New Amylase Inhibitor (S-AI) from Streptomyces diastaticus var. amylostaticus No. 2476

A one-step synthesis of dimethyl 3-substituted pyrrole-2, 4-dicarboxylates by the reaction of methyl isocyanoacetate with various aldehydes in the presence of DBU was carried out. Furthermore, the reaction mechanism was investigated; it was found that the methyl α-isocyanoacrylate compound is a key intermediate of the pyrrole compound.     

Some Properties of Amylase Inhibitor Produced by Streptomyces sp. No. 280

A strain of Streptomyces produces a new substance capable of inactivating some amylases. This has not been reported by previous workers.

This amylase inhibitor was purified by means of acetone treatment, active carbon adsorption and column chromatography on DEAE-cellulose.

It was dialyzable through a cellophane membrane and soluble in water and methyl alcohol. The inhibitor had a small molecular weight and was a peptide-like substance. The inhibiting substance was resistant to the temperatures, and acted as inhibitor of glucoamylase, bacterial saccharogenic α-amylase, salivary and pancreatic α-amylases.     

半夏胰蛋白酶抑制剂的纯化与部分性质研究

应用以胰蛋白酶为配体的亲和层析法,从生半夏蛋白粗提液的40％（NH。）：SO4（M／V）沉淀中分离纯化出一种胰蛋白酶抑制剂（Rhizoma pinelliae trypsin inhibitor,RVn）,经SDS．PAGE检测呈现单一条带,分子量为14kD左右,其N-端6个氨基酸残基顺序为DPVVDG。研究表明,其对胰蛋白酶的质量抑制比为l：4．72左右,对人低分化胃腺癌细胞系（BGC-823）的细胞增殖具有抑制作用,IC50值为121．53μg／mL。     

Further Purification of Amylase Inhibitor Produced by Streptomyces sp.

The amylase inhibitor produced by Streptomyces sp. consisted of, at least, three kinds of inhibiting materials (inhibitor A, B and C). These inhibitors were separated by the Chromatographic techniques on Amberlite CG–120 and Dowex 1 × 2, and by paper chromatography etc.

Inhibitor A, B and C seemed to contain both carbohydrates and amino acids, but the specific activities (I. U./mg glucose) of the inhibitors A, B and C were different, that is, 18,800, 45,700 and 59,000, respectively. Also their ratios of amino acids to neutral sugars (expressed by moles of leucine per those of glucose) were approximately 0.09 (inhibitor A), 2.77 (inhibitor B) and 0.58 (inhibitor C).     

苦荞种子胰蛋白酶抑制剂的分离纯化及部分性质研究

采用凝胶层析及离子交换层析等方法,从苦荞种子中分离出一组胰蛋白酶抑制剂（ＴＢＴＩ－Ⅰ、Ⅱ）．对其性质研究表明：两个组分均对胰蛋白酶有较强的抑制作用,对胰凝乳蛋白酶抑制作用较弱,其中ＴＢＴＩ－Ⅱ的抑制作用大于ＴＢＴＩ－Ⅰ,两者对胃蛋白酶、木瓜蛋白酶及枯草杆菌蛋白酶均无抑制作用．用ＳＤＳ－聚丙烯酰胺凝胶电泳和ＳｅｐｈａｄｅｘＧ－１００凝胶层析分别对纯化产物进行分析得出ＴＢＴＩ－Ⅰ和ＴＢＴＩ－Ⅱ的近似分子量分别为１５．０ｋＤ和１８．０ｋＤ．ＴＢＴＩ－Ⅰ、Ⅱ都具有较高的热稳定性,在１００℃处理１０ｍｉｎ后可保留８６％左右的抑制活性．ＴＢＴＩ在酸性环境下较为稳定,在ｐＨ２．０条件下保温１ｈ,仍保留７５％的抑制活性．用Ｌｉｎｅｖｅａｅｒ－Ｂｕｒｋ作图法得知,该抑制剂属竞争性抑制类型,ＴＢＴＩ－Ⅱ的Ｋｉ值为３．５９×１０－７ｍｏｌ／Ｌ（以ＢＡＰＮＡ为底物）,对胰蛋白酶的摩尔抑制比为１∶１．４．     

Purification and Properties of Trypsin Inhibitor from Hakuhenzu Bean (Dolichos lablab)

A highly purified trypsin inhibitor was obtained from the oriental plant Hakuhenzu bean (Dolichos lablab) by column chromatography on DEAE-Sephadex and gel-filtration on Sephadex G–75. The purified Hakuhenzu bean trypsin inhibitor (HTI) was obtained as a chemically homogeneous protein, and was stable to heat and to enzymes such as pepsin. It shows no obvious maximum at 280 nm in the ultraviolet absorption spectrum, and it contains more than 20% carbohydrate as galactose and 10% hexosamine as glucosamine. The molecular weight of this inhibitor was determined to be approximately 9,500 by gel-filtration. The protein contained 59 residures of amino acids; Lys₃, His₄, Arg₁, Asp₈, Thr₃, Ser₉, Glu₆, Pro₅, Gly₁, Ala₃, l/2Cys₁₀, Val₁, Ile₁, Leu₂, Tyr₁, Phe₁, from which a molecular weight of 6,400 is obtained. No methionine and tryptophan were found in the amino acid composition of the inhibitor. This inhibitor showed inhibitory activity against α-chymotrypsin in addition to trypsin.     

Purification and Some Properties of an Extracellular Amylase from a Moderate Halophile, Micrococcus halobius

A moderate halophile, Micrococcus halobius ATCC 21727, produced an extracellular dextrinogenic amylase when cultivated in media containing 1 to 3 M NaCl. The amylase was purified from the culture filtrate to an electrophoretically homogenous state by glycogen-complex formation, diethylaminoethyl-cellulose chromatography, and Bio-Gel P-200 gel filtration. The enzyme had maximal activity at pH 6 to 7 in 0.25 M NaCl or 0.75 M KCl at 50 to 55°C. The activity was lost by dialysis against distilled water. Molecular weight was estimated to be 89,000 by sodium dodecyl sulfate-gel electrophoresis. The action pattern on amylose, soluble starch, and glycogen showed that the products were maltose, maltotriose, and maltotetraose, with lesser amount of glucose.     

菠菜种子胰蛋白酶抑制剂的分离纯化与部分性质研究

以菠菜种子为材料,经脱脂、酸性溶液抽提、热变性、硫酸铵分部沉淀得到胰蛋白酶抑制剂粗提物。再经离子交换、亲和层析和凝胶过滤,分离得到胰蛋白酶抑制剂SOTI,纯化倍数为57．22。SDS-PAGE测定其分子量约为22kD,等电聚焦测定其等电点为4．02。SOTI具有较高的热稳定性,在100℃处理后仍然具有一定的抑制活性。     

Partial Purification and Some Properties of the Dialyzable Proteinase Inhibitor from the Eggplant Exocarp

The dialyzable proteinase inhibitor in the exocarp of the eggplant, Solanum melongena L. was partially purified by column chromatographies on DEAE-cellulose and Sephadex G–25. The inhibitor showed strong inhibitory activity on bovine trypsin [EC 3.4.4.4], Pronase and Nagarse. It also weakly inhibited α-chymotrypsin [EC 3.4.4.5], but, pepsin was not affected. The presence of three inhibitors in this preparation was demonstrated by isoelectrofocusing; their isoelectric points being pH 4.2, 4.7 and 6.3. The inhibitor with a pI value of 4.7 was present significantly more than the others. The molecular weight of the inhibitor was 5300 based on Sephadex G–75 gel filtration data. It was also very stable to heat treatment. This inhibitor was not the multi-headed type, and was gradually inactivated after a 4 hr incubation with Pronase.     

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.     

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.     

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 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.     

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 Chlorogenic Acid Oxidase from Apple (Malus pumila)

Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malus pumila cv. Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65,000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6–8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (?)-epicatechin and 4-methylcatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The K_m value for the enzyme was found to be 122μM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyl-dithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity.     

Purification and Some Properties of Metal-binding Proteins in Housefly Larvae (Musca domestica)

Cadmium and zinc-binding proteins similar to metallothionein have been isolated from housefly larvae (Musca domestica) exposed to cadmium chloride. Amino acid composition analysis found a high half-cystine content and an apparent minimum molecular weight of 5225. Metal-binding proteins of Musca domestica contained 3.9 g-atoms and 4.5 g-atoms of heavy metals per mole, respectively, and showed the spectral characteristic of cadmium-thionein, i.e., a broad shoulder at 250 nm and low residual absorption at 280 nm. The simple and specific replacement of cadmium and zinc bound to the protein with a cupric ion indicates that proteins have mercaptide bonding with a high affinity for copper. The molecular weight of the proteins modified with Ellman’s reagent was 5300 ± 250 when measured by gel filtration in the presence of 6 M guanidine hydrochloride.     

Purification and Some Properties of Milk Protease

Separation of acetic acid from palm oil mill effluent (POME) to increase its concentration by an anion exchange resin was examined as a preliminary study for its recovery from POME that had been anaerobically treated by sludge from a palm oil mill. This paper concerns the acetic acid thus separated for producing bacterial polyhydroxyalkanoate (PHA) by Alcaligenes eutrophus. It was found that sludge particles in POME strongly inhibited the adsorption of acetic acid on the anion exchange resin. Removing the sludge particles from the POME facilitated the separation of acetic acid from the POME efficiently. The concentrated acetic acid thus obtained from anaerobically treated POME could be used as a substrate in the fed-batch production of polyhydroxyalkanoate by Alcaligenes eutrophus.     

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.     

Purification and Properties of a Mitochondrial Lipoprotein Inhibitor of Sterol Synthesis

A lipoprotein inhibitor of hydroxymethylglutaryl CoA reductase (EC 1.1.1.34) and of cholesterol synthesis by rat liver homogenates, was isolated from the mitochondria of starved rats’ livers. The isolated lipoprotein complex contained a low molecular weight protein and fatty acids. The fatty acids identified were arachidonic, linoleic, oleic, stearic and palmitic. The saturated fatty acids and oleic acid did not inhibit. Inhibition of the enzyme was to a large extent related to the degree of fatty acid unsaturation.