Biophysical analysis of heat-induced structural maturation of glutamate dehydrogenase from a hyperthermophilic archaeon

Tertiary structure of the recombinant glutamate dehydrogenase from Thermococcus kodakaraensis KOD1 (Tk-rGDH) converts into an intact form induced by the heat treatment. This phenomenon, heat-induced structural maturation, means that high temperature plays an important role in the proper folding and oligomerization of Tk-rGDH. In this work, we analyzed the heat-induced structural maturation of Tk-rGDH by differential scanning microcalorimetry (DSC), circular dichroism (CD), and activity measurements. In DSC measurements, the peak of adsorption of non-heated Tk-rGDH (nh-Tk-rGDH) was two times smaller than that of Tk-rGDH heated at 70 degrees C for 30 min (h-Tk-rGDH). The transition temperature (T(m)) of h-Tk-rGDH was 115 degrees C, which was about 3 degrees C higher than that of nh-Tk-rGDH. In the presence of 0.5 M NaCl, the nh-Tk-rGDH showed two peaks at 107 degrees C and 114 degrees C, while the h-Tk-rGDH showed a single peak at 115.7 degrees C. The heat-induced conformational change process was monitored by changes in CD intensity at 222 nm, and the result showed that heat-induced structural maturation is irreversible. The heat treatment at 70 degrees C showed the highest enhancement in activity, which was 15% larger than that of heat-treated Tk-rGDH at 40 degrees C. The results indicate that heat-induced structural maturation involves an irreversible process, transforming the non-heated form to the stable and active form.     

Characterization of FtsZ homolog from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

The gene of bacterial type ftsZ homolog in hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1 (Pk-ftsZ), was identified. The gene product of the Pk-ftsZ gene is composed of 380 amino acids with a molecular mass of 41,354 Da. In the deduced amino acid sequence of the Pk-ftsZ gene, a glycine-rich sequence (Gly-Gly-Gly-Thr-Gly-Ala-Gly) implicated in GTP binding was well conserved. The Pk-ftsZ gene was overexpressed using Escherichia coli as a host and the recombinant protein was purified. The purified Pk-FtsZ protein exhibited GTPase activity with optimum temperatures higher than 80 degrees C. However, the protein showed little GTPase activity at 40 degrees C, indicating that a high reaction temperature is required for the GTPase activity in accordance with the thermophilic nature of P. kodakaraensis KOD1. The GTP-binding ability of Pk-FtsZ protein could also be detected by UV-induced cross-linking of a protein to [alpha-32P] GTP. The Pk-ftsZ gene was expressed in E. coli cells with a temperature-sensitive ftsZ mutation, E. coli ftsZ84 (ts), but its mutant phenotype of elongated cell form at a nonpermissive temperature (42 degrees C) could not be compensated, possibly because of the thermophilic nature of the Pk-FtsZ. Pk-FtsZ could form protofilaments in a GTP-dependent manner at 90 degrees C. Results of phylogenetic analysis suggest that there might be additional factors required for formation of the Z ring in P. kodakaraensis KOD1.     

Biochemical characterization of deblocking aminopeptidase from hyperthermophilic archaeon Thermococcus onnurineus NA1

A genomic analysis of the hyperthermophilic archaeon Thermoccoccus onnurineus NA1 (TNA1) revealed the presence of a deblocking aminopeptidase (DAP) gene with high similarity to the genes of DAPs from Pyrococcus furiosus (86%) and Pyrococcus horikoshii (83% identity). The optimum aminopeptidase activity of the recombinant enzyme was observed at pH 7.5 and in the range of 90 degrees C to 100 degrees C. The specific aminopeptidase and deblocking activities of the enzyme toward Leu-pNA and Ac-Leu-pNA were 18- and 3-fold higher than those of a P. horikoshii DAP (DAP2), respectively. The enzyme activity was significantly increased by Co(2+) ions. The presence of Co(2+) ions induced the activation of the enzyme with heating and changed the large oligomer to a dimer. The enzyme activated by Co(2+) ions appeared to eventually be inactivated by autodegradation, which was confirmed by mass spectrometry.     

Characterization of prolyl oligopeptidase from hyperthermophilic archaeon Thermococcus sp. NA1

The prolyl oligopeptidase TNA1_POP was found to be encoded in the genome of the hyperthermophilic archaeon Thermococcus sp. NA1 and showed high similarities to its archaeal homologs (76-83%). The enzyme was found to be a single polypeptide composed of 616 amino acids with conserved signature domains. A recombinant TNA1_POP expressed in Escherichia coli was capable of hydrolyzing succinyl-Ala-Pro-p-nitroanilide (Suc-Ala-Pro-pNA) with temperature and pH optimums of 80 degrees C and 7, respectively. TNA1_POP activity appeared to be significantly activated by pre-incubation at 80 degrees C and 90 degrees C with the optimum temperature unchanged. The heat-activated enzyme exhibited a k(cat) approximately twofold higher than that of the unheated enzyme, however, both enzymes showed the same K(m). TNA1_POP was thermostable at 80 degrees C retaining 80% of its heat-activated activity even after 23 h, but it lost its enzymatic activity at 90 degrees C with a half-life of 3 h. The loss of the enzymatic activity at 90 degrees C seemed to be caused by the autodegradation of the enzyme, not by thermal denaturation, as supported by circular dichroism spectropolarimetry. Autodegradation fragments ranging from 2 to 18 kDa were mapped by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.     

Kinetic study of thermostable L-threonine dehydrogenase from an archaeon Pyrococcus horikoshii

In the genome data base of the hyperthermophilic archaeon Pyrococcus horikoshii, an open reading frame with sequence homology to a gene encoding alcohol dehydrogenase was found. It was demonstrated that the encoded enzyme was a thermostable L-threonine dehydrogenase which can oxidize the hydroxy alkyl residue of L-threonine associated with the reduction of NAD+ or NADP+. This enzyme is a member of the zinc-containing L-threonine dehydrogenase family. One enzyme molecule contained one zinc atom, and this metal was considered to contribute to the hyperthermostablility of the enzyme. The reaction of the enzyme proceeded via a sequential mechanism. The Michaelis constants (Km) for L-threonine and NAD+ were 0.013 and 0.010 mM, respectively, and the maximum reaction rate (Vmax) was 1.75 mmol NADH formed/min/mg-protein at 65 degrees C. The Km values for both L-threonine and NADP+ were larger than those for L-threonine and NAD+ with a similar Vmax value. These results indicate that the enzyme has lower affinity to NADP+ than to NAD+, and the binding affinity for L-threonine depends on the coenzymes.     

Gene cloning and characterization of fructose-1,6-bisphosphate aldolase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

The fructose-1,6-bisphosphate (FBP) aldolase gene from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned. The gene encoding FBP aldolase (Tk-Fba) was expressed in Escherichia coli and the purified recombinant protein was characterized at high temperature. Tk-Fba is a homodecamer with a subunit molecular mass of 31,283 Da. The amino acid sequence, decameric conformation, formation of a Schiff-base intermediate, and stimulation (286%) of FBP cleavage activity by citrate suggested that Tk-Fba belonged to Class IA, a subtype of the classical Class I aldolases. The specific activity for the FBP cleavage reaction was 18.9 U/mg, which was much higher than those of other Class IA type FBP aldolases. Tk-Fba was extremely thermostable since the optimum temperature seemed to be above 100 degrees C. The optimum pH for Tk-Fba was determined to be 5.0 in the absence of citrate, while it shifted to around 7.0 in the presence of citrate. Tk-Fba accepted FBP and fructose-1-phosphate as substrates and K(m) values were determined to be 0.063 mM and 4.37 mM, respectively. In addition to citrate, phosphoenolpyruvate and pyrophosphate were also found to be potent activators of Tk-Fba, enhancing activities up to 346% and 201%, respectively. Erythrose-4-phosphate acted as an inhibitor and caused a decrease in the activity to 49%. Tk-Fba also catalyzed the condensation reaction with a similar activity level (14.9 U/mg) to that for FBP cleavage. However, none of the above compounds seemed to have a significant effect on the condensation reaction by Tk-Fba. These results suggest a regulatory function of Tk-Fba toward the catabolic direction of sugar metabolism in T. kodakaraensis KOD1.     

Indolepyruvate ferredoxin oxidoreductase: An oxygen-sensitive iron-sulfur enzyme from the hyperthermophilic archaeon Thermococcus profundus

Thermococcus profundus is a strictly anaerobic sulfur-dependent archaeon that grows optimally at 80°C by peptide fermentation. Indolepyruvate ferredoxin oxidoreductase (IOR), an enzyme involved in the peptide fermentation pathway, was purified to homogeneity from the archaeon under strictly anaerobic conditions. The maximal activity was?obtained above the boiling temperature of water (105°C), with a half-life of 62min at 100°C and 20min at 105°C. IOR was oxygen-sensitive with a half-life of 7h at 25°C under aerobic conditions. The specific activity of T.?profundus IOR was found to be dependent on the number of [4Fe-4S] clusters in the enzyme.     

Purification and characterization of the oxygen-thermostable hydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum camini

Aeropyrum camini that was isolated from a deep-sea hydrothermal vent chimney, possessed two hydrogenases (161 and 85 kDa) in its soluble fraction. The 85-kDa hydrogenase was purified to homogeneity using several chromatography columns. The specific activities of the purified hydrogenase were: 14.8 μmol methyl viologen^ox/mg/min for hydrogen oxidation, and 14.6 μmol methyl viologen^red/mg/min for proton reduction. The oxygen stabilities of hydrogenases that were purified from A. camini and the hydrogen thermophilic bacterium Persephonella hydrogeniphila, were compared. The hydrogenase purified from P. hydrogeniphila completely lost its activity following a 96-h exposure to atmosphere; however, the A. camini hydrogenase maintained 75% of its initial activity, even after a 168 h of atmospheric exposure. A. camini hydrogenase showed a half-life of 48 h at 90 °C, while P. hydrogeniphila hydrogenase showed complete denaturation after a 30 min incubation at the same temperature. Nine residues of the N-terminal amino acid sequence of A. camini hydrogenases (MARLLMIPGT) correspond to the protein sequence encoded by the hypothetical soluble hydrogenase subunit gene (APE2423) from A. pernix strain K1. A. camini hydrogenase has a high thermostability and is very tolerant to oxygen; therefore, it may be used for actual H₂ production.     

Effects of exogenous compatible solutes on growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Six known compatible solutes as well as twenty L-amino acids were individually added to a glucose minimal medium and their effects on the growth of Sulfolobus solfataricus (DSM 1617) were examined. Among the compatible solutes tested, putrescine, trehalose, and l-glutamate enhanced the growth of S. solfataricus. On the other hand, glycine betaine, choline, and L-proline showed little or no influence on cell growth. When cells were grown in the glucose medium supplemented with trehalose or L-glutamate, S. solfataricus preferentially utilized the compatible solute over glucose. The growth-enhancement effect of L-glutamate was also observed to be dependent on the glucose concentration in the medium: growth enhancement was higher when the concentration of glucose was low and gradually decreased with increasing glucose concentration. Interestingly, the effects of amino acids on cell growth differed markedly depending on the chemical nature of the amino acid added. While acidic amino acids-L-glutamate and L-aspartate-enhanced the growth rate, almost no growth was observed in the presence of glycine, L-leucine, L-valine, L-phenylalanine, L-threonine, L-methionine, or L-cysteine. Among all the low-molecular-weight solutes tested in this study, the growth-stimulation effect was most profound in the presence of L-glutamate. When S. solfataricus cells were grown in a glucose (1.0 g/l) medium supplemented with 3.0 g/l L-glutamate, the maximal cell density and growth rate were about 3.2- and 2.3-fold higher than those obtained without L-glutamate.     

Family B DNA polymerase from a hyperthermophilic archaeon Pyrobaculum calidifontis: cloning, characterization and PCR application

The 2352 bp gene coding for 783 amino acid family B DNA polymerase from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. Expression of the gene resulted in the production of Pca-Pol in soluble fraction. After heat denaturation of the host proteins, the Pca-Pol was further purified by ion exchange and hydrophobic interaction chromatographies. Activity gel analysis showed the presence of a catalytically active polypeptide of about 90 kDa. The mass of the protein, determined by Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry was found to be 89,156 Da. The isoelectric point of the enzyme was found to be 6.13. The optimal pH and magnesium ion concentration for the enzyme activity were 8.5 and 4mM, respectively. Unlike other commercially available DNA polymerases the enzyme activity of Pca-Pol was inhibited by monovalent cations such as ammonium and potassium. The half-life of the polymerase at 95 °C and 100 °C was 4.5h and 0.5h, respectively. The enzyme possessed 3'→5' exonuclease activity and was able to amplify, under suitable conditions, up to 7.5 kb DNA fragments by polymerase chain reaction which makes it a potential candidate for amplification of long DNA fragments.     

Inhibitory effect of mineral ion accumulation on high density growth of the hyperthermophilic archaeon Sulfolobus solfataricus

A fed-batch operation for high density cultivation of Sulfolobus solfataricus (DSM 1617) in a bench-top fermentor using a feed medium composed of glucose and yeast extract was investigated. The highest maximal cell density obtained in controlled fed-batch cultures was 21.7 g/l. Although higher yeast extract concentrations in the medium favored greater cell biomass yield, cell growth ceased with low cell densities. It was observed that large amounts of inorganic ions, such as sulfate, ammonium, potassium and phosphate ions, were accumulated in the culture broth at higher yeast extract concentrations. This was due to either the addition of the titrant or feeding of yeast extract during cultivation. Fed-batch cultures with additional mineral salts in the feed medium showed much lower cell biomass, indicating that accumulation of inorganic ions has a significant inhibitory effect on the growth of S. solfataricus. Inhibition of cell growth by the presence of mineral ions was further confirmed by the batch culture experiments. Some plausible mechanisms which can account for the growth inhibition at higher mineral ion concentrations have been suggested.     

明胶载体固定化乙醇脱氢酶技术研究

以明胶为载体,戊二醛为交联剂,采用包埋-交联联用法制备了明胶固定化乙醇脱氢酶。结果表明:以15%明胶为载体、4%戊二醛为交联剂、1g明胶固定1mL乙醇脱氢酶制备的固定化酶,其酶活力回收率达52.47%,所得固定化乙醇脱氢酶连续使用10次后相对活力为52.89%。     

离子交换树脂提取糖蜜酒精废液中K^＋的研究

选用5种强酸性阳离子树脂：树脂BK001、树脂S-9、树脂ZGC108、树脂D001和树脂001×7对糖蜜酒精废液中的K^＋的吸附性能进行研究。静态吸附、等温吸附及吸附和洗脱的动力学研究显示,树脂BK001和树脂ZGC108适合糖蜜酒精废液的K^＋的分离。本研究提供一种新的K^＋提取来源,为有效利用糖蜜酒精工业废弃物及实现提K^＋的工业化生产提供基础理论依据。     

Purification and characterization of alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from Geotrichum capitatum

(S)-N-Benzyl-3-pyrrolidinol is widely used in the synthesis of pharmaceuticals as a chiral building block. We produced 30 mM (S)-N-benzyl-3-pyrrolidinol (enantiometric excess > 99.9%) from the corresponding ketone N-benzyl-3-pyrrolidinone with more than 99.9% yield in 28 h of the resting-cell reaction of Geotrichum capitatum JCM 3908. NAD(+)-dependent alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from G. capitatum JCM 3908 was purified to homogeneity by ammonium sulfate fractionation and a series of DEAE-Toyopearl, Butyl-Toyopearl, Superdex 200, and Hydroxyapatite column chromatographies. The results of SDS-PAGE and HPLC showed the enzyme to be a dimer with a molecular mass of 78 kDa. The purified enzyme produced (S)-N-benzyl-3-pyrrolidinol (e.e.>99.9%) from N-benzyl-3-pyrrolidinone. The enzyme reduced 2,3-butanedione, 2-hexanone, cyclohexanone, propionaldehyde, n-butylaldehyde, n-hexylaldehyde, n-octylaldehyde, n-valeraldehyde, and benzylacetone more effectively than it did N-benzyl-3-pyrrolidinone. No activity was detected towards N-benzyl-2-pyrrolidinone or 2-pyrrolidinone. The activity towards (R)-N-benzyl-3-pyrrolidinol was not detected under the assay conditions employed. The oxidizing activity of the enzyme was higher towards 2-propanol, 2-butanol, 2-pentanol, 2-hexanol, 3-hexanol, and 1-phenyl-2-propanol than towards (S)-N-benzyl-3-pyrrolidinol. The K(m) values for N-benzyl-3-pyrrolidinone reduction and (S)-N-benzyl-3-pyrrolidinol oxidation were 0.13 and 8.47 mM, respectively. To our knowledge, this is the first time that an N-benzyl-3-pyrrolidinol/N-benzyl-3-pyrrolidinone oxidoreductase was purified from a eukaryote; moreover, this is the first report of (S)-N-benzyl-3-pyrrolidinol dehydrogenase activity in microorganisms. This enzyme showed features different from those of known prokaryotic N-benzyl-3-pyrrolidinone reductases. This enzyme will be very useful for the production of chiral compounds.     

Enterocin 416K1, an antilisterial bacteriocin produced by Enterococcus casseliflavus IM 416K1 isolated from Italian sausages

Enterococci (118) from Italian sausages were tested for the production of antimicrobial substances. Of these, 7.6% showed antibacterial activity against one or several closely related microorganisms used as indicators. Enterococcus casseliflavus IM 416K1 in particular produced a bacteriocin (Enterocin 416K1) with strong anti-listerial antagonistic activity. The bacteriocin withstood heating at 90 degrees C for 120 min and storage at 4 degrees C for 6 months. The mode of action was identified as bactericidal. The crude activity of Enterocin 416K1 was linked to a molecule with an apparent molecular weight smaller than 5 kDa. Plasmid analysis of E. casseliflavus IM 416K1 revealed the presence of four plasmids with different molecular weights (34, 11, 7 and 3.3 MDa). All the Bac- variants produced by curing experiments showed loss of the single plasmid of 34 MDa. Bacteriocin activity and immunity production may be linked to genes located on that same plasmid.     

Purification and molecular characterization of a quinoprotein alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes IFO 14464

We have cloned and verified a gene for a novel quinoprotein alcohol dehydrogenase (ADH) from Pseudogluconobacter saccharoketogenes IFO 14464 that has the ability to oxidize L-sorbose to 2-keto-L-gulonic acid (2-KLGA). The enzyme was purified from the soluble fraction of the bacterium and was estimated to be a monomeric protein with a molecular weight of 65 kDa from the analyses of SDS-PAGE and gel-filtration chromatography. An open reading frame of 1824 bp for 608 amino acid residues was estimated as the gene for ADH because of the consistency of the calculated molecular mass and the elucidated partial amino acid sequences of the native enzyme. Homology search revealed that the enzyme showed close similarity to quinoprotein alcohol dehydrogenases isolated from Methylobacterium extorquens and Acetobacter aceti, particularly in the tryptophan docking motifs in the alpha-subunits of those dehydrogenases. The ability to convert L-sorbose to 2-KLGA was found when the lysate of recombinant Escherichia coli DH10B transformed with the gene for ADH was mixed with CaCl2and pyrroloquinoline quinone (PQQ). These data indicate that the cloned DNA is the desired gene for the ADH in which CaCl2 and PQQ are essential for enzymatic activity.