Purification and characterization of two alpha-amylases from Toxoplasma gondii.

Two distinct alpha-amylases have been identified in Toxoplasma gondii. They were purified close to homogeneity from cytoplasmic and membrane fractions. The apparent molecular weight of the cytoplasmic amylase was 22,300 Da and that of the membrane enzyme was 39,600 Da by gel filtration, and 25,000 and 41,000 Da by SDS gel electrophoresis, respectively. The physicochemical and catalytic properties of both enzymes showed them to be very different. Cytoplasmic alpha-amylase had an acid isoelectric point and its optimum pH was pH 5.0; its activity was unaffected by NaCl, Ca2+, or EDTA. The membrane alpha-amylase had an isoelectric point of 7.7 and an optimum pH of 8.0. It was affected by Ca2+, inhibited by EDTA, and activated eight-fold by NaCl. Both amylases were inactivated by temperatures above 65 degrees C, but cytoplasmic amylase was more resistant to thermal denaturation.     

Purification and Properties of an Amylase from Bacillus cereus NY-14

An extracellular amylase was obtained from a culture medium of Bacillus cereus NY-14. This enzyme was purified to show a single band on disc gel electrophoresis by ammonium sulfate fractionation and Sephadex G-100 gel filtration to 1101-fold of the activity of the original culture liquor. The purified enzyme had a molecular weight of 55,000, an isoelectric point of 6.13, an optimum pH of 6.0, and an optimum temperature of 55°C. The pH-stability range was wide; the enzyme retained more than 80% of its initial activity in the range of pH 5.5 to 12. It was stable below 35°C and required calcium ions for the stabilization. The action pattern of this enzyme on amylaceous polysaccharides was unique in that the predominant product was maltopentaose. The purified amylase could also digest starch granules of such plants as rice, barley, corn, and kuzu to produce maltopentaose as the main product.     

Properties of new alkaline maltohexaose-forming amylases

Summary Three alkaline amylases have been newly discovered in a culture medium of an alkalophilic Bacillus sp. H-167 isolated from soils. These amylases produced maltohexaose as the main product from starch. All three amylases were purified to give a single band on disc electrophoresis. They had similar properties except for molecular weight (MW) and isoelectric point (pI): optimum pH, 10.5; optimum temperature, 60°C; pH stability, 7–12; heat stability, 50–55°C; MW, 59000–80000; pI, 3.5–4.3. Metal ions such as Hg²⁺, Zn²⁺, Pb²⁺, Co²⁺ and Ni²⁺ inhibited the enzyme activity. All the enzymes hydrolyzed starch to produce preferentially maltohexaose, rather than maltose and maltotetraose, in an early stage of the reaction. The yield of maltohexaose reached 25%–30% from soluble starch.     

Purification and Properties of Extracellular Amylase from the Hyperthermophilic Archaeon Thermococcus profundus DT5432

A hyperthermophilic archaeon, Thermococcus profundus DT5432, produced extracellular thermostable amylases. One of the amylases (amylase S) was purified to homogeneity by ammonium sulfate precipitation, DEAE-Toyopearl chromatography, and gel filtration on Superdex 200HR. The molecular weight of the enzyme was estimated to be 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 5.5 to 6.0 and was stable in the range of pH 5.9 to 9.8. The optimum temperature for the activity was 80(deg)C. Half-life of the enzyme was 3 h at 80(deg)C and 15 min at 90(deg)C. Thermostability of the enzyme was enhanced in the presence of 5 mM Ca(sup2+) or 0.5% soluble starch at temperatures above 80(deg)C. The enzyme activity was inhibited in the presence of 5 mM iodoacetic acid or 1 mM N-bromosuccinimide, suggesting that cysteine and tryptophan residues play an important role in the catalytic action. The amylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen to produce maltose and maltotriose of (alpha)-configuration as the main products. Smaller amounts of larger maltooligosaccharides were also produced with a trace amount of glucose. Pullulan; (alpha)-, (beta)-, and (gamma)-cyclodextrins; maltose; and maltotriose were not hydrolyzed.     

Expression and characterization of amylase encoded by a gene cloned from Cellulomonas sp. NCIM 2353

A Cellulomonas genomic fragment encoding extracellular amylase activity was isolated as a clone (ACs2) in Escherichia coli DH10B. The amylase was expressed in the absence of IPTG and in the presence of starch or maltose. This enzyme corresponded to the low mobility activity of Cellulomonas amylases as demonstrated on gel electrophoresis. Maltose, as well as lactose, xylose and xylan cross-induced the amylase of clone ACs2. Maltose-induced amylase was purified to homogeneity. ACs2-coded amylase is a 70kDa acidic protein, with a pH optimum of 7.0 at 45°C. This enzyme exhibited an endo mode of action, similar to the corresponding Cellulomonas enzyme.     

Biochemical Studies on Ricin Part X Effect of the Two Constituent Polypeptide Chains of Ricin D toward Mouse Sarcoma Ascite Tumor Cells

A maltotetraose-forming amylase from Pseudomonas stutzeri was highly purified by adsorption on starch granules and by chromatographies on Sephadex G-100 and DEAE-cellulose. The purified enzyme showed a single band in polyacrylamide gel electrophoreses with or without sodium dodecylsulfate. The optimum pH for enzyme action on starch was 6.0-6.5, and the optimum temperature was 45°C. The purified enzyme attacked starch from the non-reducing end to produce α-anomer oligosaccharides. This indicated that the enzyme was an exo-α-amylase which had not hitherto been found. The enzyme activity was markedly inhibited by the addition of Cu²⁺, Hg²⁺, N-bromosuccinimide and 2,3-butanedione. The molecular weight of the enzyme determined by the method of Weber and Osborn was about 5.7 × 10⁴. The isoelectric point of the enzyme was estimated to be 5.3 by polyacrylamide gel electrofocusing. The Km and k₀ values of this enzyme for starch, glycogen, short chain amylose and some maltooligosaccharides were calculated from Lineweaver-Burk plots.     

Isolation and some properties of a nonspecific extracellular ribonuclease of Aspergillus clavatus]

RNase has been isolated from the homogenate of the Aspergillus clavatus mycelium by gel filtration through Sephadex G-75, chromatography on CM-cellulose and DEAE-cellulose. By gel filtration and electrophoresis in polyacrylamide gel the preparation has been shown to be homogeneous. The enzyme is acid protein with the isoelectric point at pH 4.4 and molecular weight of 27,000. RNase has pH optimum at 6.0--6.2 and temperature optimum 60 degrees for RNA action. The enzyme splits RNA completely in the absence of metal ions. Ions Zn2+, Cu+2, Ag+1 and Ni+2 at a concentration of 10(-4) M are strong inhibitors of RNase activity.     

Purification and Properties of a Maltotetraose- and Maltotriose-Producing Amylase from Chloroflexus aurantiacus

A maltotetraose- and maltotriose-producing amylase which is stable at alkaline pHs and high temperatures was detected in the culture filtrate of a strain of Chloroflexus aurantiacus J-10-F1, a thermophilic, green, photosynthetic bacterium. The enzyme was purified to homogeneity, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by means of ultrafiltration, ammonium sulfate fractionation, and DEAE-cellulose, hydroxyapatite, and high-performance liquid chromatographies. The molecular mass of the purified enzyme was estimated to be about 210,000 Da. The isoelectric point of the enzyme was estimated to be 6.24 by polyacrylamide gel electrofocusing. The amylase was stable up to 55°C and at alkaline pHs of up to 12.0. The optimum pH and temperature of the enzyme activity were 7.5 and 71°C, respectively. Metal ions such as Hg²⁺, Zn²⁺, Cu²⁺, Mn²⁺, and Ni²⁺ strongly inhibited the enzyme activity. The enzyme activity was reactivated specifically by Ca²⁺ after the enzyme was treated with 1 mM EDTA. This enzyme could digest various kinds of raw-starch granules from corn, cassava, and potato. Both maltotetraose and maltotriose were formed as the main enzymatic products from soluble starch.     

Purification and Properties of Cyclodextrin Glucanotransferase from Brevibacterium sp. No. 9605

Cyclodextrin glucanotransferase (EC 2.4.1.19) from Brevibacterium sp. No. 9605 was purified to homogeneity by chromatography on butyl-Toyopearl 650M, γ-cyclodextrin-Sepharose 4B, and Toyopearl HW-55S. The molecular weight of the purified enzyme was estimated to be 75,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the purified enzyme was 2.8. The optimum pH and temperature were pH 10 and 45°C, respectively. The enzyme was stable at the range of pH 6–8 and at temperatures 50°C or less in the presence of CaCl₂. The enzyme produced mainly γ-cyclodextrin from starch in the initial stage of reaction, but later, the proportion of β-cyclodextrin was increased.     

Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes.

A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.     

Purification and properties of amylase produced by a moderately halophilic Acinetobacter sp.

A moderately halophilic Acinetobacter sp., capable of producing dextrinogenic amylase, was isolated from sea-sands. Maximum enzyme production was obtained when the bacterium was cultivated aerobically in media containing 1 to 2M NaCl or 1M KCl. Two kinds of amylase, amylases I and II were purified from the culture filtrate to an electrophoretically homogenous state by glycogen-complex formation, DEAE-Sephadex A-50 chromatography, and Sephadex G-200 gel filtration. Both enzymes had maximal activity at pH 7.0 in 0.2 to 0.6 M NaCl or KCl at 50 to 55 degrees C. The activities were lost by dialysis against distilled water. Molecular weights for amylases I and II were estimated to be 55 000 and 65 000 respectively by SDS-gel electrophoresis. The action pattern on amylose, soluble starch, and glycogen showed that the products were maltose and maltotriose.     

Purificantion and characterization of inorganic pyrophosphatase from Thiobacillus thiooxidans.

An inorganic pyrophosphatase [EC 3.6.1.1] was isolated from Thiobacillus thiooxidans and purified 975-fold to a state of apparent homogeneity. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate and no activity was found with a variety of other phosphate esters. The cation Mg2+ was required for maximum activity; Co2+ and Mn2+ supported 25 per cent and 10.6 per cent of the activity with Mg2+, respectively. The pH optimum was 8.8. The molecular weight was estimated to be 88,000 by gel filtration and SDS gel electrophoresis, and the enzyme consisted of four identical subunits. The isoelectric point was found to be 5.05. The enzyme was exceptionally heat-stable in the presence of 0.01 M Mg2+.     

Purification and Characterization of Phosphatidylinositol-specific Phospholipase C in Suspension-cultured Cells of Rice (Oryza sativa L.)

Phosphatidylinositol-specific phospholipase C was purified from the soluble fraction of suspension-cultured rice cells. The apparent molecular weight of rice enzyme was estimated to be 50,000 by both Sephadex G-100 gel filtration and SDS–polyacrylamide gel electrophoresis, indicating that the enzyme is composed of a single polypeptide. The enzyme had an isoelectric point of 6.3. The soluble phospholipase C had a high degree of specificity toward phosphatidylinositol and a weak activity toward phosphatidyl-inositol monophosphate, while the enzyme did not hydrolyze the other phospholipids or p-nitrophenylphosphorylcholine. V_max and K_m values were 5.0, μmol/min/mg protein and 0.3 mM, respectively. The pH dependency of the enzyme activity was sharp with an optimum of 5.2. In addition, the phospholipase C was a Ca²⁺ -dependent enzyme. The marked activation of enzyme was observed in the presence of 10 to 250, μM Ca²⁺ and higher Ca ²⁺ concentrations than 1 mM had a strong inhibitory effect. A possible regulation of the phospholipase C activity by pH and Ca²⁺ concentrations in the rice cells is discussed.     

Partial purification and properties of thermostable intracellular amylases from a thermophilic Bacillus sp. AK-2

Intracellular thermostable amylases from a thermophilic Baccilus sp. AK-2 have been isolated and purified. The crude enzyme, having pH optimum at 6.5. and temperature optimum at 68 degrees C was purified by DEAE-cellulose column chromatography. Three separable enzyme fractions having starch hydrolyzing property were eluted by lowering the pH from 8.5 to 7.0. Electrophoretic mobility of these fractions showed a single band. Calcium ion up to a concentration of 20 mM had an activating effect on the three fractions. The optimum temperature for the three fractions (FI, FII and FIII) was 65 degrees C and the pH optimum for each was 6.0, 6.5 and 6.0, respectively. The -SH group in the amylase molecule was essential for enzyme activity. Except for Ca2+, Mg2+, Sr2+ and Mn2+ all other metal ions studied inhibited both alpha and beta-amylase activities. EDTA showed dose dependent non-competitive inhibition. Product formation studies proved FI and FIII to be of the alpha-amylase type and FII of the beta-amylase type. The Km for the substrate (starch) in the presence or absence of EDTA was 0.8 X 10(-3) and 1.13 X 10(-3) g/ml for alpha-amylase and beta-amylase, respectively.     

黑曲霉(Aspergillus niger)β-D-葡萄糖苷酶的纯化及其性质

利用垂直板凝胶制备电泳从黑曲霉(Aspergillus niger,AS 3.316)中分离提纯了β-D-葡萄糖苷酶(EC3.2.1.21),经凝胶电泳鉴定为单一带。酶作用的最适pH为4.4,在pH4.0—6.2稳定;最适温度65℃,热稳定性较好,于60℃保温4小时,活力保留80％。此酶作用于纤维二糖的Km值为6.09mM。聚丙烯酰胺薄层等电聚焦测得其pI值为5.5;用SDS凝胶电泳测得其分子量为77000。此酶不仅能水解纤维二糖和对硝基苯-β-D-葡萄糖苷,还能微弱地水解对硝基苯β-D-半乳糖苷和β-D-木糖苷。金属离子Fe~(2+)、Hg~(2+)、Cu~(2+)、Al~(3+)、Hg~+和Ag~+等对此酶有不同程度的抑制作用,蛋白质侧链修饰剂N-溴代琥珀酰亚胺对此酶有较强的抑制作用,2-羟基-5-硝基溴苯对酶也有一定的抑制作用,推测色氨酸残基对β-D-葡萄糖苷酶的活力是非常必要的。     

Purification and Properties of Three Types of Xylanases Induced by Methyl β-Xyloside from Streptomyces sp.

When mycelia of Streptomyces sp. No. 3137 were cultivated in a medium containing methyl β-xyloside, xylanases (EC 3.2.1.8) were inductively produced into the medium. Three types of enzyme from the culture filtrate have been purified by ultrafiltration with DIAFLO UM-10, chromatography on DEAE-Sephadex A-25, gel filtration on Bio Gel P-100, and isoelectric focusing with Servalyt 6~8 or 9~11. The three purified enzymes, tentatively named X-I, X-II-A, and X-II-B, were homogeneous by Polyacrylamide gel electrophoresis at pH 4.3. The molecular weight of X-I was about 50,000 by SDS-polyacrylamide gel electrophoresis or gel filtration on Bio Gel P-100. The molecular weight of X-II-A and X-II-B were both approximately 25,000 by SDS-polyacrylamide gel electrophoresis and that of X-II-B was 25,680 by the sedimentation-equilibrium method. X-I had an isoelectric point at 7.10, and X-II-A and X-II-B had different isoelectric points, 10.06 and 10.26, respectively. The three enzymes were optimally active at 60~65°C and stable to 55°C. The optimal pH of X-I, X-II-A, and X-II-B were pH 5.5~6.5, 5.0~6.0, and 5.0~6.0, respectively. The ranges of two X-I’s pH stability (pH 1.5 ~ 11.5) were wider than that of X-I’s (pH 3.0 ~ 10.5). These purified preparations hydrolyzed xylotriose, xylotetraose, and xylan but not xylobiose, cellobiose, maltose, carboxymethyl cellulose, or soluble starch. Their actions were inhibited by Hg²⁺ and Fe³⁺ ions, sodium dodecyl sulfate, and N-bromosuccinimide.     

Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

Purification and characterization of choline oxidase from Arthrobacter globiformis

Choline oxidase was purified from the cells of Arthrobacter globiformis by fractionations with acetone and ammonium sulfate, and column chromatographies on DEAE-cellulose and on Sephadex G-200. The purified enzyme preparation appeared homogeneous on disc gel electrophoresis. The enzyme was a flavoprotein having a molecular weight of approx. 83,000 (gel filtration) or approx. 71,000 (sodium dodecyl sulfate--polyacrylamide disc gel electrophoresis) and an isoelectric point (pI) around pH 4.5. Identification of the reaction products showed that the enzyme catalyzed the following reactions: choline + O2 leads to betaine aldehyde + H2O2, betaine aldehyde + O2 + H2O leads to betaine + H2O2. The enzyme was highly specific for choline and betaine aldehyde (relative reaction velocities: choline, 100%; betaine aldehyde, 46%; N,N-dimethylaminoethanol, 5.2%; triethanolamine, 2.6%; diethanolamine, 0.8%; monoethanolamine, N-methylaminoethanol, methanol, ethanol, propanol, formaldehyde, acetaldehyde, and propionaldehyde, 0%). Its Km values were 1.2 mM for choline and 8.7 mM for betaine aldehyde. The optimum pH for the enzymic reaction was around pH 7.5.     

Characterization of Proenkephalin-Cleaving Proteinases in Bovine Adrenal Chromaffin Granules Using [35S]Proenkephalin Copolymerized into Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Proteinases capable of cleaving proenkephalin into smaller peptides have been identified in bovine adrenal chromaffin granules using [35S]methionine-labeled recombinant rat proenkephalin as a selective substrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis proteinase radiozymography. This technique was used for the screening of subcellular fractions, general characterization of pH optima, and the mechanistic characterization of proteinases with both reversible and irreversible inhibitors. Two enzymes with approximate molecular masses of 76 and 30 kDa were shown to be localized to the highest-density fractions of chromaffin granules by sucrose density gradient fractionation. Both were enriched in a 1 M NaCl wash of purified chromaffin granule membranes, were active at high pH, and were characterized as serine proteinases based on inhibition by soybean trypsin inhibitor. The 30-kDa enzyme was also inhibited by diisopropyl fluorophosphate, D-Phe-Pro-Arg-CH2Cl, and D-Val-Phe-Lys-CH2Cl and appeared to be the previously described adrenal trypsin-like enzyme. A third enzyme, of 66 kDa, was also associated with the 1 M NaCl wash of purified chromaffin granule membranes but was not localized exclusively to chromaffin granules in sucrose gradients. This proteinase was found to be Ca2+ activated and inhibited by EDTA but not diisopropyl fluorophosphate, soybean trypsin inhibitor, p-chloromercuriphenylsulfonic acid, 1,10-phenanthroline, or pepstatin.     

Purification and characterization of the hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae.

1. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400-fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation. 2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G-100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51,000. 3. The enzyme requires Mg2+ and has its pH optimum at 8.5. 4. Isoelectric focussing as well as gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1. 5. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 microns, 18 microns, and 50 microns respectively.