Purification and Some Properties of Two Boticins

Two bacteriocins (boticins) were elaborated without induction by strain S5, a nontoxigenic variant of Clostridium botulinum type E. After separation of the two active entities by gel filtration on Sephadex G-50, a large particle with boticin activity was isolated by density gradient ultracentrifugation, and a small soluble boticin was purified by continuous curtain electrophoresis and chromatography on sulfoethyl-Sephadex. Large and small boticins were purified 200- and 3,000-fold, respectively, with yields of 50% or more. The small boticin, a basic substance with molecular weight under 30,000, was the predominant species; the large boticin, a negatively charged particle with particle weight greater than 40 x 10(6), represented less than 20% of the total activity. Both purified boticins were resistant to heat and were attacked by proteolytic enzymes, but the large boticin was less thermostable and less sensitive to proteolytic enzymes than was the smaller variety. The activity of the large boticin was not reduced by treatment with urea or deoxyribonuclease. Both boticins exhibited sporostatic and bactericidal activities for C. botulinum type E, strain 070. A suspension of type E strain 070 vegetative cells was rendered nonviable within 9 min by the small boticin. The lethal action of this bacteriocin was not reversed by trypsin.     

Partial Purification and Some Properties of Two Novel Deoxyribonucleases from Bacillus subtilis

Optically active guaiane dienes, 3 and 4, related to sclerosporene 2 were synthesized from (—)-carvone and (—)-α-santonin. The mass spectra of 3 and 4 were not identical with that of natural sclerosporene.     

Purification of Two Dipeptidyl Aminopeptidases II from Rat Brain and Their Action on Proline-Containing Neuropeptides

From the soluble and membrane fractions of rat brain homogenate, two enzymes that liberate dipeptides of the type Xaa-Pro from chromogenic substrates were purified to homogeneity. The two isolated dipeptidyl peptidases had similar molecular and catalytic properties: For the native proteins, molecular weights of 110,000 were estimated; for the denatured proteins, the estimate was 52,500. Whereas the soluble peptidase yielded one band of pI 4.2 after analytical isoelectric focusing, two additional enzymatic active bands were detected between pI 4.2 and 4.3 for the membrane-associated form. As judged from identical patterns after neuraminidase treatment, both peptidases contained no sialic acid. A pH optimum of 5.5 was estimated for the hydrolysis of Gly-Pro- and Arg-Pro-nitroanilide. Substrates with alanine instead of proline in the penultimate position were hydrolyzed at comparable rates. Acidic amino acids in the ultimate N-terminal position of the substrates reduced the activities of the peptidases 100-fold as compared with corresponding substrates with unblocked neutral or, especially, basic termini. The action of the dipeptidyl peptidase on several peptides with N-terminal Xaa-Pro sequences was investigated. Tripeptides were rapidly hydrolyzed, but the activities considerably decreased with increasing chain length of the peptides. Although the tetrapeptide substance P 1-4 was still a good substrate, the activities detected for the sequential liberation of Xaa-Pro dipeptides from substance P itself or casomorphin were considerably lower. Longer peptides were not cleaved. The peptidases hydrolyzed Pro-Pro bonds, e.g., in bradykinin 1-3 or 1-5 fragments, but bradykinin itself was resistant. The enzymes were inhibited by serine protease inhibitors, like diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride, and by high salt concentrations but not by the aminopeptidase inhibitors bacitracin and bestatin. Based on the molecular and catalytic properties, both enzymes can be classified as species of dipeptidyl peptidase II (EC 3.4.14.2) rather than IV (EC 3.4.14.5). However, some catalytic properties differentiate the brain enzyme from forms of dipeptidyl peptidase II of other sources.     

Purification and Some Properties of Two Carboxypeptidases from the Hepatopancreas of the Crab Paralithodes camtschatica

High activity of carboxypeptidases was detected in the hepatopancreas of the crab Paralithodes camtschatica, while aminopeptidase activity in this tissue was low. Two crab carboxypeptidases were purified by chromatography on DEAE-cellulose, phenyl-Sepharose, and Sephadex G-75 to homogeneity. The molecular weight values of carboxypeptidases I and II were 40,000 and 37,000, respectively. The isoelectric point value for both carboxypeptidases was 4.5. The crab carboxypeptidases were activated by NaCl, with maximal activity of carboxypeptidases I and II at 1.0 M and 0.6 M NaCl, respectively. Using 19 N-blocked dipeptides with the general structures Bz-Gly-X and Z-Gly-X, broad substrate specificity of the purified enzymes was demonstrated. Under optimal conditions the values of K _m and k _cat for the hydrolysis of Bz-Gly-l-Phe, Bz-Gly-l-Arg, and Bz-Gly-l-Lys by crab carboxypeptidases were determined. Inhibition data led to classification of the crab enzymes as metallopeptidases. Both carboxypeptidases were stable under neutral and mildly alkaline conditions. In addition, the presence of 1 M NaCl decreased the thermostability of the crab carboxypeptidases. Received August 13, 1999; accepted November 19, 1999.     

Purification and Properties of Two Galactanases from Penicillium citrinum

Two galactanases (I and II) were purified to homogeneous states from water extracts of a wheat bran culture of Penicillium citrinum. Although these enzymes were separable by affinity chromatography and distinct on Polyacrylamide gel electrophoresis, they were similar in physical and enzymatic nature. They had almost the same molecular weight (3.5 } 10⁴) and isoelectric point (pH 4.2), and similar amino acid compositions and carbohydrate contents (3%). Alanine and leucine were detected for both enzymes as the N- and C-terminal amino acids, respectively.

These enzymes were most active at pH 4.5 and 55%C, and were stable between pH 4 and 10 (at 15%C for 24hr), and below 55%C (at pH 5.5 for 10min). The enzymes hydrolyzed soybean arabinogalactan to produce galactose and several galac-tooligosaccharides, but did not attack coffee bean arabinogalactan. Therefore, these enzymes were suggested to be endo-l,4-²-D-galactanases.

The enzymes also attacked ONPG and PNPG, and lag phases were observed at the beginning of the reactions.

Among the compounds examined, Hg²⁺ and Fe³⁺ inhibited the enzyme actions. ONPG-hydrolyzing activities of the enzymes were inhibited by some sugars such as lactose, galactose, arabinose, glucose and xylose.

Galactobiose, -triose and -tetraose prepared from soybean arabinogalactan with purified galactanase I were found to be further hydrolyzed by the enzymes. A lag phase was also observed in the time course of hydrolysis of galactobiose.     

Purification and Some Properties of Chitinase from Momordica charantia

Chitinase was purified from Momordica charantia L. by affinity chromatography. The purified enzyme showed single band on sodium dodecyl sulfate polyacrylamicle gel electrophoresis and the molecular weight was estimated as 35 kD. The enzyme was stable at temperatures up to 50℃ or less than 10 % loss of activity in 1 h. Its optimum temperature was about 45 ℃. Its suitable pH had a rather wide range from pH 4.4 to pH 6.8 and the optimum pH was about 6.2. The activity of the enzyme was similar in root and stem. In the lower leaves,the activity was higher than that of the upper.     

Purification and Some Properties of Cellulases from Aspergillus niger

Four types of cellulases, FI-1-b, FI-2-b, FI-2-c, and FII, were obtained from a commercial crude cellulase preparation produced from a water extract of a culture of A. niger.

Ammonium sulfate fractionation and column chromatography using DEAE-Sephadex A-25, Amberlite IRC-50 and Hydroxylapatite were employed for the purification of these cellulases.

Some properties of these enzymes were investigated: the optimum pH for the hydrolysis of glycol cellulose by FI-2-b and FI-2-c was pH 4.0 to 5.0, while that of FI-1-b was pH 2.3 to 2.5, and the optimum temperature for the activity of FI-2-b and FI-2-c was 40°C, but that of FI-1-b was 65°C.

The FII seemed to be most active toward cellobiose. Studies of the mode of action on glycol cellulose indicated that A. niger cellulases seemed not to be capable of attacking highly polymerized cellulose.     

蟾蜍细胞溶素的纯化及其生化特性研究

利用阴离子交换和凝胶过滤柱层析等方法对蟾蜍卵黄外被细胞溶素进行了分离纯化,获得了高纯度的样品．该酶的质量为３２ｋＤ,其特异性ＭＣＡ－人工合成底物为Ｂｏｃ－Ｇｌｎ－Ａｒｇ－Ａｒｇ－ＭＣＡ,能被ＤＦＰ、ＳＢＴＩ、ｌｅｕｐｅｐｔｉｎ和ｐ－ＡＭＰＳＦ等蛋白酶抑制剂所强烈抑制,但不受ｃｈｙｍｏｓｔａｔｉｎ、ｂｅｓｔａｔｉｎ、Ｅ－６４和ＥＤＴＡ等的影响,表明该酶是一种丝氨酸类型的蛋白酶     

大鼠肝脏过氧化氢酶的分离纯化及性质

根据过氧化氢酶的催化特性,采用盐析,透析,离心等技术,从大鼠肝脏中分离纯化过氧化氢酶,提纯倍数达到26倍,酶活性回收率为57%。为一般实验室分离纯化大鼠过氧化氢酶提供了一种有效的方法。酶学性质研究表明,该酶最适温度为37℃,最适pH值为7.5,在此条件下,以过氧化氢为底物的Km值为56 mmol.L-1。     

Purification and Some Properties of Alcohol Acetyltransferase from Banana Fruit

Isoamyl acetate, one of the major characteristic aroma compoundsof banana fruit (Musa sapientum L.), was synthesized by thecondensation of acetyl-CoA and isoamyl alcohol under the actionof alcohol acetyltransferase, which was found to be localizedin the soluble fraction of pulp cells. The activity of thisenzyme increased with the ripening of banana fruit. The enzyme was purified about 62-fold. The purified enzyme wasvery labile at pHs lower than pH 7.0 but relatively stable atpH 7.5{small tilde}9. The enzyme was most active at 30C andpH 8.5. The molecular weight was estimated to be about 40,000by gel filtration. Its K_m values for acetyl-CoA and isoamylalcohol were 50 µM and 0.4 mM, respectively. (Received January 28, 1985; Accepted May 30, 1985)     

Partial Purification and Some Properties of Polyphenoloxidases from Sago Palm

Two polyphenoloxidases (PPO I and PPO III, EC 1.10.3.1) were extracted and partially purified from sago palm pith by hydroxylapatite chromatography, DEAE-cellulose chromatography and gel filtration. Both purified isozymes gave a single activity band on polyacrylamide gel electrophoresis. The molecular weights of both enzymes were estimated to be 40,000. They had the same pH optima of 6.5 but different temperature optima, 35°C for PPO I and 45°C for PPO III. PPO I was stable at neutral to alkaline pH and PPO III at acidic pH. PPO III was somewhat more stable than PPO I when incubated at various temperatures for 15 min. PPO I and PPO III oxidized well DL-epicatechin and d-catechin, respectively. Both enzymes were strongly inhibited by KCN, Na-diethyldithiocarbamate and NaHSO₃.     

N-氨甲酰基-D-氨基酸酰胺水解酶的快速纯化及性质

通过硫酸铵分级沉淀、疏水层析及阴离子交换层析等三步 ,有效地从一菌株NO .2 2 6 2中纯化了N 氨甲酰基 D 氨基酸酰胺水解酶。结果表明 ,酶活性回收约 2 0 %,纯化了 8 4倍。天然PAGE与SDS PAGE分析表明 ,该酶分子为同源四聚体 ,单体分子量约为 3 5kD。酶催化反应的最适pH为 7 7～ 8 0 ,最适温度为 45℃。以N 氨甲酰 DL 丙氨酸为底物时 ,Km =1 3×1 0 - 3 mol L ,Vmax=0 .3 3mol min。二价金属离子Ni2 + 有激活作用 ,Zn2 + 有明显的抑制作用 ,而Co2 + 对酶活无影响。该酶N 末端 8个氨基酸残基依次为TRQKILAF。     

Purification and Some Properties of Plant Endonuclease from Scallion Bulbs

A plant endonuclease with 3′-nucleotidase activity was purified from scallion bulbs to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 38,000 by SDS-PAGE and 40,000 by Sephadex G-100 gel filtration. The enzyme rapidly hydrolyzed yeast RNA and denatured calf thymus DNA to acid-soluble substances, and hydrolyzed the plasmid pBR322 to yield small DNA fragments at low enzyme concentrations. These four activities were eliminated by treatments with EDTA and tetraethylenepentamine. The enzyme had maximum activity at pH 8.5-9.0 for 3′-AMP, 3′-GMP, and 3′-UMP, at pH 6.5 for 3′-CMP and yeast RNA, and at pH 6.0 for denatured calf thymus DNA and pBR322. During digestion of yeast RNA by the enzyme at pH 6.5, 5′-GMP was released most rapidly, followed by 5′-UMP, 5′-AMP, and 5′-CMP. These properties were different from those of endonucleases isolated from other sources such as mung bean sprouts and wheat seedlings.     

Purification and Some Physical Properties of Acid-cellulase from Aspergillus niger

A cellulase preparation which exhibits the highest activity at a lower pH range, 2.3 to 2.5, was purified from a commercial cellulase preparation from a culture filtrate of Asp. niger and referred to as acid-cellulase.

The purification involves ammonium sulfate fractionation, gel filtration and ion-exchange and adsorption chromatographies. The purified enzyme was revealed to be homogenous in ultracentrifugation and disc as well as ampholine electrophoreses and to be an acidic protein of which isoelectric point lied at pH 3.3. The sedimentation coefficient and molecular weight were determined to be 3.27 S and 46,000, respectively. The optical properties were also studied.     

Purification and Some Properties of a Protease from Streptomyces limosus

Streptomyces limosus was selected because it secreted a novel protease that catalyzed the synthetic reaction forming Pro-Pro-Pro from Pro-Pro. The protease was purified to an electrophoretically homogeneous state and an activity of more than about 20,000-fold that of the culture broth. The molecular mass of the enzyme was estimated to be 50 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was most active in alkaline pH for the synthetic reaction producing Pro-Pro-Pro from Pro-Pro, although for the hydrolytic reaction forming proline it was most active in neutral pH. The enzyme was inhibited by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and diazoacetyl-DL-norleucine methyl ester (DAN). It can be considered that this enzyme belongs to the class of aspartic proteases. The substrate specificity indicates that this enzyme has a strong affinity for proline as a N-terminal amino acid of peptides.     

Purification and Some Properties of Cyclomaltodextrin Glucanotransferase from Bacillus circulans

Cyclomaltodextrin glucanotransferase was purified from B. circulans C31 through two successive steps of starch and Biogel column chromatography. The enzyme was purified up to 90-fold with a 30% yield. Its molecular weight was around 103,000. The purified enzyme converted 28% of the soluble starch to β-cyclodextrin at pH 7.0 and a substrate concentration of 5%. The optimum pH for the enzyme was found to be 5.5. The optimum temperature was 60°C. The enzyme optimum was stable from pH 5.5~9.0 and up to 50°C.     

Purification and Some Properties of Thiosulfate Dehydrogenase from Acidithiobacillus ferrooxidans

Abstract

Thiosulfate dehydrogenase was purified from Acidithiobacillus ferrooxidans using three purification steps. The purification procedure involved ammonium sulfate fractionation, ion‐exchange chromatography, and gel permeation chromatography. Specific activity of the purified enzyme (after IEC) was 3.26 nkat/mg, and yield of the enzyme was 78%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is a tetramer composed of four probably identical subunits of relative molecular weight 45,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 3.0. The isoelectric point of the enzyme was 8.3. Enzyme activity was found to be particularly sensitive to the histidine‐selective reagent diethylpyrocarbonate. Reagents selective for arginine, cysteine, and tryptophane had no effect on enzyme activity.     

Purification and Some Properties of Tetanolysin

Tetanolysin was purified from the culture fluid of a strain of Clostridium tetani by ammonium sulfate fractionation, acetone precipitation and repeated gel filtration. Two hemolysins with different molecular weights were separated by gel filtration, and the smaller one, tetanolysin, was further purified. The purification raised the specific activity of tetanolysin 1,050-fold to 500 HU/μg of protein. The purified preparation gave a single, relatively broad band on polyacrylamide gel electrophoresis, in which the activity was roughly parallel with the protein concentration. However, on sodium dodecylsulfate-gel electrophoresis it gave two bands with nearly equal amounts of proteins, showing molecular weights of 53,000 and 48,000±3,000. Furthermore, isoelectric focusing revealed four peaks of the activity whose isoelectric pHs were 6.1, 5.6, 5.3, and 6.6 in decreasing order of the activity. These findings suggest that the preparation contains four hemolysins with different pis, which are classifiable into two groups by molecular size. The preparation was completely free of tetanus neurotoxin and proteases. Tetanolysin was more strongly inhibited by cholesterol and more rapidly adsorbed onto erythrocytes than θ-toxin of Cl. perfringens.