Characterization of phytase produced byAspergillus niger

The extracellular activity ofAspergillus niger phytase at the end of the growth phase was 132 nkat/mL in a laboratory bioreactor. The purified enzyme has molar mass approximately 100 kDa, pH optimum at 5.0, temperature optimum at 55°C and high pH and temperature stability. TheK _m for dodecasodium phytate, calcium phytate and 4-nitrophenyl phosphate are 0.44, 0.45 and 1.38 mmol/L, respectively. The enzyme is noncompetively inhibited by inorganic monophosphate (K _i=2.85 mmol/L) and by Cu²⁺, Zn²⁺, Hg²⁺, Sn²⁺, Cd²⁺ ions and strongly by F⁻ ones; it is activated by Ca²⁺, Mg²⁺ and Mn²⁺ ions. The substrate specificity of phytase is broad with the highest affinity to calcium phytate.     

Purification and properties of extracellular phytase from Bacillus sp. KHU-10

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40°C without 10 mM CaCl₂ and pH 6.0-9.5 and 60°C with 10 mM CaCl₂. About 50% of its original activity remained after incubation at 80°C or 10 min in the presence of 10 mM CaCl₂. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The K _m value for sodium phytate was 50 M. Its activity was inhibited by EDTA and metal ions such as Ba²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, and Mn²⁺ ions.     

Purification and Biochemical Characterization of Phytase Enzyme from <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> (<Emphasis Type="Italic">MH121153</Emphasis>)

Phytase (myo-inositol hexaphosphate phosphohydrolase) belongs to phosphatases. It catalyzes the hydrolysis of phytate to less-phosphorylated inorganic phosphates and phytate. Phytase is used primarily for the feeding of simple hermit animals in order to increase the usability of amino acids, minerals, phosphorus and energy. In the present study, phytase isolation from the Lactobacillus coryniformis strain, isolated from Lor cheese sources, phytase purification and characterization were studied. The phytase was purified in simple three steps. The enzyme was obtained with 2.60% recovery and a specific activity of 202.25 (EU/mg protein). The molecular mass of the enzyme was determined to be 43.25 kDa with the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The optimum temperature and pH for the enzyme were found as 60 °C and 5.0 and respectively. To defined the substrate specificity of the phytase, the hydrolysis of several phosphorylated compounds by the purified enzyme was studied and sodium phytate showed high specificity. Furthermore, the effects of Ca²⁺, Ag⁺, Mg²⁺, Cu²⁺, Co²⁺, Pb²⁺, Zn²⁺ and Ni²⁺ metal ions on the enzyme were studied.     

Mononuclear and dinuclear mechanisms for catalysis of phosphodiester cleavage by alkaline earth metal ions in aqueous solution

In biological systems, enzymes often use metal ions, especially Mg²⁺, to catalyze phosphodiesterolysis, and model aqueous studies represent an important avenue of examining the contributions of these ions to catalysis. We have examined Mg²⁺ and Ca²⁺ catalyzed hydrolysis of the model phosphodiester thymidine-5′-p-nitrophenyl phosphate (T5PNP). At 25 °C, we find that, despite their different Lewis acidities, these ions have similar catalytic ability with second-order rate constants for attack of T5PNP by hydroxide (k_OH) of 4.1 × 10⁻⁴ M⁻¹s⁻¹ and 3.7 × 10⁻⁴ M⁻¹s⁻¹ in the presence of 0.30 M Mg²⁺ and Ca²⁺, respectively, compared to 8.3 × 10⁻⁷ M⁻¹s⁻¹ in the absence of divalent metal ion. Examining the dependence of k_OH on [M²⁺] at 50 °C indicates different kinetic mechanisms with Mg²⁺ utilizing a single ion mechanism and Ca²⁺ operating by parallel single and double ion mechanisms. Association of the metal ion(s) occurs prior to nucleophilic attack by hydroxide. Comparing the k_OH values reveals a single Mg²⁺ catalyzes the reaction by 1800-fold whereas a single Ca²⁺ ion catalyzes the reaction by only 90-fold. The second Ca²⁺ provides an additional 10-fold catalysis, significantly reducing the catalytic disparity between Mg²⁺ and Ca²⁺.     

Adsorption immobilization of Escherichia coli phytase on probiotic Bacillus polyfermenticus spores

The immobilization of enzymes on edible matrix supports is of great importance for developing stabilized feed enzymes. In this study, probiotic Bacillus spores were explored as a matrix for immobilizing Escherichia coli phytase, a feed enzyme releasing phosphate from phytate. Because Bacillus spore is inherently resistant to heat, solvents and drying, they were expected to be a unique matrix for enzyme immobilization. When mixed with food-grade Bacillus polyfermenticus spores, phytases were adsorbed to their surface and became immobilized. The amount of phytase attached was 28.2 ± 0.7 mg/g spores, corresponding to a calculated activity of 63,960 U/g spores; however, the measured activity was 41,120 ± 990.1 U/g spores, reflecting a loss of activity upon adsorption. Immobilization increased the half life (t_1/2) of the enzyme three- to ten-fold at different temperatures ranging from 60 to 90 °C. Phytase was bound to the spore surface to the extent that ultrasonication treatment was not able to detach phytases from spores. Desorption of spore-immobilized phytase was only achieved by treatment with 1 M NaCl, 10% formic acid in 45% acetonitrile, SDS, or urea, suggesting that adsorption of phytase to the spore might be via hydrophobic and electrostatic interactions. We propose here that Bacillus spore is a novel immobilization matrix for enzymes that displays high binding capacity and provides food-grade safety.     

Purification and characterisation of a bifunctional alginate lyase from novel Isoptericola halotolerans CGMCC 5336

A novel halophilic alginate-degrading microorganism was isolated from rotten seaweed and identified as Isoptericola halotolerans CGMCC5336. The lyase from the strain was purified to homogeneity by combining of ammonium sulfate fractionation and anion-exchange chromatography with a specific activity of 8409.19 U/ml and a recovery of 25.07%. This enzyme was a monomer with a molecular mass of approximately 28 kDa. The optimal temperature and pH were 50 °C and pH 7.0, respectively. The lyase maintained stability at neutral pH (7.0–8.0) and temperatures below 50 °C. Metal ions including Na⁺, Mg²⁺, Mn²⁺, and Ca²⁺ notably increased the activity of the enzyme. With sodium alginate as the substrate, the K_m and V_max were 0.26 mg/ml and 1.31 mg/ml min, respectively. The alginate lyase had substrate specificity for polyguluronate and polymannuronate units in alginate molecules, indicating its bifunctionality. These excellent characteristics demonstrated the potential applications in alginate oligosaccharides production with low polymerisation degrees.     

Temperature-dependent activity in early life stages of the stone crab Paralomis granulosa (Decapoda, Anomura, Lithodidae): A role for ionic and magnesium regulation?

Marine brachyuran and anomuran crustaceans are completely absent from the extremely cold (− 1.8 °C) Antarctic continental shelf, but caridean shrimps are abundant. This has at least partly been attributed to low capacities for magnesium excretion in brachyuran and anomuran lithodid crabs ([Mg²⁺]_HL = 20-50 mmol L^− 1) compared to caridean shrimp species ([Mg²⁺]_HL = 5-12 mmol L^− 1). Magnesium has an anaesthetizing effect and reduces cold tolerance and activity of adult brachyuran crabs. We investigated whether the capacity for magnesium regulation is a factor that influences temperature-dependent activity of early ontogenetic stages of the Sub-Antarctic lithodid crab Paralomis granulosa. Ion composition (Na⁺, Mg²⁺, Ca²⁺, Cl⁻, SO₄²⁻) was measured in haemolymph withdrawn from larval stages, the first and second juvenile instars (crabs I and II) and adult males and females. Magnesium excretion improved during ontogeny, but haemolymph sulphate concentration was lowest in the zoeal stages. Neither haemolymph magnesium concentrations nor Ca²⁺:Mg²⁺ ratios paralleled activity levels of the life stages. Long-term (3 week) cold exposure of crab I to 1 °C caused a significant rise of haemolymph sulphate concentration and a decrease in magnesium and calcium concentrations compared to control temperature (9 °C). Spontaneous swimming activity of the zoeal stages was determined at 1, 4 and 9 °C in natural sea water (NSW, [Mg²⁺] = 51 mmol L^− 1) and in sea water enriched with magnesium (NSW + Mg²⁺, [Mg²⁺] = 97 mmol L^− 1). It declined significantly with temperature but only insignificantly with increased magnesium concentration. Spontaneous velocities were low, reflecting the demersal life style of the zoeae. Heart rate, scaphognathite beat rate and forced swimming activity (maxilliped beat rate, zoea I) or antennule beat rate (crab I) were investigated in response to acute temperature change (9, 6, 3, 1, − 1 °C) in NSW or NSW + Mg²⁺. High magnesium concentration reduced heart rates in both stages. The temperature-frequency curve of the maxilliped beat (maximum: 9.6 beats s^− 1 at 6.6 °C in NSW) of zoea I was depressed and shifted towards warmer temperatures by 2 °C in NSW + Mg²⁺, but antennule beat rate of crab I was not affected. Magnesium may therefore influence cold tolerance of highly active larvae, but it remains questionable whether the slow-moving lithodid crabs with demersal larvae would benefit from an enhanced magnesium excretion in nature.     

Production and partial characterization of two types of phytase from Aspergillus niger NCIM 563 under submerged fermentation conditions

Novel extracellular phytase was produced by Aspergillus niger NCIM 563 under submerged fermentation conditions at 30 °C in medium containing dextrin and glucose as carbon sources along with sodium nitrate as nitrogen source. Maximum phytase activity (41.47 IU/mL at pH 2.5 and 10.71 IU/mL at pH 4.0) was obtained when dextrin was used as carbon source along with glucose and sodium nitrate as nitrogen source. Nearly 13 times increase in phytase activity was observed when phosphate in the form of KH₂PO₄ (0.004 g/100 mL) was added in the fermentation medium. Physic-chemical properties of partially purified enzyme indicate the possibility of two distinct forms of phytases, Phy I and Phy II. Optimum pH and temperature for Phy I was 2.5 and 60 °C while Phy II was 4.0 and 60 °C, respectively. Phy I was stable in the pH range 1.5–3.5 while Phy II was stable in the wider pH range, 2.0–7.0. Molecular weight of Phy I and Phy II on Sephacryl S-200 was approximately 304 kDa and 183 kDa, respectively. Phy I activity was moderately stimulated in the presence of 1 mM Mg²⁺, Mn²⁺, Ca²⁺ and Fe³⁺ ions and inhibited by Zn²⁺ and Cd²⁺ ions while Phy II activity was moderately stimulated by Fe³⁺ ions and was inhibited by Hg²⁺, Mn²⁺ and Zn²⁺ ions at 1 mM concentration in reaction mixture. The Km for Phy I and II was 3.18 and 0.514 mM while Vmax was 331.16 and 59.47 μmols/min/mg protein, respectively.     

Effect of metal ion substitutions in anticoagulation factor I from the venom of Agkistrodon acutus on the binding of activated coagulation factor X and on structural stability

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X (FXa)-binding protein that binds in a Ca²⁺-dependent fashion with marked anticoagulant activity. The thermodynamics of the binding of alkaline earth metal ions to ACF I and the effects of alkaline earth metal ions on the guanidine hydrochloride (GdnHCl)-induced unfolding of ACF I and the binding of ACF I to FXa were studied by isothermal titration calorimetry, fluorescence, circular dichroism, and surface plasmon resonance, respectively. The results indicate that the ionic radii of the cations occupying Ca²⁺-binding sites in ACF I crucially affect the binding affinity of ACF I for alkaline earth metal ions as well as the structural stability of ACF I against GdnHCl denaturation. Sr²⁺ and Ba²⁺, with ionic radii larger than the ionic radius of Ca²⁺, can bind to Ca²⁺-free ACF I (apo-ACF I), while Mg²⁺, with an ionic radius smaller than that of Ca²⁺, shows significantly low affinity for the binding to apo-ACF I. All bindings of Ca²⁺, Sr²⁺, and Ba²⁺ ions in two sites of ACF I are mainly enthalpy-driven and the entropy is unfavorable for them. Sr²⁺-stabilized ACF I exhibits slightly lower resistance to GdnHCl denaturation than Ca²⁺–ACF I, while Ba²⁺-stabilized ACF I exhibits much lower resistance to GdnHCl denaturation than Ca²⁺–ACF I. Mg²⁺ and Sr²⁺, with ionic radii close to that of Ca²⁺, can bind to FXa and therefore also induce the binding of ACF I to FXa, whereas Ba²⁺, with a much larger ionic radius than Ca²⁺, cannot support the binding of ACF I with FXa. Our observations suggest that bindings of Ca²⁺, Sr²⁺, and Ba²⁺ ions in two sites of ACF I increase the structural stability of ACF I, but these bindings are not essential for the binding of ACF I with FXa, and that the binding of Mg²⁺, Ca²⁺, and Sr²⁺ ions to FXa may be essential for the recognition between FXa and ACF I.     

Purification and characterization of a thiol amylase over produced by a non-cereal non-leguminous plant, Tinospora cordifolia

A 43 kDa α-amylase was purified from Tinospora cordifolia by glycogen precipitation, ammonium sulfate precipitation, gel filtration chromatography, and HPGPLC. The enzyme was optimally active in pH 6.0 at 60 °C and had specific activity of 546.2 U/mg of protein. Activity was stable in the pH range of 4-7 and at temperatures up to 60 °C. PCMB, iodoacetic acid, iodoacetamide, DTNB, and heavy metal ions Hg²⁺ > Ag⁺ > Cd²⁺ inhibited enzyme activity while Ca²⁺ improved both activity and thermostability. The enzyme was a thiol amylase (3 SH group/mole) and DTNB inhibition of activity was released by cysteine. N-terminal sequence of the enzyme had poor similarity (12-24%) with those of plant and microbial amylases. The enzyme was equally active on soluble starch and amylopectin and released maltose as the major end product.     

Non-covalent (iso)guanosine-based ionophores for alkali(ne earth) cations

Different (iso)guanosine-based self-assembled ionophores give distinctly different results in extraction experiments with alkali(ne earth) cations. A lipophilic guanosine derivative gives good extraction results for K⁺, Rb⁺, Ca²⁺, Sr²⁺, and Ba²⁺ and in competition experiments it clearly favors the divalent Sr²⁺ (and Ba²⁺) cations. 1,3-Alternate calix[4]arene tetraguanosine hardly shows any improvement in the extraction percentages compared to its reference compound 1,3-alternate calix[4]arene tetraamide. This indicates that one G-quartet does not provide efficient cation complexation under these conditions. In the case of the lipophilic isoguanosine derivative there is a cation size dependent affinity for the monovalent cations (Cs⁺ ? Rb⁺ ? K⁺), but not for the divalent cations (Ca²⁺ > Ba²⁺ > Sr²⁺ > Mg²⁺). In competition experiments the isoguanosine derivative, unlike guanosine, does not discriminate between monovalent and divalent cations, giving an almost equal extraction of Cs⁺ and Ba²⁺.     

Interaction of calcium with the human divalent metal-ion transporter-1

Iron deficiency is the most prevalent micronutrient deficiency worldwide. Whereas dietary calcium is known to reduce the bioavailability of iron, the molecular basis of this interaction is not understood. We tested the hypothesis that divalent metal-ion transporter-1 (DMT1)—the principal or only mechanism by which nonheme iron is taken up at the intestinal brush border—is shared also by calcium. We expressed human DMT1 in RNA-injected Xenopus oocytes and examined its activity using radiotracer assays and the voltage clamp. DMT1 did not mediate ⁴⁵Ca²⁺ uptake. Instead, we found that Ca²⁺ blocked the Fe²⁺-evoked currents and inhibited ⁵⁵Fe²⁺ uptake in a noncompetitive manner (K_i ≈ 20 mM). The mechanism of inhibition was independent of voltage and did not involve intracellular Ca²⁺ signaling. The alkaline-earth metal ions Ba²⁺, Sr²⁺, and Mg²⁺ also inhibited DMT1-mediated iron-transport activity. We conclude that Ca²⁺ is a low-affinity noncompetitive inhibitor—but not a transported substrate—of DMT1, explaining in part the effect of high dietary calcium on iron bioavailability.     

Phytase production by the yeast, Pichia anomala

Pichia anomala, isolated from dried flower buds of Woodfordia fruticosa, produced a high activity of an intracellular phytase, at 68 U per g dry biomass, when grown at 20 °C for 24 h in a medium containing glucose (40 g l^–1) and beef extract (10 g l^–1) supplemented with Fe²⁺ (0.15 mM). Partially purified phytase was optimally active at 60 °C and pH 4 with a half life of 7 days at 60 °C. It retained 85% of its activity at 80 °C for 15 min. The enzyme is suitable for supplementing animal feeds to improve the availability of phosphate from phytate.     

Inhibition of plasma membrane Ca-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca-occluded state

The bidentate complex of ATP with Cr³⁺, CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca²⁺-ATPase and the Na⁺,K⁺-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca²⁺ and Na⁺, respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca²⁺-ATPase. The complex inhibited with similar efficiency the Ca²⁺-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T_1/2 = 30 min at 37 °C) with a K_i = 28 ± 9 μM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg²⁺ but unaltered when Ca²⁺ was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca²⁺ occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La³⁺ with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca²⁺ at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca²⁺ promoted by the plasma membrane Ca²⁺-ATPase goes through an enzymatic phospho-intermediate that maintains Ca²⁺ ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.     

Characteristics of extracellular proteases produced by Bacillus laterosporus and Flavobacterium sp. isolated from gelatinfactory effluents

Forty bacterial isolates from the effluents of a gelatin factory (Jabalpur, India) were screened for protease activity and the two most potent producers were identified as Bacillus laterosporus and a Flavobacterium sp. The enzymes of both isolates were optimal at pH 8 and 60°C, with maximum activity after 90 min. The enzyme activity of B. laterosporus was suppressed by Fe²⁺, Mg²⁺, Mn²⁺ and Zn²⁺ ions but was enhanced by Ba²⁺ and Ca²⁺. That of Flavobacterium sp. was suppressed by Mg²⁺ and Mn²⁺ ions but enhanced by Ba²⁺, Ca²⁺ and Fe²⁺. The enzyme activity of the former was strongly inhibited by KCN, whereas that of the latter was only slightly inhibited by 8-hydroxyquinoline.     

Assimilation of Phytate-phosphorus by the Extracellular Phytase Activity of Tobacco (Nicotiana tabacum) is Affected by the Availability of Soluble Phytate

Phytate, the major organic phosphorus in soil, is not readily available to plants as a source of phosphorus (P). It is either complexed with cations or adsorbed to various soil components. The present study was carried out to investigate the extracellular phytase activities of tobacco (Nicotiana tabacum variety GeXin No.1) and its ability to assimilate external phytate-P. Whereas phytase activities in roots, shoots and growth media of P_i-fed 14-day-old seedlings were only 1.3–4.9% of total acid phosphatase (APase) activities, P starvation triggered an increase in phytase secretion up to 914.9 mU mg⁻¹ protein, equivalent to 18.2% of total APase activities. Much of the extracellular phytase activities were found to be root-associated than root-released. The plants were not able to utilize phytate adsorbed to sand, except when insoluble phytate salts were preformed with Mg²⁺ and Ca²⁺ ions for supplementation. Tobacco grew better in sand supplemented with Mg-phytate salts (31.9 mg dry weight plant⁻¹; 0.68% w/w P concentration) than that with Ca-phytate salts (9.5 mg plant⁻¹; 0.42%), presumably due to its higher solubility. We conclude that insolubility of soil phytate is the major constrain for its assimilation. Improving solubility of soil phytate, for example, by enhancement of citrate secretion, may be a feasible approach to improve soil phytate assimilation.     

A Thermostable phytase from <Emphasis Type="Italic">Neosartorya spinosa</Emphasis> BCC 41923 and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its K _m and V _max for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe²⁺, Fe³⁺, and Al³⁺. When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).     

Production of constitutive, thermostable, hyper active exo-pectinase from Bacillus GK-8

Summary A new alkalophilic Bacillus GK-8 overproduced three thermostable extracellular pectinases. The temperature optima was 60°C for all the three enzymes which retained full activity for 2 h. They had pH optima 5.4, 7.0 and 10.4 and were designated as PI, PII and PIII respectively. The half life at 80°C of PI, PII and PIII was 18 min., 12 min. and 12 min., respectively. The enzyme activity was stimulated by Mg²⁺, Ca²⁺, Zn²⁺, Co²⁺ and Mn²⁺ ions.     

Purification and characteristics of Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases from spermatozoa of the sea urchin Strongylocentrotus intermedius

Ca²⁺,Mg²⁺- and Ca²⁺,Mn²⁺-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca²⁺,Mg²⁺-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mg²⁺) > Mn²⁺ = (Ca²⁺ + Mn²⁺) > (Mg²⁺ + EGTA) > Ca²⁺. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn²⁺ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca²⁺,Mn²⁺-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mn²⁺) > (Ca²⁺ + Mg²⁺) > Mn²⁺ > (Mg²⁺ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed.