Glutamate Fermentation Research Articles

A glutamate mutase is involved in the biosynthesis of the lipopeptide antibiotic friulimicin in Actinoplanes friuliensis.

Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin. This antibiotic is active against gram-positive bacteria such as multiresistant Enterococcus and Staphylococcus strains. It consists of 10 amino acids that form a ring structure and 1 exocyclic amino acid to which an acyl residue is attached. By a reverse genetic approach, biosynthetic genes were identified that are required for the nonribosomal synthesis of the antibiotic. In close proximity two genes (glmA and glmB) were found which are involved in the production of methylaspartate, one of the amino acids of the peptide core. Methylaspartate is synthesized by a glutamate mutase mechanism, which was up to now only described for glutamate fermentation in Clostridium sp. or members of the family ENTEROBACTERIACEAE: The active enzyme consists of two subunits, and the corresponding genes overlap each other. To demonstrate enzyme activity in a heterologous host, it was necessary to genetically fuse glmA and glmB. The resulting gene was overexpressed in Streptomyces lividans, and the fusion protein was purified in an active form. For gene disruption mutagenesis, a host-vector system was established which enables genetic manipulation of Actinoplanes spp. for the first time. Thus, targeted inactivation of biosynthetic genes was possible, and their involvement in friulimicin biosynthesis was demonstrated.

Structure of the mammalian CoA transferase from pig heart.

Ketoacidosis affects patients who are deficient in the enzyme activity of succinyl-CoA:3-ketoacid CoA transferase (SCOT), since SCOT catalyses the activation of acetoacetate in the metabolism of ketone bodies. Thus far, structure/function analysis of the mammalian enzyme has been predicted based on the three-dimensional structure of a CoA transferase determined from an anaerobic bacterium that utilizes its enzyme for glutamate fermentation. To better interpret clinical data, we have determined the structure of a mammalian CoA transferase from pig heart by X-ray crystallography to 2.5 A resolution. Instrumental to the structure determination were selenomethionine substitution and the use of argon during purification and crystallization. Although pig heart SCOT adopts an alpha/beta protein fold, resembling the overall fold of the bacterial CoA transferase, several loops near the active site of pig heart SCOT follow different paths than the corresponding loops in the bacterial enzyme, accounting for differences in substrate specificities. Two missense mutations found associated with SCOT of ketoacidosis patients were mapped to a location in the structure that might disrupt the stabilization of the amino-terminal strand and thereby interfere with the proper folding of the protein into a functional enzyme.

POLYELECTROLYTE CONDITIONING FOR IRON-HYDROXIDE-CONTAINING SLUDGE PRODUCED FROM ELECTROFLOCCULATION OF FERMENTATION WASTEWATER

ABSTRACT The purpose of this study is to investigate the application of chemical conditioning method for the post-electroflocculation wastewater sludge treatment with polyelectrolytes of varying ionic characters, namely anionic, cationic and nonionic flocculating agents. Electroflocculation, a combination of the processes of electroflotation and electroprecipitation, was found effective to remove the organic and color components in monosodium glutamate fermentation wastewater. The settleability and dewaterability of the separated sludge, containing 15–20% of iron hydroxides, increased 35% and 60% due to polymer conditioning through a jar-test apparatus. The influence of shear conditions and polymer dose on the floc size, zeta potential, filterability and dewatering performance, and shear resistance of sludges was investigated. Experimental results showed that the anionic polymer acts as a better flocculating agent than both cationic and nonionic polymers, and the optimal polymer doses fall between 4.0 and 10.0 mg/g Fe sludge under corresponding shear rates of 200–300 rpm. This study also indicated that both charge neutralization and polymer bridging mechanisms operate in the sludge conditioning process.

Identification of the Omega4514 regulatory region, a developmental promoter of Myxococcus xanthus that is transcribed in vitro by the major vegetative RNA polymerase.

Omega4514 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. DNA upstream of the insertion site was cloned, and the promoter was localized. The promoter resembles vegetative promoters in sequence, and sigma(A) RNA polymerase, the major form of RNA polymerase in growing M. xanthus, initiated transcription from this promoter in vitro. Two complete open reading frames were identified downstream of the promoter and before the Omega4514 insertion. The first gene product (ORF1) has a putative helix-turn-helix DNA-binding motif and shows sequence similarity to transcriptional regulators. ORF2 is most similar to subunit A of glutaconate coenzyme A (CoA) transferase, which is involved in glutamate fermentation. Tn5 lac Omega4514 is inserted in the third codon of ORF3, which is similar to subunit B of glutaconate CoA-transferase. An orf1 disruption mutant exhibited a mild sporulation defect, whereas neither a disruption of orf2 nor insertion Omega4514 in orf3 caused a defect. Based on DNA sequence analysis, the three genes are likely to be cotranscribed with a fourth gene whose product is similar to alcohol dehydrogenases. ORF1 delays and reduces expression of the operon during development, but relief from this negative autoregulation does not fully explain the regulation of the operon, because expression from a small promoter-containing fragment is strongly induced during development of an orf1 mutant. Also, multiple upstream DNA elements are necessary for full developmental expression. These results suggest that transcriptional activation also regulates the operon. Omega4514 is the first example of a developmentally regulated M. xanthus operon that is transcribed by the major vegetative RNA polymerase, and its regulation appears to involve both negative autoregulation by ORF1 and positive regulation by one or more transcriptional activators.

Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586.

We present a complete DNA sequence and metabolic analysis of the dominant oral bacterium Fusobacterium nucleatum. Although not considered a major dental pathogen on its own, this anaerobe facilitates the aggregation and establishment of several other species including the dental pathogens Porphyromonas gingivalis and Bacteroides forsythus. The F. nucleatum strain ATCC 25586 genome was assembled from shotgun sequences and analyzed using the ERGO bioinformatics suite (http://www.integratedgenomics.com). The genome contains 2.17 Mb encoding 2,067 open reading frames, organized on a single circular chromosome with 27% GC content. Despite its taxonomic position among the gram-negative bacteria, several features of its core metabolism are similar to that of gram-positive Clostridium spp., Enterococcus spp., and Lactococcus spp. The genome analysis has revealed several key aspects of the pathways of organic acid, amino acid, carbohydrate, and lipid metabolism. Nine very-high-molecular-weight outer membrane proteins are predicted from the sequence, none of which has been reported in the literature. More than 137 transporters for the uptake of a variety of substrates such as peptides, sugars, metal ions, and cofactors have been identified. Biosynthetic pathways exist for only three amino acids: glutamate, aspartate, and asparagine. The remaining amino acids are imported as such or as di- or oligopeptides that are subsequently degraded in the cytoplasm. A principal source of energy appears to be the fermentation of glutamate to butyrate. Additionally, desulfuration of cysteine and methionine yields ammonia, H(2)S, methyl mercaptan, and butyrate, which are capable of arresting fibroblast growth, thus preventing wound healing and aiding penetration of the gingival epithelium. The metabolic capabilities of F. nucleatum revealed by its genome are therefore consistent with its specialized niche in the mouth.

Unusual enzymes involved in five pathways of glutamate fermentation.

Anaerobic bacteria from the orders Clostridiales and Fusobacteriales are able to ferment glutamate by at least five different pathways, most of which contain enzymes with radicals in their catalytic pathways. The first two pathways proceed to ammonia, acetate and pyruvate via the coenzyme B12-dependent glutamate mutase, which catalyses the re-arrangement of the linear carbon skeleton to that of the branched-chain amino acid (2S,3S)-3-methylaspartate. Pyruvate then disproportionates either to CO2 and butyrate or to CO2, acetate and propionate. In the third pathway, glutamate again is converted to ammonia, CO2, acetate and butyrate. The key intermediate is (R)-2-hydroxyglutaryl-CoA, which is dehydrated to glutaconyl-CoA, followed by decarboxylation to crotonyl-CoA. The unusual dehydratase, containing an iron-sulfur cluster, is activated by an ATP-dependent one-electron reduction. The remaining two pathways require more then one organism for the complete catabolism of glutamate to short chain fatty acids. Decarboxylation of glutamate leads to 4-aminobutyrate, which is fermented by a second organism via the fourth pathway to acetate and butyrate, again mediated by an unusual dehydratase which catalyses the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The fifth pathway is the only one without decarboxylation, since the gamma-carboxylate of glutamate is reduced to the amino group of delta-aminovalerate, which then is fermented to acetate, propionate and valerate. The pathway involves the oxidative dehydration of 5-hydroxyvaleryl-CoA to 2,4-pentadienoyl-CoA followed by reduction to 3-pentenoyl-CoA and isomerisation to 2-pentenoyl-CoA.

The ecology of non-starter lactic acid bacteria (NSLAB) and their use as adjuncts in New Zealand Cheddar

This paper reviews the recent New Zealand studies on non-starter lactic acid bacteria (NSLAB) in Cheddar cheese. In New Zealand, cheese adventitious NSLAB were usually strains of homofermentative lactobacilli and occasionally pediococci and heterofermentative lactobacilli. In Cheddar cheese, manufactured in six factories at different times and ripened over 24 months, over 140 NSLAB strains were identified using pulse field gel electrophoresis profiles and fermentation patterns on 22 carbohydrates. The majority of NSLAB isolates identified belong to two species: Lactobacillus paracasei subsp. paracasei and Lactobacillus rhamnosus. The composition of the NSLAB strains in a cheese was variable and dependent on the manufacturing factory, the date of manufacture and the age of the cheese. In most cheeses, no single NSLAB strain predominated during ripening. Up to six strains formed the majority (about 90%) of the NSLAB population, the remainder was represented by up to 15 other strains. In only about 10% of the cheeses investigated was one strain predominant throughout the 24 month ripening period. The acid and salt tolerance, effect of temperature, ability to produce biogenic amines and the lipolytic and proteolytic activities of the NSLAB strains were studied to screen strains for use as adjuncts. Sixty suitable strains were selected for further screening in a cheese model system. The NSLAB were incubated at 30°C for 10 days in the model system and the flavour and biochemical changes were assessed. Twenty-three strains were eliminated as potential adjuncts because they produced defective flavours and a further 13 strains were eliminated on the basis of undesirable biochemical properties (e.g. excess fermentation of glutamate, lactate racemisation). The 24 remaining NSLAB strains that had acceptable microbiological biochemical and flavour properties were used as adjuncts in Cheddar trials. Seven of the 24 adjunct combinations studied (made up from 2–4 strains of NSLAB) provided consistent flavour benefits. Analysis of the cheeses during ripening in these trials provided significant understanding of the ripening mechanisms of the NSLAB adjuncts.

Importance of Phosphoenolpyruvate Carboxylase of Corynebacterium glutamicum during the Temperature Triggered Glutamic Acid Fermentation

To give clues about the respective importance of phosphoenolpyruvate carboxylase (PEPc) and pyruvate carboxylase (Pc) in Corynebacterium glutamicum metabolism during a temperature triggered glutamic acid fermentation, PEPc activity was genetically amplified and Pc activity was suppressed by biotin limitation in the culture medium. In absence of Pc activity, glutamate production was dramatically reduced whereas lactate excretion was strongly increased. Whereas PEPc amplification in excess of biotin (4mg/L) only slightly modified the cell kinetics, under biotin limiting conditions this amplification strongly improved the glutamate production (4μg/L). When Pc was absent, PEPc activity was sufficient to allow up to 70% of the maximal glutamate production rate and seemed to have an important anaplerotic role, especially at the beginning of the production phase. In contrast, Pc was predominant during the remainder of the glutamate fermentation.

Natural and Artificial Functionalized Biopolyesters I. Chemoenzymatic Mediated Synthesis of Chiral Precursors

Design of optically active polymeric materials for temporary therapeutic and environmental applications requires the working-out of functionalized polymers with structures and properties adjusted to the considered applications. Biocompatibility of selected polymers is a required property in regard to the interactions between living organisms and macromolecular systems. It is therefore important to prepare highly optically active monomers and their corresponding hydrolyzable and biocompatible polymers. Bacterium Clostridium tetanomorphum is an useful source of enzymes for bioconversion and particularly in the chemoenzyrnatic route to optically active alkylmalolactonic acid esters and their optically pure stereoisomers. 3-Methylaspartase, involved in the glutamate fermentation pathway, is a very interesting enzyme, which can provide chiral precursors with high optically purity. This chemoenzymatic strategy can be used to prepare the four stereoisomers of 3-methylmalolaconic acid esters, by enzymatic resolution of natural and artificial stereoisomers of 3-methylaspartic acid. A series of 3-alkymalolactonic acid esters with alkyl equal to ethyl or isopropyl, have been also synthesized and transformed into corresponding optically active polyesters.

Biosensors for Fermentation Process Controla

Abstract: Two biosensors for fermentation process control have been introduced, which were developed in our lab recently. One is an enzyme electrode‐based on‐line monitoring system for glutamate fermentation process control and the other is an H+‐ISFET‐based ENFET for penicillin G fermentation process control.

Metabolic flux distribution for the optimized production of l-glutamate

A comprehensive metabolic network was proposed for glutamic acid bacteria and used in a stoichiometrically based flux balance model for l-glutamate production. Theoretical metabolic pathways leading to optimized glutamate overproduction were determined for several specific cell growth rates; variation in the fluxes was obtained. The off-line extracellular analyses throughout the batch fermentation with Brevibacterium flavum showed the accumulation of arginine, aspartate, lysine, alanine, proline, lactate, α-ketoglutarate, succinate, pyruvate, and gluconate in the medium in addition to glutamate. Metabolic flux distributions in the cells throughout the fermentation were determined using the model in combination with the extracellular analyses of the metabolites. The flux distribution maps showed that the cells utilized the TCA cycle in part whereas the glyoxylate bypass was active throughout the fermentation. The results also indicated that the phosphate pentose shunt played an important role in the glutamate fermentation. These diversions in the pathways and certain metabolic reactions depending on the fermentation periods and conditions are also presented in this paper.

3-Methylaspartate ammonia-lyase as a marker enzyme of the mesaconate pathway for (S)-glutamate fermentation in Enterobacteriaceae.

The enzyme 3-methylaspartase (3-methylaspartate ammonia-lyase, EC 4. 3.1.2) was found in the cells of enteric bacteria, especially in the genera Citrobacter and Morganella, that were grown under anoxic and oxygen-limited conditions. The enzymes were purified to homogeneity from the cell-free extracts of 18 active strains and had similar enzymological properties such as action on columns, specific activity, molecular weight, subunit structure, and N-terminal amino acid sequence similarity. The production of the enzyme was dependent on the limitation of oxygen during growth and was arrested by aeration. The addition of external electron acceptors such as dimethylsulfoxide could support cell growth and production of the enzyme. Activities of glutamate mutase (EC 5.4.99.1) and (S)-citramalate hydrolyase (EC 4.2.1.34), key enzymes of the mesaconate pathway of (S)-glutamate fermentation in the genus Clostridium, were detected in the cells of the active strains grown under oxygen-limited conditions. Based on the results, the mesaconate pathway is proposed to explain the (S)-glutamate fermentation process observed in Enterobacteriaceae, and 3-methylaspartase could be a marker enzyme for this pathway.

Sodium ion-dependent hydrogen production in Acidaminococcus fermentans.

Acidaminococcus fermentans is able to ferment glutamate to ammonia, CO2, acetate, butyrate, and H2. The molecular hydrogen (approximately 10 kPa; E' = -385 mV) stems from NADH generated in the 3-hydroxybutyryl-CoA dehydrogenase reaction (E degrees ' = -240 mV) of the hydroxyglutarate pathway. In contrast to growing cells, which require at least 5 mM Na+, a Na+-dependence of the H2-formation was observed with washed cells. Whereas the optimal glutamate fermentation rate was achieved already at 1 mM Na+, H2 formation commenced only at > 10 mM Na+ and reached maximum rates at 100 mM Na+. The acetate/butyrate ratio thereby increased from 2.0 at 1 mM Na+ to 3.0 at 100 mM Na+. A hydrogenase and an NADH dehydrogenase, both of which were detected in membrane fractions, are components of a model in which electrons, generated by NADH oxidation inside of the cytoplasmic membrane, reduce protons outside of the cytoplasmic membrane. The entire process can be driven by decarboxylation of glutaconyl-CoA, which consumes the protons released by NADH oxidation inside the cell. Hydrogen production commences exactly at those Na+ concentrations at which the electrogenic H+/Na+-antiporter glutaconyl-CoA decarboxylase is converted into a Na+/Na+ exchanger.

Fermentation of trans-aconitate via citrate, oxaloacetate, and pyruvate by Acidaminococcus fermentans.

Growing cells of Acidaminococcus fermentans (DSM 20731 and ATCC 25085) fermented trans-aconitate via citrate, oxaloacetate, and pyruvate to approximately 2 CO2, 1.8 acetate, 0.1 butyrate and 0.9 H2. The carbon and electron recoveries were close to 100%. On citrate no growth was observed and washed cells were unable to ferment this tricarboxylate. In cell-free extracts, however, citrate as well as trans-aconitate were readily fermented to CO2 and acetate. Under these conditions, also cis-aconitate, oxaloacetate, and pyruvate were formed, whereas butyrate and intermediates of glutamate fermentation, 2-oxoglutatrate and glutaconate, could not be detected. Citrate Si-lyase, a Mg2+-dependent oxaloacetate decarboxylase, and pyruvate synthase were present in quantities that corresponded to the growth rate of the organism.

A new method for the accurate and rapid determination of the concentrations of intracellular metabolites in cells during fermentation

A method based on density gradient centrifugation for the accurate and rapid determination of concentrations of intracellular metabolites was developed. The new method was applied to determination of intracellular levels of lactate during lactate fermentation and of intracellular levels of glutamate during glutamate fermentation. The method gave satisfactory results, showing good reproducibility and reliability with a probability of 95%. This method will allow basic information to be obtained about the transport of metabolites from within cells to the culture broth and about dynamic changes in metabolism.

Identification of glutamate β54 as the covalent-catalytic residue in the active site of glutaconate CoA-transferase from Acidaminococcus fermentans

In the course of glutamate fermentation by Acidaminococcus fermentans glutaconate coenzyme A-transferase catalyzes the transfer of CoAS − from acetyl-CoA to ( R)-2-hydroxyg;utarate, forming ( R)-2-hydroxyglutaryl-CoA. Glutamate (E) 54 of the β-subunit was postulated to be directly involved in catalysis by formation of a CoASH ester intermediate [(1994) Eur. J. Biochem., in press]. In order to prove this preliminary result, the following mutations, βE54A, βE64A, βE54Q and βE54D, were introduced by mismatch oligonucleotide priming. As expected, βE54A was inactive (0.02% of the wild-type), whereas βE64A and βE54D were active, 30% and >7%, respectively. However, no CoASH intermediate was detected in the latter mutant, indicating a change in the catalytic mechanism. The activity of the βE54Q mutant increased from 1% to almost 100% upon incubation with acetyl-CoA and glutaconate at 37°C within 40 h. Hence, the substrates induced the conversion of the mutant glutamine residue into the glutamate residue of the wild-type enzyme.

Characterization of the Coenzyme‐B12–Dependent Glutamate Mutase from Clostridium cochlearium Produced in Escherichia coli

The glutamate mutase dependent on adenosylcobalamin (coenzyme B12) catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-3-methylaspartate, the first step of the glutamate fermentation pathway of the anaerobic bacterium Clostridium cochlearium. The enzyme consists of two protein components, E, a dimer epsilon 2 (epsilon, 53.5 kDa) and S, a monomer (sigma, 14.8 kDa). The corresponding genes (glmE and glmS) were cloned, sequenced and over-expressed in Escherichia coli. The genes glmS and glmE are separated by glmL encoding a protein of unknown function. The deduced amino acid sequence of GlmL contains an ATP-binding motif which is common to chaperones of the HSP70-type, actin and procaryotic cell-cycle proteins. Both components of glutamate mutase were purified with excellent yields from cell-free extracts of E. coli carrying the corresponding genes. In contrast to component E, component S was shown to bind coenzyme B12. This observation strongly supports the idea that significant similarities of the amino acid sequences of component S and several other cobamide-dependent enzymes represent a common binding motif. Incubation of pure components E and S with coenzyme B12 resulted in the formation of a fully active glutamate mutase heterotetramer (epsilon 2 sigma 2) containing one molecule of coenzyme B12. EPR spectra of recombinant glutamate mutase, now available in sufficiency large amounts, were recorded after incubation of the enzyme with coenzyme B12 and (S)-glutamate. The EPR signals (gx,y approximately 2.1, gz = 1.985) were of much better resolution than observed earlier with the clostridial enzyme. Their typical hyperfine splitting is clearly derived from Co(II), which is involved in the formation of the paramagnetic species but is different from cob(II)alamin (gx,y = 2.25). The spin concentration was 34-50% of the concentration of the enzyme (epsilon 2 sigma 2) coenzyme complex. The competitive inhibitors (2S, 4S)-4-fluoroglutamate and 2-methyleneglutarate induced similar but not identical signals with spin concentrations of 134-148% of the enzyme concentration. Even (S)-[2,3,3,4,4-2H5]glutamate induced a signal significantly different to that of (S)-glutamate with an intensity of only 7%. These data suggest an involvement of the Co(II)-containing paramagnetic species in catalysis, the concentration of which reflects a steady state between its formation and decomposition. The large difference in the spin concentrations observed with (S)-glutamate as compared to the predeuterated glutamate is probably due to a kinetic isotope effect and indicates a cleavage of a C-H bond during formation of the paramagnetic species.(ABSTRACT TRUNCATED AT 400 WORDS)

Location of the Two Genes Encoding Glutaconate Coenzyme A‐Transferase at the Beginning of the Hydroxyglutarate Operon in Acidaminococcus fermentans

Glutaconate coenzyme A‐transferase (Gct) from Acidaminococcus fermentans consists of two subunits (GctA, 35725 Da and GctB, 29168 Da). The N‐termini sequences of both subunits were determined. DNA sequencing of a subgenomic fragment of A. fermentans revealed that the genes encoding glutaconate CoA‐transferase (gctAB) are located upstream of a gene cluster formed by gcdA, hgdC, hgdA and hgdB in this order. Further upstream of gctA, a DNA sequence was detected showing significant similarities to σ70‐type promoters from Escherichia coli. Primer‐extension analysis revealed that this specific DNA sequence was indeed the location of transcription initiation in A. fermentans. The entire gene cluster, 7.3 kb in length, comprising gctAB, gcdA and hgdCAB, has tentatively been named the hydroxyglutarate operon, since the enzymes encoded by these genes are involved in the conversion of (R)‐2‐hydroxyglutarate to crotonyl‐CoA in the pathway of glutamate fermentation by A. fermentuns. The genes gctAB were expressed together in E. coli. Cell‐free extracts of a transformant E. coli strain contained glutaconate CoA‐transferase at a specific activity of up to 30 U/mg protein. The recombinant enzyme was purified to homogeneity with a specific activity of 130 U/mg protein by ammonium sulfate fractionation and crystallisation. The amino acid residue directly involved in catalysis was tentatively identified as E54 of the small subunit of the enzyme (GctB).

Location of the two genes encoding glutaconate coenzyme A-transferase at the beginning of the hydroxyglutarate operon in Acidaminococcus fermentans.

Glutaconate coenzyme A-transferase (Gct) from Acidaminococcus fermentans consists of two subunits (GctA, 35725 Da and GctB, 29168 Da). The N-termini sequences of both subunits were determined. DNA sequencing of a subgenomic fragment of A. fermentans revealed that the genes encoding glutaconate CoA-transferase (gctAB) are located upstream of a gene cluster formed by gcdA, hgdC, hgdA and hgdB in this order. Further upstream of gctA, a DNA sequence was detected showing significant similarities to sigma 70-type promoters from Escherichia coli. Primer-extension analysis revealed that this specific DNA sequence was indeed the location of transcription initiation in A. fermentans. The entire gene cluster, 7.3 kb in length, comprising gctAB, gcdA and hgdCAB, has tentatively been named the hydroxyglutarate operon, since the enzymes encoded by these genes are involved in the conversion of (R)-2-hydroxyglutarate to crotonyl-CoA in the pathway of glutamate fermentation by A. fermentans. The genes gctAB were expressed together in E. coli. Cell-free extracts of a transformant E. coli strain contained glutaconate CoA-transferase at a specific activity of up to 30 U/mg protein. The recombinant enzyme was purified to homogeneity with a specific activity of 130 U/mg protein by ammonium sulfate fractionation and crystallisation. The amino acid residue directly involved in catalysis was tentatively identified as E54 of the small subunit of the enzyme (GctB).

Cloning, sequencing and expression inEscherichia coliof the gene encoding component S of the coenzyme B12-dependent glutamate mutase fromClostridium cochlearium

Adenosylcobalamin (coenzyme B12) dependent glutamate mutase catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-methylaspartate. This is the first step of the fermentation of glutamate by the strict anaerobic bacterium Clostridium cochlearium. The enzyme consists of the two protein components E and S. The gene encoding component S (glmS) was cloned in Escherichia coli and its nucleotide sequence was determined. The nucleotide sequence and the deduced amino acid sequence showed very strong identities to the sequence of the glmS (also called mutS) gene (80%) and to component S (82%) from the related C. tetanomorphum, respectively. Cell-free extracts of E. coli carrying the glmS gene showed glutamate mutase activity which was strictly dependent on the addition of coenzyme B12 and component E purified from C. cochlearium. Enzyme activity of the recombinant protein was achieved up to 2200 nkat/g wet cells which is due to a ten-fold overexpression compared with the activities determined in cell-free extracts of C. cochlearium. This is the first report of overexpression of an active component of glutamate mutase. A rapid purification procedure consisting only of ammonium sulfate precipitation and a gel filtration step was developed to obtain large amounts of pure component S in a short time.