Purification and characterization of phosphoribulokinase from the marine chromophytic alga Heterosigma carterae

In this study we characterized phosphoribulokinase (PRK, EC 2.7.1. 19) from the eukaryotic marine chromophyte Heterosigma carterae. Serial column chromatography resulted in approximately 300-fold purification of the enzyme. A polypeptide of 53 kD was identified as PRK by sequencing the amino terminus of the protein. This protein represents one of the largest composite monomers identified to date for any PRK. The native holoenzyme demonstrated by flow performance liquid chromatography a molecular mass of 214 +/- 12.6 kD, suggesting a tetrameric structure for this catalyst. Because H. carterae PRK activity was insensitive to NADH but was stimulated by dithiothreitol, it appears that the enzyme may require a thioredoxin/ferredoxin rather than a metabolite mode of regulation. Kinetic analysis of this enzyme demonstrated Michaelis constant values of ribulose-5-phosphate (226 microM) and ATP (208 microM), respectively. In summary, H. carterae PRK is unique with respect to holoenzyme structure and function, and thus may represent an alternative evolutionary pathway in Calvin-cycle kinase development.     

Purification and characterization of cytochrome c6 from the unicellular green alga Scenedesmus obliquus

Purification of a soluble cytochrome c6 from the unicellular green alga Scenedesmus obliquus by a simple and rapid method is described. The purification procedure includes ammonium sulfate precipitation and non-denaturating PAGE. The N-terminal sequence of the first 20 amino acids was determined and shows 85% similarity and 75% identity to the sequence of cytochrome c6 from the green alga Monoraphidium braunii. The ferrocytochrome shows typical UV/VIS absorption peaks at 552.9, 521.9 and 415.7 nm. The apparent molecular mass was estimated to be 12 kDa by SDS-PAGE. EPR-spectroscopy at 20 K shows resonances indicative for two distinct low-spin heme forms.     

Purification and characterization of oxygen-evolving photosystem II core complexes from the green alga Chlamydomonas reinhardtii

Oxygen-evolving photosystem II complexes were isolated from the green alga Chlamydomonas reinhardtii by selective solubilization of thylakoid membranes with dodecyl maltoside followed by density gradient centrifugation and anion-exchange chromatography. In the presence of CaCl2 and K3[Fe(CN)6] the complexes evolved oxygen at rates exceeding 1000 mumol (mg of chl)-1 h-1. The particles contained 40 chlorophylls a and had properties very similar to those of PSII isolated from higher plants. Chlamydomonas reinhardtii is now the first organism which can be used for both site-directed mutagenesis and detailed biochemical and biophysical characterization of oxygen-evolving photosystem II. It seems therefore to be an ideal model organism for investigation of structure-function relationships in photosynthetic oxygen evolution.     

Purification and spectroscopic characterization of a recombinant chloroplastic hemoglobin from the green unicellular alga Chlamydomonas eugametos

Hemoglobins (Hb), which have the important task of delivering molecular oxygen by facilitating its reversible binding to the heme, are now thought to have evolved in all groups of organisms including prokaryotes, fungi, plants and animals. Our recent finding of a light-inducible chloroplastic Hb in the green unicellular alga Chlamydomonas eugametos has further extend this idea, while raising questions about the function that an Hb could play in a high oxygen environment such as in the chloroplast. In order to understand the role played by this new Hb, we have undertaken its biochemical characterization. To facilitate the characterization of Chlamydomonas Hb, which represents less than 0.01% of the soluble protein in the green alga, the protein has been expressed in Escherichia coli and purified to apparent homogeneity. The purified recombinant protein possesses a non-covalently bound iron-protoporphyrin IX heme. The oxy form of the recombinant Hb. purified directly from bacterial cells, is very stable, with a measured half-life of 7 days at pH 8 and has an ultraviolet/visible spectrum similar to those of the related cytoplasmic Hbs of the ciliated protozoa Paramecium and Tetrahymena and of the cyanobacterium Nostoc commune. In contrast to what has been reported for oxymyoglobins and oxyhemoglobins, the dioxygen molecule bound to the L1637 Hb can be reduced by the electron-transfer mediator phenazine methosulfate in the presence of NADPH, indicating that the heme pocket of Chlamydomonas Hb may be more accessible to small molecules. With regard to this we found that when the small reducing agent sodium dithionite is used to reduce the met form, it must be removed anaerobically from the Hb prior to oxygenation of the protein to stably produce the oxy form. Otherwise, the oxy form is obtained readily from the met form under an oxygenic atmosphere when ferredoxin and ferredoxin NADP+ reductase are used to enzymically reduce the Hb. Finally, the spectra of the deoxy and met forms were unusual, the heme being partly low-spin at physiological pH. These results confirm the existence of a reversible oxygen-binding protein in the chloroplast of C. eugametos. The unusual spectral and biochemical properties of the protein may reflect a specialized function for this Hb.     

The discovery of a novel R-phycoerythrin from an antarctic red alga

A novel biliprotein, named R-phycoerythrin IV, has been discovered. It absorbs blue light better than any other known red algal biliprotein. The protein was found in Phyllophora antarctica, a benthic macroalga, which grows beneath the coastal waters of McMurdo Sound, Antarctica. Fluorescence emission and fluorescence excitation polarization spectroscopy demonstrated that R-phycoerythrin IV behaved as a typical R-phycoerythrin in the functioning of energy migration and has an emission maximum at 577 nm. The circular dichroism (CD) spectrum of the chromophores was compared with visible absorption spectrum, and both were deconvoluted. This process showed the energy states of various individual chromophores. The molecular weight of the protein suggested a alpha6beta6gamma polypeptide structure, and far UV CD studies revealed polypeptides with highly alpha-helical secondary structures. Dynamic light scattering indicated that the protein had a 5.54 nm radius, and its shape was nonspherical. R-phycoerythrin was also purified from a second benthic Antarctic red alga, Iridaea cordata. Its spectroscopic properties were similar to those of some R-phycoerythrins from nonpolar regions. The unique spectroscopic properties of R-phycocerythrin IV may help enable the alga to occupy its niche deeper in the water column than the red alga that has the typical R-phycoerythrin.     

Cloning and characterization of the nuclear gene and cDNAs for triosephosphate isomerase of the marine red alga Gracilaria verrucosa

cDNAs and an intronless single-copy nuclear gene (TPI1) encoding triosephosphate isomerase have been cloned and sequenced from the marine red alga Gracilaria verrucosa. The predicted amino-acid sequence of TPI1 is readily alignable with those of other known TPIs; 26 of 27 active-site residues and 19 of 26 intersubunit-contact residues are identical between TPIs of G. verrucosa and/or animals and green plants. A partial cDNA sequence of a second TPI gene (TPI2), presumably encoding plastid-localized TPI, was recovered by PCR and demonstrated by phylogenetic analysis to be red algal; no TP12 cDNA or genomic clones could be recovered. Genomic Southern analysis demonstrated that at least two TPI-like genes are present in the nuclear DNA of G. verrucosa.     

Purification and characterization of an isoaspartyl dipeptidase from Escherichia coli

We have identified a gene (iadA) in Escherichia coli encoding a 41-kDa polypeptide that catalyzes the hydrolytic cleavage of L-isoaspartyl, or L-beta-aspartyl, dipeptides. We demonstrate at least a 3000-fold purification of the enzyme to homogeneity from crude cytosol. From the amino-terminal amino acid sequence obtained from this preparation, we designed an oligonucleotide that allowed us to map the gene to the 98-min region of the chromosome and to clone and obtain the DNA sequence of the gene. Examination of the deduced amino acid sequence revealed no similarities to other peptidases or proteases, while a marked similarity was found with several dihydroorotases and imidases, reflecting the similarity in the structures of the substrates for these enzymes. Using an E. coli strain containing a plasmid overexpressing this gene, we were able to purify sufficient amounts of the dipeptidase to characterize its substrate specificity. We also examined the phenotype of two E. coli strains where this isoaspartyl dipeptidase gene was deleted. We inserted a chloramphenicol cassette into the disrupted coding region of iadA in both a parent strain (MC1000) and a derivative strain (CL1010) lacking pcm, the gene encoding the L-isoaspartyl methyltransferase involved in the repair of isomerized proteins. We found that the iadA deletion does not result in reduced stationary phase or heat shock survival. Analysis of isoaspartyl dipeptidase activity in the deletion strain revealed a second activity of lower native molecular weight that accounts for approximately 31% of the total activity in the parent strain MC1000. The presence of this second activity may account for the absence of an observable phenotype in the iadA mutant cells.     

Purification and characterization of an alkaline elastase from Myxococcus xanthus

An extracellular elastase, termed Myxococcus xanthus alkaline protease 1 (MAP1), has been purified from M. xanthus DK1622 culture supernatants by a combination of ion-exchange and affinity chromatographies. It consists of a single peptide chain of 39 kDa. The elastolytic activity was totally suppressed by 10 mM 1,10-phenanthroline and the enzyme may then be classified as a metalloprotease. Its pH optimum was estimated to be 8.2 with both elastin-orcein and succinyl-Ala3 p-nitroanilide as substrates. Despite its low pI (5.2), MAP1 was adsorbed on elastin at 80%, a result which privileges hydrophobic interactions between MAP1 and elastin rather than salt bridges, as for known basic elastases. About 80% of the original amidasic and elastolytic activities were conserved after a 30-min prior incubation of the enzyme at 40 degrees C; however, 70% of the amidasic activity is measured, instead of 15% for the elastolytic activity, after 30 min at 50 degrees C. Thermal denaturation at this temperature may prevent adsorption of the enzyme on elastin without any important change of the elastase structure. MAP1 readily hydrolyzes the Gly23-Phe24 bond in the oxidized insulin B chain; the peptide bonds Ala14-Leu15, Leu15-Tyr16, Phe24-Phe25, Phe25-Tyr26 are also cleaved, suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at the first amino acid towards the C-terminus from the cleavage site (P'1 position) [Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. This hypothesis is consistent with the fact that Ala2-Phe-Ala and Ala3-Phe-Ala are hydrolyzed even though tri-alanine to hexa-alanine oligomers are not. The evidence of an elastase with the same molecular mass and pI as MAP1 is given during fruiting body development in submerged culture of M. xanthus. The fact that aromatic amino acids have been found to be the most representative of A-signal [Kuspa, A., Plamann, L. & Kaiser, D. (1992) J. Bacteriol. 174, 3319-3326] is consistent with the hypothesis that, regarding its specificity, MAP1 is likely to play a role in development of myxobacteria.     

Purification and characterization of an endo-1,3-beta-glucanase from Aspergillus fumigatus

An endo-1,3-beta-glucanase was purified from a cell wall autolysate of Aspergillus fumigatus. This beta-glucanase activity was associated with a glycosylated 74-kDa protein. Using a sensitive colorimetric assay and a high-performance anion-exchange chromatography with a pulsed electrochemical detector for product analysis, it was shown that the endoglucanase hydrolysed exclusively linear 1,3-beta-glucan chains, had an optimum pH of 7.0 and an optimum temperature of 60 degrees C. A substrate kinetic study gave a Km value of 0.3 mg/ml for soluble (laminarin and laminari-oligosaccharides) and 1.18 mg/ml for insoluble (curdlan) 1,3-beta-glucan. Laminari-oligosaccharide degradation, analysed by HPLC, showed that the endoglucanase bind to the subtrate at several positions and suggested that the active site of the enzyme recognized five glucose units linked by a 1,3-beta bond. The association of the present endo-1,3-beta-glucanase with the cell wall of A. fumigatus suggests a putative role for this enzyme during cell-wall morphogenesis.     

Purification and partial characterization of an acid proteinase from Dirofilaria immitis

Tumour necrosis factor-alpha (TNF-alpha) is a pluripotent cytokine with its receptors distributed throughout many different cell types. Because of the diverse effects of the cytokine, it is difficult to clearly define its role in infection and immunity, and appreciate its clinical therapeutic value. We have identified peptides derived from the primary amino acid sequence of human TNF-alpha that have neutrophil-stimulating activity, as measured by enhanced chemiluminescence and superoxide production, and peptides which are both directly cytotoxic for tumour cells (WEHI-164) in vitro and also prevent TNF binding to tumour cells. However, only one of these neutrophil-stimulating peptides was toxic for tumour cells in vitro. Our results indicate that the region of amino acids 54-94 of human TNF-alpha has previously undescribed human neutrophil-stimulatory activity, while peptides encompassing the regions 43-68 and 132-150, which are in close proximity, as indicated in the recently determined three-dimensional structure of human TNF-alpha, have in vitro anti-tumour activity. These peptides also slowed tumour growth or induced tumour regression in WEHI-164 tumour-bearing mice. The peptide 73-94, which activated neutrophils but which was not cytotoxic for tumour cells in vitro, also caused in vivo tumour regression, presumably by activating neutrophils with the consequent release of free radicals at the tumour site. Peptide 63-83, which was able to activate neutrophils in vitro, did not possess tumour regression activity in vivo. The TNF peptides described in this report did not elicit procoagulant activity in cultured bovine aortic endothelial cells and as such are devoid of at least one of the potentially lethal side-effects of elevated TNF levels in vivo.     

Purification and characterization of an NADH-rubredoxin oxidoreductase involved in the utilization of oxygen by Desulfovibrio gigas

An NADH--rubredoxin oxidoreductase previously isolated from Desulfovibrio gigas [LeGall, J. (1968) Ann. Inst. Pasteur 114, 109-115] has now been fully purified and further characterized. It contains two subunits of 27 kDa and 32 kDa. With two mid-point redox potentials of -295 mV and -325 mV, this FMN- and FAD-containing protein can induce the specific reduction of D. gigas rubredoxin. In contrast, rubredoxins from the other Desulfovibrio species or desulforedoxin from D. gigas show very low reaction rates with the same enzyme. The phylogenetic significance of the narrow specificity of the enzyme toward the rubredoxin from the same organism is discussed. The purified enzyme has NADH oxidase activity with H2O2 as a final product of O2 reduction. The reaction is half-inhibited by 4.2 microM p-chloromercuribenzoate, whereas cyanide and azide are not significant inhibitors in this reaction. The role of this protein as a part of the enzymic equipment that allows the formation of ATP in the presence of oxygen from the degradation of carbon reserves is discussed.     

Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus

Cell-free extracts of cellobiose-grown cells of the hyperthermophile Pyrococcus furiosus contain very high activities (19.8 U/mg) of a beta-glucosidase. The cytoplasmic enzyme was purified 22-fold to apparent homogeneity, indicating that the enzyme comprises nearly 5% of the total cell protein. The native beta-glucosidase has a molecular mass of 230 +/- 20 kDa, composed of 58 +/- 2-kDa subunits. The enzyme has a pI of 4.40. Thiol groups are not essential for activity, nor is the enzyme dependent on divalent cations or a high ionic strength. The enzyme shows optimum activity at pH 5.0 and 102-105 degrees C. From Lineweaver-Burk plots, Vmax values of 470 U/mg and 700 U/mg were found for cellobiose (Km = 20 mM) and p-nitrophenyl-beta-D-glucopyranoside (Km = 0.15 mM), respectively. The purified enzyme also exhibits high beta-galactosidase activity and beta-xylosidase activity, but shows no activity towards alpha-linked disaccharides or beta-linked polymers, like cellulose. The purified beta-glucosidase shows a remarkable thermostability with a half life of 85 h at 100 degrees C and 13 h at 110 degrees C.     

Purification and characterization of an NAD-malic enzyme from Bradyrhizobium japonicum A1017

An NAD-malic enzyme was purified to homogeneity from Bradyrhizobium japonicum A1017, and its molecular characteristics were surveyed. The enzyme exhibited native and subunit molecular masses of 388 and 85 kDa, respectively, suggesting that it exists as a homotetramer, and was activated by metabolic intermediates in glycolysis. The role of the enzyme in bacteroids' carbon metabolism is discussed.     

Purification and partial characterization of an ostrich alpha 1-antichymotrypsin-like serum inhibitor

alpha 1-Antichymotrypsin, a member of the serpins, is the predominant plasma inhibitor of neutrophil cathepsin G. The aim of this study was to purify ostrich alpha 1-antichymotrypsin and to compare its biochemical properties with those of other species. Ostrich alpha 1-antichymotrypsin was purified from serum by ammonium sulphate fractionation, QAE-Sephadex C-50 and phenyl-Toyopearl chromatography. N-terminal sequence, amino acid composition, molecular mass, isoelectric point and reaction with cathepsin G, elastase and chymotrypsin were determined. SDS-PAGE revealed a M, of 55,000 for ostrich alpha 1-antichymotrypsin and pI values of 6.8 and 4.1-4.3 were obtained. The amino acid composition revealed 444 residues and the N-terminal sequence of the first 20 residues revealed a homology of 30% when compared with several other alpha 1-antichymotrypsin sequences. Total inhibition of cathepsin G by ostrich alpha 1-antichymotrypsin was found at a 4:1 molar ratio of inhibitor to enzyme which was similar to that found for commercial alpha 1-antichymotrypsin. Immunological studies highlighted the lack of cross-reactivity between ostrich and human alpha 1-antichymotrypsin. The study indicated that ostrich alpha 1-antichymotrypsin-like molecule exhibited similar properties to human alpha 1-antichymotrypsin although there were notable differences.     

Purification and characterization of an allergy-induced melanogenic stimulating factor in brownish guinea pig skin

We have demonstrated recently that phenylazonaphthol (PAN) allergy-induced hyperpigmentation in brownish guinea pig skin is associated with the concomitant appearance of a melanogenic soluble factor(s) that activates the intracellular signal transduction system, including phosphatidylinositol turnover subsequent to ligand-receptor binding in cultured guinea pig melanocytes. In this study we have purified and characterized the PAN-induced melanogenic stimulating factor (PIMSF) that occurs in allergy-associated hyperpigmented skin. By successive column chromatography on TSK 2000SW, Mono Q, and octadecyl-NPR, the PIMSF was purified to homogeneity with a single band of apparent molecular mass of 7.9 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific bioactivity of PIMSF increased by 5,195-fold over the original skin homogenate. In cultured guinea pig melanocytes, this purified PIMSF had the potential of activating an intracellular signal transduction system such as inositol 1,4,5-trisphosphate formation and intracellular calcium levels through a pertussis toxin-sensitive G protein-coupled receptor. PIMSF consistently caused a rapid translocation of cytosolic protein kinase C (PKC) to membrane-bound PKC within 5 min of treatment with a return to the basal level after 120 min. The stimulating effects of PIMSF on proliferation and melanization of cultured guinea pig melanocytes were abolished completely by a PKC down-regulating agent (phorbol 12,13-dibutyrate). PIMSF was similar in molecular mass to rat growth-related oncogene alpha (GRO-alpha; molecular mass of 7.9 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had immunocross-reactivity with GRO-alpha upon Western immune blotting analysis. Further, the stimulatory effect of purified PIMSF on DNA synthesis of cultured guinea pig melanocytes was suppressed markedly by the addition of anti-rat GRO-alpha antibody, implying that the PIMSF is apparently identical to GRO-alpha. These findings suggest that PAN allergy provides a new mechanism of hyperpigmentation in which biological factors such as the GRO-alpha superfamily generated within allergy-induced skin stimulate melanocytes through activation of the PKC-related signal transduction pathway.     

Purification and characterization of diacylglycerol acyltransferase from the lipid body fraction of an oleaginous fungus

BACKGROUND AND PURPOSE: Ischemic cerebral infarction (CI) is a serious complication of acute myocardial infarction (MI). Little information exists on CI after thrombolytic therapy for MI. METHODS: Of 3924 MI patients treated with recombinant tissue plasminogen activator (rt-PA) and heparin, 29 (0.7%) developed CI after treatment. All CI patients had detailed neurological evaluations, and 27 (93%) had CT scans centrally reviewed. RESULTS: Age range was 40 to 74 years (mean, 60 years); 25 patients (86%) were men, and 22 (76%) were white. The electrocardiographic location of MI was anterior in 22 (76%) and nonanterior in 7 (24%). Five CIs occurred within 6 hours, 4 between 6 to 24 hours, 8 during the remainder of the first week, 10 during the second week, and 2 others distributed over the 4 weeks after study entry. Six of 29 CIs did not involve the cerebral cortex; 9 patients (31%) had multiple CIs. Of 28 CIs thought to be embolic in origin, 17 showed strong evidence for at least one cardiac abnormality (mural clot, wall-motion abnormality, aneurysm, or atrial fibrillation) known to be associated more specifically with embolism than MI. Eight of 27 CIs (30%) with CT scans had hemorrhagic transformation of varying degrees; 5 were symptomatic. CONCLUSIONS: The time of occurrence and sites of CI after rt-PA and heparin therapy for acute MI are similar to those reported during the prethrombolytic era.     

Purification and characterization of an oxygen-labile, NAD-dependent alcohol dehydrogenase from Desulfovibrio gigas

A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (Km, 0.15 mM) and 1-propanol (Km, 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as Zymomonas mobilis ADH2 and Bacillus methanolicus MDH.     

Molecular characterization of a single mitochondria-associated double-stranded RNA in the green alga Bryopsis

OBJECTIVE: To evaluate the effect of a moderate dose of fish oil on glycemic control and in vivo insulin action in type 2 diabetic men with elevated plasma triacylglycerols and to determine the effect of the same treatment on gene expression of GLUT4, lipoprotein lipase (LPL), and hormone-sensitive lipase (HSL) in the abdominal adipose tissue. RESEARCH DESIGN AND METHODS: A total of 12 type 2 diabetic men were randomly allocated to 2 months of 6 g daily of either fish oil or sunflower oil, separated by a 2-month washout interval, in a double-blind crossover design. RESULTS: For glucose metabolism, 2 months of fish oil supplementation compared with sunflower oil led to similar fasting plasma insulin, glucose, and HbA1c. Basal hepatic glucose production did not increase after fish oil. There was no difference in insulin suppression of hepatic glucose production nor in insulin stimulation of whole-body glucose disposal measured by the euglycemic-hyperinsulinemic clamp. Fish oil did not ameliorate the low mRNA level of GLUT4 in adipose tissue of these patients. For lipid profile, fish oil lowered plasma triacylglycerol more than sunflower oil (P < 0.05) and tended to increase the amount of mRNA of both LPL and HSL in adipose tissue. CONCLUSIONS: A moderate dose of fish oil did not lead to deleterious effects on glycemic control or whole-body insulin sensitivity in type 2 diabetic men, with preserved triacylglycerol-lowering capacities.     

Purification and characterization of the human interleukin-18 receptor

Interleukin (IL)-18 was identified as a molecule that induces IFN-gamma production and enhances NK cell cytotoxicity. In this paper, we report upon the purification and characterization of human IL-18 receptor (hIL-18R). We selected the Hodgkin's disease cell line, L428, as the most strongly hIL-18R-expressing cell line based on the results of binding assays. This binding was inhibited by IL-18 but not by IL-1beta. The dissociation constant (Kd) of 125I-IL-18 binding to L428 cells was about 18.5 nM, with 18,000 binding sites/cell. After immunizing mice with L428 cells and cloning, a single monoclonal antibody (mAb) against hIL-18R was obtained (mAb 117-10C). Sequentially, hIL-18R was purified from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-extracted L428 cells by wheat germ lectin-Sepharose 4B chromatography and mAb 117-10C-Sepharose chromatography. The internal amino acid sequences of hIL-18R all matched those of human IL-1 receptor-related protein (IL-1Rrp), the ligand of which was unknown to date. When expressed in COS-1 cells, the cDNA of IL-1Rrp conferred IL-18 binding properties on the cells and the capacity for signal transduction. From these results, we conclude that a functional IL-18 receptor component is IL-1Rrp.     

Purification and characterization of the D-hydantoinase from Bacillus circulans

A D-hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity from Bacillus circulans. Purification of two hundred forty-three-fold was achieved with an overall yield of 12%. The relative molecular mass of the native enzyme is 212,000 and that of the subunit is 53,000. This enzyme is an acidic protein with an isoelectric point of 4.55. The enzyme is sensitive to thiol reagent and requires metal ions for its activity. The optimal conditions for the hydantoinase activity are pH 8.0-10.0 and a temperature of 75 degrees C. The enzyme is the most stable in a pH range of 8.5-9.5 and up to 60 degrees C. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturant SDS. These remarkable properties are used for the purification procedure.