Purification and characteristics of an alginase from Alginovibrio aquatilis.

An exocellular inducible alginase from a strain of Alginovibrio aquatilis was purified 61-fold by ammonium sulfate precipitation and column chromatography on Sephadex G-150 and diethylaminoethyl-cellulose. The purified enzyme was more resistant than the crude enzyme to elevated temperatures. The monovalent cations Cs+, Rb+, K+, Na+, and Li+, in order of decreasing enzyme activation, were required for activity. The pH optimum of the purified alginase was 8.0 and its molecular weight from exclusion chromatography on Sephadex G-150 was 110,000.     

Purification and properties of Pichia guilliermondii yeast alkaline nucleotide pyrophosphatase hydrolyzing flavin adenine dinucleotide

Alkaline nucleotide pyrophosphatase was isolated from the Pichia guilliermondii Wickerham ATCC 9058 cell-free extracts. The enzyme was 740-fold purified by saturation of ammonium sulphate, gel-chromatography on Sephadex G-150 and ion-exchange chromatography on DEAE-cellulose. Nucleotide pyrophosphatase is the most active at pH 8.3 and 49 degrees C. The enzyme catalyzes the hydrolysis of FAD, NAD+, NADH, NADPH, GTP. The Km value for FAD is 2.4 x 10(-4) M and for NAD+--5.7 x 10(-6) M. The hydrolysis of FAD was inhibited by NAD+, NADP+, ATP, AMP, GTP, PPi and Pi. The Ki for NAD+, AMP and Na4P2O7 was 1.7 x 10(-4) M, 1.1 x 10(-4) M and 5 x 10(-5) M, respectively. Metal chelating compounds, 8-oxyquinoline, o-phenanthroline and EDTA, inhibited completely the enzyme activity. The EDTA effect was irreversible. The molecular weight of the enzyme determined by gel-filtration on Sephadex G-150 and thin-layer gel-filtration chromatography was 78000 dalton. Protein-bound FAD of glucose oxidase is not hydrolyzed by the alkaline nucleotide pyrophosphatase. The enzyme is stable at 2 degrees C in 0.01 M tris-HCl-buffer (pH 7.5).     

Purification and characterization of a collagenase from the mackerel,Scomber japonicus

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.     

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.     

Purification and properties of the riboflavin kinase of the yeast Pichia guilliermondii]

Riboflavin kinase (E.C.2.7.1.26) was isolated from the cells of the yeast Pichia guilliermondii. The enzyme was 680-fold purified uzing ammonium sulphate fractionation, chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50 and gel-filtration through Sephadex G-75. Purified enzyme preparation was free from phosphatases and FAD-synthetase. The pH optimum was 8,7, the temperature optimum-45 degrees C. The enzyme was activated by Zn2+, Mg2+ and Co2+ ions. Km for riboflavin was 1,0x10(-5) M, for ATP -- 6,7X10(-6) M. Riboflavin kinase catalyzed the phosphorylation of riboflavin analogues with the substitution of methyl groups at positions 7 and 8. UTP, GTP, ADP and CTP, besides ATP, were phosphate donors. AMP inhibited the enzyme activity. Molecular weight of the enzyme was 28000, as estimated by gel-filtration through Sephadex G-150. Purified riboflavin kinase was stable under storage.     

华丽曲霉Z58有机磷农药降解酶的纯化和性质

华丽曲霉(Aspergillus ornatus)Z58有机磷农药降解酶经硫酸铵分级沉淀、Sephadex G100凝胶过滤、DEAE52离子交换层析得到了分离纯化,用聚丙烯酰胺凝胶电泳(PAGE)鉴定为单一组分。凝胶过滤法测得分子量为67 000,提纯倍数为34.2,收率为17.8%。该酶的最适反应温度45℃,最适反应pH72,对热较稳定,并且能在pH6～10范围保持活性。重金属Cu2+对该酶具有明显的促进作用,而SDS对酶具有抑制作用。此酶对所试的有机磷农药都有较好降解作用。     

A galactokinase of Mycobacterium sp. 279 galactose mutant

A galactokinase and the other enzymes of a galactose catabolic pathway were found in Mycobacterium sp. 279 galactose mutant. The galactokinase was partially purified in a procedure involving ammonium sulfate precipitation, Sephadex G-100 filtration and DEAE-cellulose chromatography. The enzyme was 170-fold purified with 25% of recovery. It was most active at pH 7.8-8.0 in the presence of Mg2+, CO2+, Mn2+ or Fe2+ ions. The molecular weight of the enzyme as determined by Sephadex G-100 filtration amounted to 41,700. The apparent Michaelis constants for galactose and ATP in spectrophotometric test were 1.0 mM and 0.29 mM, respectively. Mercuric compounds at concentration of 0.4 mM completely blocked the enzyme. The galactokinase was quite stable during storage at moderatory temperatures and neutral pH but underwent rapid inactivation on heating above 50 degrees C.     

粘质赛氏菌胞外蛋白酶的提取、纯化及部分性质研究

经过硫酸铵３０％～５０％分级沉淀、二步柱层析可获聚丙烯酰胺凝胶电泳均一的粘质赛氏菌胞外蛋白酶制品,收率可达５３％,并制备了酶的结晶,该酶以ＳｅｐｈａｄｅｘＧ１００柱层析及ＳＤＳ－ＰＡＧＥ测得分子量约为８１０００,该酶的最适ｐＨ为７．０,最适温度为４５℃,Ｚｎ２＋、Ｍｎ２＋、Ｆｅ２＋、Ｃｕ２＋、Ｃｏ２＋等重金属离子不同程度地抑制酶活性。     

Hepatic tyrosine aminotransferase from the rainbow lizard Agama agama: purification and some properties

Tyrosine aminotransferase, induced by dexamethasone in the liver of the rainbow lizard, Agama agama, was extracted under optimal conditions which yield the native undegraded enzyme; purified by heat treatment at 65 degrees C, ammonium sulfate precipitation, chromatography on DEAE-Sephacel and Sephadex G-150-120 and then characterized. The enzyme was purified over 2000-fold to a specific activity of 2653 units/mg of protein. It had an optimum pH of 7.6 in potassium phosphate buffer, KmTyr: 1.0 mM; K alpha-KGm: 0.32 mM; Vmax: 1.33 nmol/min and a molecular weight of about 130,000. It was inhibited by L-glutamate (competitively, Ki, 2.5 mM), and by metal ions Ca2+, Mn2+, Zn2+, Hg2+ and Ag2+, but was unaffected by chelating agents and other divalent cations. Lizard hepatic cytosolic tyrosine aminotransferase was specific for L-tyrosine and alpha-ketoglutarate as substrates sensitive to sulfhydryl inactivation and to protection from thermal lability by alpha-ketoglutarate and pyridoxal phosphate.     

Purification of an Apoplastic β-N-Acetyl-D-Hexosaminidase from Tomato Leaves Infected with Tobacco Mosaic Virus

Systematic infection of tomato (Lycopersicon esculenturn) leaves by tobacco mosaic virus (TMV) increased the levels of β-N-acetyl-D-hexosaminidase activity. The enzyme was purified from intercellular fluid by --20℃ acetone precipitation, CM-Sephadex C-25 ion exchange chromatography, Polybuffer Exchanger 94 chromatofocusing and Sephadex G-150 gel filtration column to homogeneity. The molecular weight obtained by SDS-PAGE and Sephadex G-150 gel filtration was 75 kD and 145 kD respectively. The enzyme hydrolysed p-nitrophenyl-N-acetyl-β-glucosaminide and p-nitrophenyl-N-acetyl-β-galaetosaminide, it was a glycoprotein. Most of the enzyme activity in the TMV-infected tomato leaves was found in the intercellular spaces.     

Purification and properties of a DNA ligase from sea urchin embryos

DNA ligase was purified about 2,000-fold from blastulae of sea urchin, Hemicentrotus pulcherrimus, by means of 1 M KCl-extraction, phosphocellulose, DEAE-cellulose, Sepharose CL-6B, and double-stranded DNA cellulose column chromatography. The purified DNA ligase had a molecular weight of 80,000 (determined by Sephadex G-150) and a sedimentation coefficient of 4.1S (by glycerol gradient centrifugation). The purified enzyme required ATP and Mg2+ (or Mn2+) as cofactors for activity, and was inhibited by N-ethylmaleimide. Apparent Km values for ATP, Mg2+, and Mn2+ were 4 microM, 2.7 mM, and 0.3 mM, respectively.     

球孢白僵菌胞外壳聚糖酶的纯化和性质*

球孢白僵菌Beauveria bassiana 1316-V1的培养上清液经硫酸铵分级沉淀,Sephadex G-75凝胶过滤,Chitosan-bead亲和层析,第二次Sephadex G-75凝胶过滤, 得到电泳纯的一种胞外壳聚糖酶,比活力达到45u/mg 。此酶的分子量为36 kD; 最适酶反应温度为60℃;最适pH为4.0;最适离子强度为 0.25mol/L NaCl; 37℃以下,pH 2.0~5.0之间稳定性好; Cu2+、Hg2+、Pb2+、Ni2+ 对该酶有强烈抑制作用;Ag+、Mn2+也有较强抑制作用;Fe2+有轻微激活作用。该壳聚糖酶是一种糖蛋白,含糖约为12.6%。酶的最适底物为脱乙酰度为90%的胶体壳聚糖;也能轻微水解CMC、DEAE-Cellulose和胶体几丁质;但不能水解片状的壳聚糖和几丁质。     

Studies on phospholipase C from Pseudomonas aureofaciens. I. Purification and some properties of phospholipase C.

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Pseudomonas aureofaciens was purified 3600-fold from the culture filtrate with a recovery of 1.6%. Purification was performed with the useof (NH4)2SO4 precipitation, Sephadex G-100 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50. The purified enzyme appeared to be homogeneous as revealed by polyacrylamide disc gel electrophoresis at pH 9.3. The molecular weight was estimated to be 35 000 by gel filtration on Sephadex G-75. Under our experimental conditions, phosphatidylethanolamine was more rapidly hydrolysed than phosphatidylcholine. Lyso forms of these two phosphatides were poor substrates. Phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin and sphingomyelin were not hydrolysed. The enzyme activity with phosphatidylcholine as substrate was slightly stimulated by Ca2+, Mg2+, and Mn2+. However, these cations inhibited the activity with phosphatidylethanolamine as substrate. An anionic detergent, sodium deoxycholate, slightly enhanced the activity when phosphatidylcholine and phosphatidylethanolamine were used as substrates. A cationic detergent, cetyltrimethylammonium bromide, inhibited enzyme activity. EDTA and o-henanthroline inhibited the activity of the enzyme to a marked degree.     

Purification and characterization of arylamidase from monkey brain.

Arylamidase [EC3.4.11.2] was isolated from monkey brain extract and purified about 2100-fold in approximately 11% yield by a six-step procedure comprising extraction from monkey brain homogenate, ammonium sulfate fractionation, first hydroxylapatite chromatography, DEAE-cellulose chromatography, Sephadex G-200 gell filtration and second hydroxylapatite chromatography. The enzyme showed a single band on polyacrylamide disc electrophoresis and consisted of a single polypeptide chain, as judged by disc electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was strongly inhibited by PCMB, TPCK, and puromycin. Puromycin competitively inhibited the enzyme and the Ii value was about 5 x 10(-7)M. Treatment with EDTA resulted in a loss of enzyme activity. The enzyme activity was restored by addition of Zn2+, Co2+, Mn2+. Among various amino acid beta-naphthylamides, L-alanine beta-naphthylamide was most rapidly hydrolyzed and N-carbobenzoxyl-L-leucine beta-naphthylamide was not hydrolyzed by this enzyme preparation. The molecular weight of the enzyme was 92,000 as determined by gel filtration on Sephadex G-200.     

Purification and properties of a beta-1,6-clucosidase from Flavobacterium.

An intracellular beta-1,6-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced semiconstitutively by Flavobacterium M64. This enzyme was purified 180-fold by fractionation with ammonium sulfate followed by chromatographies on carboxymethylcellulose, hydroxyapatite and Sephadex G-100. The final preparation appeared homogeneous on disc electrophoresis on polyacrylamide gel. The molecular weight of the enzyme was determined to be ca. 59 000 by Sephadex G-100 gel filtration and sodium dodecylsulfate-polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 5.8 and the optimum temperature was 40 degrees C. The enzyme readily hydrolyzed oligomers with beta-a,6-glucosidic linkages, converting them to glucose. The Km values for gentio-biose, -triose, -tetraose and -pentaose were 2.8, 3.0, 4.2 and 4.6 times 10- minus 4 M, respectively. The rates of their hydrolyses decreased with increase in their chain lengths. The enzyme was concluded to be a beta-1,6-glucosidase from its substrate specificity, production of glucose, transferring ability and inhibition by glucono-delta-lactone. The enzyme activity was inhibited by Hg-2+, Cu-2+, Ag-+, Fe-3+, p-chloromercuribenzoate, N-ethylmaleimide, glucose and trishydroxyaminomethane (Tris) but not by ethylenediaminetetraacetic acid.     

β-葡聚糖酶的分离纯化和特性研究*

对里氏木霉所产β-葡聚糖酶粗酶液通过饱和硫酸铵沉淀、Sephadex G-100 柱层析和DEAE-Sephadex A-50 柱层析进行纯化,比活提高14.60倍,活力回收6.62%。酶特性研究表明,最适温度和pH分别为60℃和5.0,在pH低于5.0时酶较稳定,酶的热稳定性在60℃以下。 Cu～2+、 Mn～2+ 、Mg～2+ 、Fe～3+ 和K+对酶有抑制作用, Zn～2+、Ca～2+、 Co2+和 Fe～2+ 有激活作用。     

Purification and characterization of 7 beta-hydroxysteroid dehydrogenase from Ruminococcus sp. of human intestine

7 beta-Hydroxysteroid dehydrogenase (7 beta-HSD) was produced by Ruminococcus sp. PO1-3 obtained from among human intestinal bacteria. The enzyme was purified from a crude extract by ammonium sulfate fractionation, and Butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A and Octyl-Sepharose chromatographies. The purified enzyme was obtained as a single band on polyacrylamide gel electrophoresis with enzyme activity staining and as one band corresponding to a molecular weight of 30,000 on SDS-polyacrylamide gel electrophoresis. On gel filtration, its apparent molecular weight was estimated to be 60,000. The enzyme had a sulfhydryl group(s) in its active site. Substrate specificity studies revealed that the enzyme showed absolute specificity for the beta-configuration of a hydroxyl group at the 7 position of bile acids, and required NADP+ and NADPH as cosubstrates. The Km values for ursodeoxycholic acid, 7-k etolithocholic acid, NADP+, and NADPH were 5.0, 8.5, 7.7, and 24 microM, respectively.     

Partial purification and properties of geranyl pyrophosphate synthase from Lithospermum erythrorhizon cell cultures

A prenyltransferase (EC 2.5.1.1) was isolated from cell cultures of Lithospermum erythrorhizon. The enzyme was purified 92-fold by subsequent chromatography on DEAE-Sephacel, phenyl-Sepharose, and Sephadex G-150. Geranyl pyrophosphate (GPP) was the sole product of the enzymatic reaction with dimethylallyl pyrophosphate and isopentenyl pyrophosphate as the substrates. The enzyme showed a molecular weight of 73,000, estimated by gel chromatography on Sephadex G-150, and an isoelectric point at pH 4.95, determined by analytical isoelectric focusing. It had an absolute requirement for a divalent cation with Mg2+ and Mn2+ being most effective. The enzyme was soluble rather than membrane-bound. The physiological role of this prenyltransferase probably is to supply GPP for the biosynthesis of shikonin. It is the first chain-length specific geranyl pyrophosphate synthase reported from eukaryotic cells.     

Complete purification and general characterization of FAD synthetase from rat liver

Flavin adenine dinucleotide synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2) was purified about 10,000-fold from the high-speed supernatant of rat liver by a sequence of ammonium sulfate fractionation and column chromatographies on DEAE-Sephadex (A-50), chromatofocusing, FMN-agarose affinity, and Sephadex G-200. The specific activity of the purified enzyme was 133 units (nanomoles of FAD formed per min at 37 degrees C)/mg of protein. This preparation was free from contaminating FAD pyrophosphatase. The apparent molecular weight was estimated to be 97,000 by gel filtration on Sephadex G-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 53,000. Hence, the enzyme is a dimer of approximately 100,000. The enzyme was found most active at pH 7.1, requires Mg2+, and is essentially irreversible in the direction of FAD formation. Kinetic analysis gave Km values of 9.6 microM for FMN and 53 microM for ATP.     

β-葡聚糖酶的分离纯化和特性研究

对里氏木霉所产β-葡聚糖酶粗酶液通过饱和硫酸铵沉淀、Sephadex G-100 柱层析和DEAE-Sephadex A-50 柱层析进行纯化,比活提高14.60倍,活力回收6.62%。酶特性研究表明,最适温度和pH分别为60℃和5.0,在pH低于5.0时酶较稳定,酶的热稳定性在60℃以下。 Cu～2+、 Mn～2+ 、Mg～2+ 、Fe～3+ 和K+对酶有抑制作用, Zn～2+、Ca～2+、 Co2+和 Fe～2+ 有激活作用。