⁵⁶, Trp³² and Trp.²²⁰ residues. These assignments have been made not only to further study the importance of these 3 amino acid residues in protein-ligand and protein-protein interactions associated with the active transport ofl-glutamine across the cytoplasmic membrane ofEscherichia coli, but also to serve as the starting points in the sequence-specific backbone assignment. The assignment of H₂ of His¹⁵⁶ refines the earlier, model where this particular proton formas an intermolecular hydrogen bond to the -carbonyl ofl-glutamine, while assignments of both Trp³² and Trp²²⁰ show the variation in local structures which ensure the specificity in ligand binding and protein-protein interaction. Using 3D NOESY-HMQC NMR, amide connectivities can be traced along 8–9 amino acid residues at a time. This paper illustrates the usefulness of combining¹⁵N isotopic labeling and multinuclear, multidimensional NMR techniques for a structural investigation of a protein with a molecular weight of 25 000.     

Production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyhexanoate) with flexible 3-hydroxyhexanoate content in<Emphasis Type="Italic"> Aeromonas hydrophila</Emphasis> CGMCC 0911

Aeromonas hydrophila CGMCC 0911 isolated from lake water was found to be able to synthesize a polyhydroxyalkanoate (PHA) copolymer (PHBHHx) consisting of 3-hydroxybutyrate (HB) and 4–6 mol% 3-hydroxyhexanoate (HHx). The wild-type bacterium accumulated 49% PHBHHx containing 6 mol% HHx in terms of cell dry weight (CDW) when grown on lauric acid for 48 h. When A. hydrophila CGMCC 0911 expressed the Acyl-CoA dehydrogenase gene (yafH) of Escherichia coli, the recombinant strain could accumulate 47% PHBHHx, while the HHx content reached 17.4 mol%. The presence of changing glucose concentration in the culture changed the HHx content both in wild type and recombinant A. hydrophila CGMCC 0911. When 5 g l^–1 glucose was added to a culture containing 5 g l^–1 lauric acid as co-substrate, 45% PHBHHx/CDW consisting of 8.8 mol% HHx was produced by wild-type A. hydrophila CGMCC 0911 compared with only 5% in the absence of glucose. When the recombinant A. hydrophila CGMCC 0911 was grown on a mixed substrate containing lauric acid and 8–10 g l^–1 glucose, the HHx content could be further increased to 35.6 mol%. When the glucose concentration exceeded 10 g l^–1, cell growth, PHA content and mole percentages of HHx in PHBHHx were significantly reduced.     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.     

The outer membrane TolC is involved in cysteine tolerance and overproduction in <Emphasis Type="Italic">Escherichia coli</Emphasis>

l-Cysteine is an important amino acid in terms of its industrial applications. We previously found marked production of l-cysteine directly from glucose in recombinant Escherichia coli cells by the combination of enhancing biosynthetic activity and weakening the degradation pathway. Further improvements in l-cysteine production are expected to use the amino acid efflux system. Here, we identified a novel gene involved in l-cysteine export using a systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection). Among the 3,985 nonessential gene mutants, tolC-disrupted cells showed hypersensitivity to l-cysteine relative to wild-type cells. Gene expression analysis revealed that the tolC gene encoding the outer membrane channel is essential for l-cysteine tolerance in E. coli cells. However, l-cysteine tolerance is not mediated by TolC-dependent drug efflux systems such as AcrA and AcrB. It also appears that other outer membrane porins including OmpA and OmpF do not participate in TolC-dependent l-cysteine tolerance. When a low-copy-number plasmid carrying the tolC gene was introduced into E. coli cells with enhanced biosynthesis, weakened degradation, and improved export of l-cysteine, the transformants exhibited more l-cysteine tolerance and production than cells carrying the vector only. We concluded that TolC plays an important role in l-cysteine tolerance probably due to its export ability and that TolC overexpression is effective for l-cysteine production in E. coli. Natthawut Wiriyathanawudhiwong and Iwao Ohtsu contributed equally to this work.     

Comparative studies of fructose 1,6-diphosphate aldolase fromEscherichia coli 518 andLactobacillus casei var.rhamnosus ATCC 7469

A comparative study has been carried out with FDP aldolases fromEscherichia coli 518 andLactobacillus casei ATCC 7469, which had been purified 17.6- and 65-fold, respectively. The aldolase ofL.casei was stable only in the presence of mercaptoethanol, whereas that ofE.coli was strongly inhibited at low (1.0×10^–4 m) and activated at high concentrations (2.0×10^–1 m) of the same compound.p-Chloromercuric benzoic acid inhibited both aldolases, with 40% inhibition at 2×10^–5 m withE.coli aldolase against at 2×10^–4 m withL.casei aldolase. Significant differences were also observed in pH optima and Km values.E.coli aldolase exhibited a maximal activity at pH 9.0 and gave a Km value of 1.76×10^–3 m FDP with strong substrate inhibition above 7×10^–3 m, against pH 6.8–7.0 and a Km of 7.04×10^–3 m FDP forL.casei aldolase. Strong resistance ofL.casei aldolase against inhibition by EDTA, Ca²⁺ and Mn²⁺ was observed compared with complete inhibition at concentrations of 20mm, 40mm and 20mm, respectively, withE. coli aldolase. Polyacrylamide gel electrophoresis did not reveal any differences between the two enzyme preparations.The differences of the properties of FDP aldolases from different bacterial genera are discussed in relation to other Class II aldolases.     

Na+-independentl-aspartate binding sites in chick brain

1. One binding component with aK _d value of 200×10^–9 M and half-life of the ligand binding component of 30 min was found. 2. Chloride ions produced a significant increase ofl-[³H]aspartate andl-[³H]glutamate binding. 3.l-Glutamate,l-ibotenate,l-quisqualate, anddl-homocysteic acid were potent inhibitors ofl-[³H]aspartate binding. 4. In all brain regions major increases of binding were observed during the third week of the in ovo period of life.     

Metabolism of d(+)-carnitine by Escherichia coli

Summary Escherichia coli 044 K74 grown under anaerobic conditions in the presence of l(–)-carnitine is able to convert d(+)-carnitine into the l(–)-enantiomer. This activity is repressed by electron acceptors such as oxygen and nitrate as well as by glucose. d(+)-Carnitine shows no effect on the induction or repression of the corresponding enzyme or enzyme system. Resting cells of E. coli 044 K74 were used for the formation of l(–)-carnitine from d(+)-carnitine. The maximum obtained yield was 50%. -Butyrobetaine was formed as a by-product. Offprint requests to: H. Jung     

Proton-linked sugar transport systems in bacteria

The cell membranes of various bacteria contain proton-linked transport systems ford-xylose,l-arabinose,d-galactose,d-glucose,l-rhamnose,l-fucose, lactose, and melibiose. The melibiose transporter ofE. coli is linked to both Na⁺ and H⁺ translocation. The substrate and inhibitor specificities of the monosaccharide transporters are described. By locating, cloning, and sequencing the genes encoding the sugar/H⁺ transporters inE. coli, the primary sequences of the transport proteins have been deduced. Those for xylose/H⁺, arabinose/H⁺, and galactose/H⁺ transport are homologous to each other. Furthermore, they are just as similar to the primary sequences of the following: glucose transport proteins found in a Cyanobacterium, yeast, alga, rat, mouse, and man; proteins for transport of galactose, lactose, or maltose in species of yeast; and to a developmentally regulated protein of Leishmania for which a function is not yet established. Some of these proteins catalyze facilitated diffusion of the sugar without cation transport. From the alignments of the homologous amino acid sequences, predictions of common structural features can be made: there are likely to be twelve membrane-spanning -helices, possibly in two groups of six, there is a central hydrophilic region, probably comprised largely of -helix; the highly conserved amino acid residues (40–50 out of 472–522 total) form discrete patterns or motifs throughout the proteins that are presumably critical for substrate recognition and the molecular mechanism of transport. Some of these features are found also in other transport proteins for citrate, tetracycline, lactose, or melibiose, the primary sequences of which are not similar to each other or to the homologous series of transporters. The glucose/Na⁺ transporter of rabbit and man is different in primary sequence to all the other sugar transporters characterized, but it is homologous to the proline/Na⁺ transporter ofE. coli, and there is evidence for its structural similarity to glucose/H⁺ transporters in Plants.In vivo andin vitro mutagenesis of the lactose/H⁺ and melibiose/Na⁺ (H⁺) transporters ofE. coli has identified individual amino acid residues alterations of which affect sugar and/or cation recognition and parameters of transport. Most of the bacterial transport proteins have been identified and the lactose/H⁺ transporter has been purified. The directions of future investigations are discussed.     

Transport of malic acid in Leuconostoc oenos strains defective in malolactic fermentation: a model to evaluate the kinetic parameters

Two Leuconostoc oenos mutant strains unable to metabolize malic acid were differentiated by [U-¹⁴C]-labelled L-malate transport assays into a malolactic-enzyme-deficient mutant and a malate-transport-defective mutant. A mathematical analysis of the data from L-malic acid uptake at three pH values (5.2, 4.5, and 3.2) in the malolactic-enzyme-deficient strains suggest two simultaneous uptake mechanisms, presumably a carrier-mediated transport and a passive diffusion for the anionic and the undissociated forms of the acid, respectively. The apparent affinity constant (K _m t) and the maximal rate (V _m t) values for L-malate active transport were, 12 mM and 43 mol L-malate·mg^–1·s^–1, respectively. Active transport was constitutive and strongly inhibited by protonophores and by ATPase inhibitors. L-Lactic acid appeared to inhibit L-malic acid transport, suggesting an L-lactate/L-malate exchange. At pH values of 4.5 or above, the passive diffusion of L-malic acid was negligible. However, at pH 3.2, the mean pH of wine, the permeability of the cells to the undissociated acid by simple diffusion could represent more than 50% of total L-malic acid uptake, with a diffusion constant (K _D) of 0.1 s^–1. Correspondence to: C. Divies     

Improvement of production of l-aspartic acid using immobilized microbial cells

Summary In our laboratory, EAPc-7 a strain having higher aspartase activity was derived from Escherichia coli ATCC 11303. For the improvement of l-aspartic acid productivity using EAPc-7 cells immobilized in -carrageenan, it was necessary to eliminate the fumarase activity which converts fumaric acid to l-malic acid. Several treatments for specifically eliminating fumarase activity from EAPc-7 cells were tested and it was found that when EAPc-7 cells were treated in a culture broth (pH 4.9) containing 50 mM l-aspartic acid at 45° C for 1 h, fumarase activity was almost completely eliminated without inactivation of the aspartase.The treated cells, immobilized in -carrageenan, were used for continuous production of l-aspartic acid from ammonium fumarate. The formation of l-malic acid was negligible and the half-life of the immobilized preparation was 126 days.Productivity of immobilized preparation of treated EAPc-7 cells in l-aspartic acid production was six times of that of the parent cell preparation.     

Evaluation ofl-lactic acid production in batch, fed-batch, and continuous cultures ofRhizopus sp. MK-96-1196 using an airlift bioreactor

Various processes which producel-lactic acid using ammonia-tolerant mutant strain,Rhizopus sp. MK-96-1196, in a 3 L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose concentration at 30 g/l, more than 140 g/l ofl-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200 g/l of initial glucose concentration, 121 g/L ofl-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture, 1.5 g/l/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of 0.024 h⁻¹. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam, and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively, compared to that of batch culture. The fed-batch culture with highl-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production ofl-lactic acid.     

Fed-batch production of <Emphasis Type="SmallCaps">d</Emphasis>-ribose from sugar mixtures by transketolase-deficient <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SPK1

d-Ribose, a five-carbon sugar, is used as a key intermediate for the production of various biomaterials, such as riboflavin and inosine monophosphate. A high d-ribose-producing Bacillus subtilis SPK1 strain was constructed by the chemical mutation of the transketolase-deficient strain, B. subtilis JY1. Batch fermentation of B. subtilis SPK1 with 20 g l^–1 xylose and 20 g l^–1 glucose resulted in 4.78 g l^–1 dry cell mass, 23.0 g l^–1d-ribose concentration, and 0.72 g l^–1 h^–1 productivity, corresponding to a 1.5- to 1.7-fold increase when compared with values for the parental strain. A late-exponential phase was chosen as the best point for switching to a fed-batch process. Optimized fed-batch fermentation of B. subtilis SPK1, feeding a mixture of 200 g l^–1 xylose and 50 g l^–1 glucose after the late-exponential phase reduced the residual xylose and glucose concentrations to less than 7.0 g l^–1 and gave the best results of 46.6 g l^–1d-ribose concentration and 0.88 g l^–1 h^–1 productivity which were 2.0- and 1.2-fold higher than the corresponding values in a simple batch fermentation.     

A high cell density fermentation strategy to produce recombinant malarial antigen in <Emphasis Type="Italic">E. coli</Emphasis>

A high cell density cultivation method was developed to produce recombinant PvRII, a malaria vaccine candidate, in E. coli for use in vaccine studies. Cells were grown in completely defined media and glucose was fed to achieve a specific growth rate of 0.12 h^–1 until cells reached 55 g dry wt l^–1. Culture was then induced with 1 mm IPTG and cells were further grown for 4 h to reach 85 g dry wt l^–1 at 0.1 h^–1. Recombinant PvRII was purified from inclusion bodies under denaturing conditions using metal affinity chromatography which yielded 10 mg PvRII g^–1 dry wt. After refolding, PvRII was greater than 98% pure, homogeneous and functionally active in that it specifically bound Duffy positive human red cells.Revisions requested 21 September 2004; Revisions received 29 October 2004