Two uncommon phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.)

Phospholipase D (PLD) has been detected in seedlings of Papaver somniferum L. cv. Lazúr (Papaveraceae). Purification of the enzyme revealed the existence of two forms of PLD (named as PLD-A and PLD-B). The two enzymes strongly differ in their catalytic properties. The pH optima were found at pH 8.0 for PLD-A and at pH 5.5 for PLD-B. While both enzymes show hydrolytic activity toward phosphatidylcholine (PC) and phosphatidyl-p-nitrophenol (PpNP), PLD-B only was able to catalyze the exchange of choline in PC by glycerol. Both enzymes were activated by Ca²⁺ ions with an optimum concentration of 10 mM. In contrast to PLDs from other plants, PLD-B was still more activated by Zn²⁺ ions with an optimum concentration of 5 mM. The apparent molecular masses of PLD-A and PLD-B, derived from sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), were estimated to be 116.4 and 114.1 kDa. N-terminal protein sequencing indicated N-terminal blockage in both cases. The isoelectric points were found to be 8.7 for PLD-A and 6.7 for PLD-B. Both enzymes were shown to be N-linked glycoproteins. This paper is the first report on PLD in poppy and indicates some important differences of the two enzyme forms to other PLDs known so far.     

A novel fibrinolytic enzyme from <Emphasis Type="Italic">Cordyceps militaris</Emphasis>, a Chinese traditional medicinal mushroom

A novel fibrinolytic enzyme from Cordyceps militaris was purified and partially characterized for the first time, which was designated C. militaris fibrinolytic enzyme (CMase). This extracellular enzyme from C. militaris was isolated by ammonium sulphate fraction, and purified to electrophoretic homogeneity using gel filtration chromatography. The apparent molecular mass of the purified enzyme was estimated to be 27.3 kDa by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 6.0 and 25 °C, respectively. In the presence of metal ions such as Mg²⁺ and Fe²⁺ ions the activity of the enzyme increased, whereas EDTA and Cu²⁺ ion inhibited the enzyme activity. Interestingly the N-terminal amino acid sequences of the enzyme is extremely similar to those of the trypsin proteinases from insects, and has no significant homology with those of the fibrinolytic enzyme from other medicinal mushroom. In conclusion, C. militaris produces a strong fibrinolytic enzyme CMase and may be considered as a new source for thrombolytic agents.     

Purification and Characterization of a Fibrinolytic Protease from a Culture Supernatant of Flammulina velutipes Mycelia

In this study we purified a fibrinolytic enzyme from the culture supernatant of Flammulina velutipes mycelia by ion exchange and gel filtration chromatographies, it was designated as F. velutipes protease (FVP-I). This purification protocol resulted in 18.52-fold purification of the enzyme at a final yield of 0.69%. The molecular mass of the purified enzyme was estimated to be 37 kDa by SDS–PAGE, fibrin-zymography and size exclusion by FPLC. This protease effectively hydrolyzed fibrin, preferentially digesting α-chain over β-and γ–γ chain. Optimal protease activity was found to occur at a pH of 6.0 and a temperature of 20 to 30 °C. The protease activity was inhibited by Cu²⁺, Fe²⁺ and Fe³⁺ ions, but was found to be enhanced by Mn²⁺ and Mg²⁺ ions. Furthermore, FVP-I activity was potently inhibited by EDTA and EGTA, and it was found to exhibit a higher specificity for chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 20 amino acid residues of the N-terminal sequence of FVP-I were LTYRVIPITKQAVTEGTELL. They had a high degree of homology with hypothetical protein CC1G_11771, GeneBank Accession no. EAU86463.     

High activity of NADP-dependent malic enzyme in mitochondria from abdomen muscle of the crayfish Orconectes limosus.

1. Mitochondria isolated from abdomen muscle of crayfish Orconectes limosus exhibit malic enzyme activity in the presence of L-malate, NADP and Mn2+ ions after addition of Triton X-100. Under optimal conditions about 230 nmole of reduced NADP and an equivalent amount of pyruvate are produced per min per mg of mitochondrial protein. 2. The pH optimum for decarboxylation of L-malate is about 7.5. 3. The apparent Km for L-malate, NADP and Mn2+ ions was found to be 0.66, 0.012, and 0.0025 mM, respectively. 4. The requirement for Mn2+ can be replaced by Mg2+, Co2+ and Ni2+ ions; however, higher concentrations of these ions than Mn2+ are required for a full stimulation of malic enzyme activity. 5. Oxaloacetate and pyruvate inhibited the enzyme activity in a competitive manner with apparent Ki values of 0.05 mM and 5.4 mM, respectively.     

Purification and properties of an intracellular leucine aminopeptidase from the fungus,Penicillium citrinum strain IFO 6352

An intracellular leucine aminopeptidase (LAP) fromPenicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 45–50°C whenl-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60°C. The Michaelis-Menten constants forl-Leu-pNA andl-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co²⁺ ions. The activity of EDTA-treated enzyme was restored by addition of Zn²⁺, but reconstitution with Ca²⁺, Mg²⁺ or Mn²⁺ restored some enzyme activity. It is likely that Co²⁺ ions play an important role in the catalysis or stability of thePenicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.     

Purification and characterization of a fibrinolytic protease from a culture supernatant of Flammulina velutipes mycelia

In this study we purified a fibrinolytic enzyme from the culture supernatant of Flammulina velutipes mycelia by ion exchange and gel filtration chromatographies, it was designated as F. velutipes protease (FVP-I). This purification protocol resulted in 18.52-fold purification of the enzyme at a final yield of 0.69%. The molecular mass of the purified enzyme was estimated to be 37 kDa by SDS-PAGE, fibrin-zymography and size exclusion by FPLC. This protease effectively hydrolyzed fibrin, preferentially digesting alpha-chain over beta-and gamma-gamma chain. Optimal protease activity was found to occur at a pH of 6.0 and a temperature of 20 to 30 degrees C. The protease activity was inhibited by Cu2+, Fe2+ and Fe3+ ions, but was found to be enhanced by Mn2+ and Mg2+ ions. Furthermore, FVP-I activity was potently inhibited by EDTA and EGTA, and it was found to exhibit a higher specificity for chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 20 amino acid residues of the N-terminal sequence of FVP-I were LTYRVIPITKQAVTEGTELL. They had a high degree of homology with hypothetical protein CC1G_11771, GeneBank Accession no. EAU86463.     

An amylase from fresh fruiting bodies of the monkey head mushroom Hericium Erinaceum

An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40°C. The enzyme activity was enhanced by Mn²⁺ and Fe³⁺ ions, but inhibited by Hg²⁺ ions.     

假单胞菌N-13中丝氨酸羟甲基转移酶的酶学性质研究

从产L-丝氨酸菌株假单胞菌N-13中纯化了丝氨酸羟甲基转移酶,并对其性质进行了研究.结果表明,丝氨酸羟甲基转移酶酶活力在pH=7.0～9.0间稳定,最适宜pH=8.0;酶的最适温度为35℃,在30～40℃水浴30 min酶活力未见明显下降.磷酸吡哆醛的最适添加浓度为25 μmol·L-1.研究了不同金属离子对酶活力的影...     

Characterization of L-asparaginase from Bacillus sp. isolated from an intertidal marine alga (Sargassum sp.)

B.R. MOHAPATRA, R.K. SANI AND U.C. BANERJEE. 1995. The bacterial flora associated with an intertidal marine alga ( Sargassum sp.) were screened for the presence of extracellular L-asparaginase; one out of five Bacillus strains was found positive. The maximum L-asparaginase activity was found at 37°C and pH 8.0. The optimum NaCl concentration for enzyme activity was found to be 2% (w/v). The enzyme activity was not affected by the addition of different metal ions (Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺and Ni²⁺) at 10 mmol 1^-1, but was strongly inhibited by EDTA.     

Purification and characterization of 4-N-trimethylamino-1-butanol dehydrogenase of Pseudomonas sp. 13CM

A new enzyme, NAD+-dependent 4-N-trimethylamino-1-butanol dehydrogenase from Pseudomonas sp. 13CM, was purified 526-fold to apparent homogeneity in 5 chromatographic steps. The enzyme had a molecular mass of 45 kDa and appeared to be a monomer enzyme. The isoeletric point was found to be 4.8. The optimum temperature was 50 degrees C, and the optimum pHs for the oxidation and reduction reactions were 9.5 and 6.0 respectively. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters, and amino acid terminal sequence. The Km values for trimethylamino-1-butanol and NAD+ were 0.54 mM and 0.22 mM respectively. In the reduction reaction, the apparent Km values for trimethylaminobutylaldehyde and NADH were 0.67 mM and 0.04 mM, respectively. The enzyme was inhibited by SH reagents, chelating reagents, and heavy metal ions. The N-terminal 12 amino acid residues were sequenced.     

Purification, enzymatic properties of a recombinant d-hydantoinase and its dissociation by zinc ion

Summary A d-hydantoinase was expressed in the soluble form by a recombinant E. coli strain, pE-HDT/E. coli BL21 in LB medium. The enzymatic activity of cultured cells reached 5.2–6.5 IU/ml culture at a cell turbidity of 10 at 600 nm. The expressed enzyme was efficiently purified by three steps, ammonium sulfate fractionation, Phenyl-Sepharose hydrophobic interaction chromatography and Sephacryl S-200 size-exclusion chromatography. With the above purification process, the enzyme was purified to more than 95% purity as estimated by SDS-PAGE. The overall recovery of enzymatic activity was 54.4% and the specific activity for substrate dl-hydantoin achieved 48 U/mg. The purified enzyme appeared as a dimer with a molecular mass of 103 kDa, as measured by size-exclusion chromatography. The enzyme was stable from pH 6 to 12 with an optimum pH at 9.5 The optimum temperature of the enzyme was 45 °C and it activity was rapidly lost over 55 °C. Divalent metal ions, including Co²⁺, Mn²⁺ and Ni ²⁺ ions obviously enhanced the enzymatic activity, while Zn²⁺ ion had a slight inhibitory effect. In addition, the dissociation of purified enzyme into its subunits occurred in the presence of 1 mM Zn²⁺ ion. The effect of different metal ions on the d-hydantoinase activation/attenuation was discussed.     

大凉疣螈碱性磷酸酶的分离纯化及部分性质

碱性磷酸酶 (alkaline phosphatase,AKP)在生物界的分布很广 ,动物、植物、微生物中均广泛存在 .提纯的 AKP常被应用于对核酸等的研究 ,是基因工程常用的工具酶 ,也是酶标免疫测定技术的常用工具酶之一 .人类血清中的 AKP在不同疾病状态下有显著变化 ,临床上将血清 AKP变化指标作为诊断某些疾病的依据 .对于细菌和高等动物的 AKP已有广泛的研究 ,但国内外对两栖爬行类动物 AKP的研究报导却很少 ,仅有蛇毒中 AKP的研究报导 [1,2 ] .本文对大凉疣螈皮肤的 AKP进行了分离纯化 ,并对其部分性质进行了初步研究 .1　材料和方法1 .1　材…     

黑曲霉F044脂肪酶的分离纯化及酶学性质研究

黑曲霉F044脂肪酶发酵上清液经硫酸铵沉淀、透析、DEAESepharoseFastFlow阴离子交换层析和SephadexG-75凝胶过滤层析得到电泳纯的脂肪酶,纯化倍数为73·71倍,活性回收率为34%。对纯化脂肪酶性质研究表明:该脂肪酶分子量约为35~40kD,水解橄榄油的最适温度和最适pH分别为45℃和7·0,在60℃以下和pH2·0~9·0之间有很好的稳定性。该脂肪酶的水解活性对Ca2 表现明显的依赖性,而Mn2 、Fe2 和Zn2 对脂肪酶则有显著的抑制作用。在最适条件下水解pNPP的Km和Vmax分别为7·37mmol/L和25·91μmol/(min·mg)。其N-端的15个氨基酸序列为Ser(Glu/His)-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln。     

Isolation and properties of tripolyphosphatase from Neurospora crassa]

Homogenous preparation of tripolyphosphatase from Neurospora crassa is obtained. The enzyme is found to consist of two equal subunits with molecular weight of 40 000 and to have pH optimum 7.0 and temperature optimum 50 degrees C. Bivalent metal ions are required for its catalytical activity, the hest activators being Co2+, Mg2+ and Mn2+. Strict specificity of the enzyme to tripolyphosphate is demonstrated, Km being 5.9-10(-4) M. The enzyme hydrolyses tripolyphosphate to equimolar mixture of ortho- and pyrophosphate. The enzyme activity depends on orthophosphate and pyrophosphate concentrations in the incubation medium.     

Partial purification and characterization of a soluble protein kinase from Leishmania donovani promastigotes

A soluble protein kinase from the promastigote form of the parasitic protozoon Leishmania donovani was partially purified using DEAE-cellulose, Sephadex G-200 and phosphocellulose columns. The enzyme preferentially utilized protamine as exogenous phosphate acceptor. The native molecular mass of the enzyme was about 85 kDa. Mg2+ ions were essential for enzyme activity; other metal ions, e.g. Ca2+, Co2+, Zn2+ and Mn2+, could not substitute for Mg2+. cAMP, cGMP, Ca2+/calmodulin and Ca2+/phospholipid did not stimulate enzyme activity. The pH optimum of the enzyme was 7.0-7.5, and the temperature optimum 37 degrees C. The apparent Km for ATP was 60 microM. Phosphoamino acid analysis revealed that the protein kinase transferred the gamma-phosphate of ATP to serine residues in protamine. The thiol reagents p-hydroxymercuribenzoic acid, 5-5'-dithio-bis(2-nitrobenzoic acid) and N-ethylmaleimide inhibited enzyme activity; the inhibition by p-hydroxymercuribenzoic acid and 5-5'-dithio-bis(2-nitrobenzoic acid) was reversed by dithiothreitol.     

Isopentenyl pyrophosphate isomerase from pig liver

Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) from pig liver has been purified 197-fold. The preparation was estimated to contain less than 10% of contaminating protein. The molecular weight determined by gel filtration was 82,500 ± 3,000 and the isoelectric point from isoelectric focusing was in the range 6.0–6.2. N-terminal analysis showed the presence of both leucine and proline. The pH optimum of the enzyme preparation was 6.3. After dialysis against EDTA, activity was restored by either Mn²⁺ or Mg²⁺, the former being more effective. At the optimum pH and concentration of Mn²⁺, K_m and V were 2.7 μm and 6.7 μmol min^?1 mg^?1, respectively. The enzyme was partially inhibited by a variety of terpene mono- and pyrophosphate esters, by inorganic phosphate ions, and by acetate ions; essentially complete inhibition by sulfhydryl-blocking reagents was observed. ATP partially inhibited, the degree of inhibition showing a sigmoid dependence on ATP concentration. Monothiols and dithiothreitol activated the enzyme, as did mevalonic acid.     

Solubilization and characterization of RNA polymerase from a higher plant

RNA polymerase has been solubilized from sugar beet chromatin. With calf thmus or sugar beet DNA as template enzyme activity was linear with respect to protein concentration and required the presence of all four nucleoside triphospahates, added DNA and divalent metal ions. The enzyme exhibited a sharp Mn²⁺ optimum of 1·25 mM and a Mg²⁺ optimum at 10mM. The Mn²⁺/Mg²⁺ activity ratio (activity at optimum concentrations) was 2·0 with an optimum salt concentration of 50 mM. Based on data including inhibition with α-amanitin (0·025 μg/ml), the majority of the total activity appeared to be RNA polymerase I. Subsequent fractionation by DEAE-Sephadex column chromatography resulted in one peak of activity eluted with 0·18 M (NH₄)₂SO₄.     

Purification and characterization of bovine brain gamma-aminobutyraldehyde dehydrogenase.

gamma-Aminobutyraldehyde dehydrogenase was purified to homogeneity from bovine brain. The molecular weight of the native enzyme and subunit were 230,000 and 58,000, respectively. The Km value for gamma-aminobutyraldehyde and NAD+ were 154 microM and 53 microM, respectively. The optimum pH and temperature were 8.0 and 37 degrees C, respectively. N-terminal sequence of the enzyme is as follows: NH2-S-A-A-T-Q-A-V-P-T-P-N-Q-Q-COOH. The enzyme migrates on isoelectric focusing with pI = 6.5. Enhancement of the enzyme activity by polyamine, Mn2+, Mg2+ and inhibition by gamma-aminobutyric acid and Zn2+ will enhance the limited information on regulation of the gamma-ABALDH activity and GABA metabolism to some extent.     

假单胞杆菌D-海因酶的纯化及酶学性质

D-海因酶是工业上生产D-型氨基酸的关键酶,用热变性,硫酸铵沉淀及Sepharose Q fast flow,Phenyl-Sepharose fast flow,Superose 12等柱层析步骤从pseudomonas2262菌体中分离纯化了该酶,纯化倍数约为60,活力回收约为16%.该酶为同源二聚体,分子量约为109kD,亚基分子量约为53.7kD,反应最适pH为8.0,最适温度为70℃,在pH6.0～10.0和温度60℃以下稳定,该酶对巯基试剂敏感,大多数二价金属离子如镁、锰离子等能促使酶活提高,但高浓度锌离子能抑制酶活,以二氢尿嘧啶为底物的米氏常数Km=2.5×10-2mol/L.该酶的N末端10个氨基酸残基依次为MDKLIKNGTI.     

Purification and properties of an acid endo-1,4-beta-glucanase from Bacillus sp. KSM-330

A novel acid cellulase (endo-1,4-beta-glucanase, EC 3.2.1.4) was found in a culture of Bacillus sp. KSM-330 isolated from soil. One-step chromatography on a column of CM-Bio-Gel A yielded a homogeneous enzyme, as determined by silver staining of both sodium dodecyl sulphate (SDS) and nondenaturing gels. The enzyme had a molecular mass of 42 kDa, as determined by SDS-polyacrylamide gel electrophoresis. The isoelectric point was higher than pH 10. The N-terminal amino acid sequence of the enzyme was Val-Ala-Lys-Glu-Met-Lys-Pro-Phe-Pro-Gln-Gln-Val-Asn-Tyr-Ser-Gly-Ile-Leu- Lys-Pro . This enzyme had an optimum pH for activity of 5.2, being active over an extremely narrow range of pH values, from 4.2 to 6.9; below and above these pH values no activity was detectable. The optimum temperature at pH 5.2 was around 45 degrees C. The enzyme efficiently hydrolysed carboxymethylcellulose (CMC) and lichenan, but more crystalline forms of cellulose, curdlan, laminarin, 4-nitrophenyl-beta-D-glucopyranoside and 4-nitrophenyl-beta-D-cellobioside were barely hydrolysed. The enzymic activity was inhibited by Hg2+ but was not affected by other inhibitors of thiol enzymes, such as 4-chloromercuribenzoate. N-ethylmaleimide and monoiodoacetate. N-Bromosuccinimide abolished the enzymic activity, and CMC protected the enzyme from inactivation by this tryptophan-specific oxidant. It is suggested that a tryptophan residue(s) is involved in the mechanism of action of the Bacillus cellulase and that the inhibition of enzymic activity by Hg2+ is ascribable to interactions with the tryptophan residue(s) rather than with thiol group(s).