Purification and Some Properties of Crystalline Acid Protease from Acrocylindrium sp.

A crystalline acid protease produced by a strain of Acrocylindrium in a submerged culture was prepared by treatment with acetone (60%), salting out with ammonium sulfate (saturated) and, after chromatography on Duolite GS-101 column, dialysis against distilled water. This preparation was homogeneous on sedimentation analysis, starch-gel electrophoresis and gel filtration with Sephadex G-75. The optimum pH was 2.0 for milk casein digestion and the pH stability was for 2.0~5.0 at 30°C for one day. The crystalline enzyme was completely stable below 50°C, but lost the activity at 70°G in ten minutes. The acid protease was almost equal to pepsin on specific activity when milk casein solution (pH 2.0) was used as substrate.     

Purification and Properties of Catalasc from a Methanol-utilizing Yeast,Candida sp. N-16

Defatted soybean extract was fractionated into protein fractions and low molecular weight fractions with gel filtration. NAD-dependent aldehyde dehydrogenase from bovine liver mitochondria and from yeast was found to oxidize aldehyde in both fractions. These enzymes, therefore, were used to determine the quantity of aldehyde. When the protein fraction obtained by gel filtration was subjected to gel filtration again, aldehyde was recovered in the protein fractions. The level of aldehyde in the protein fractions was unchanged before and after digestion of the protein with pepsin. When the soybean extract was incubated beforehand with aldehyde dehydrogenase and NAD⁺ and the subjected to gel filtration, no aldehyde was detected in the protein fractions. These results indicate that aldehyde dehydrogenase acts on the soybean protein-bound aldehyde. Alcohol dehydrogenase from horse liver in the presence of NADH did not convert the bound aldehyde to alcohol.

A large portion of the aldehyde in the extract was separated from the protein by acid precipitation of the protein. Aldehyde dehydrogenase acts on the aldehyde remaining in the protein after acid precipitation. Thus acid precipitation helps to save NAD⁺ required for complete removal of aldehyde from the soybean protein by aldehyde dehydrogenase.     

Purification and Some Properties of a New Levanase from Bacillus sp. No. 71

A levanase from Bacillus sp. was purified to a homogeneous state. The enzyme had a molecular weight of 135,000 and an isoelectric point of pH 4.7. The enzyme was most active at pH 6.0 and 40°C, stable from pH 6.0 to 10.0 for 20 hr of incubation at 4°C and up to 30°C for 30 min of incubation at pH 6.0. The enzyme activity was inhibited by Ag ⁺, Hg^{2 +}, Cu^{2 +}, Fe^{3 +}, Pb²⁺, and p-chloromercuribenzoic acid. The enzyme hydrolyzed levan and phlein endowise to produce levanheptaose as a main product. The limit of hydrolysis of levan and phlein were 71% and 96%, respectively.     

毛壳霉内切菊粉酶的纯化与性质

毛壳霉 (Chaetomiumsp .)C34发酵液经硫酸铵分级沉淀、DEAE 纤维素 11离子交换层析、Q SepharoseFastFlow离子交换层析、SephacrylS 2 0 0凝胶过滤、PhenolSepharoseTM HP疏水层析 ,得到电泳纯的内切菊粉酶组分 ,纯化倍数为 30 8倍 ,活力回收率为 7 7%。用SDS PAGE测得该酶亚基的分子量为 6 6kD。菊粉酶的最适pH为 6 0 ,最适温度为 5 0～ 5 5℃。菊粉酶在 5 0℃以下 ,pH5 0～ 8 0时较稳定。Cu2 完全抑制酶的活性 ,Mn2 、Zn2 、Fe2 、EDTA以及NBS(N bromosuccinimide ,N 溴代丁二酰亚胺 )对该酶有很强的抑制作用。该酶对菊粉有较强底物专一性 ,产物主要为低聚果糖 ,也可作用于蔗糖 ,I S值为 2 0。以菊粉为底物时 ,Km 为 0 199mmol L ,Vmax为 115 μmol (mg·min)。     

Purification and characterization of alginate lyase from marine Vibrio sp. YWA

Extracellular alginate lyase secreted by marine Vibrio sp.YWA,isolated from decayedLaminaria japonica,was purified by a combination of ammonium sulfate precipitation and diethylaminoethyl-Sephacel column chromatography.The results show that the molecular mass of alginate lyase wasapproximately 62.5 kDa,with an optimal pH and temperature at pH 7.0 and 25℃,respectively.K_m wasapproximately 72.73 g/L.The activity of the enzyme was enhanced by EDTA and Zn~(2 ),but inhibited by Ba~(2 ).The substrates specificity analysis shows that it was specific for hydrolyzing poly-β-D-1,4-mannuronate inalginate.     

Purification and Properties of an Alcohol Dehydrogenase Isozyme from a Methanol-using Yeast,Candida sp. N-16

The distribution coefficients of glucose, maltose, and maltotriose on cation-exchange resin in Na⁺ form with a divinylbenzene content of 4% were determined in the temperature range of 5 to 60°C. The coefficients increased with decreasing temperature. The temperature dependence of the distribution coefficient was analyzed based on the swelling pressure of the resin.     

Purification and characterization of a novel beta-agarase from Vibrio sp. AP-2

beta-Agarase was purified from the culture fluid of a porphyran-decomposing marine bacterium (strain AP-2) by ammonium sulfate precipitation, successive column chromatography and DNase and RNase treatment. The final enzyme preparation appeared to be homogeneous on polyacrylamide gel electrophoresis. The enzyme had a molecular mass of 20 kDa, a pH optimum of 5.5, and was stable in the pH region 4.0-9.0 and at temperatures below 45 degrees C. The beta-agarase was a novel endo-type enzyme which hydrolyzed neoagarotetraose, larger neoagarooligosaccharides and agar to give neoagarobiose [3,6-anhydro-alpha-L-galactopyranosyl-(1----3)-D-galactose] as the predominant product. The enzyme did not act on kappa-carrageenan. According to the criteria of Bergey's Manual of Systematic Bacteriology, the strain was assigned to the genus Vibrio.     

Purification and Characterization of Two Epoxide Hydrolases from Corynebacterium sp. Strain N-1074

Enzymes II_a and II_b, which catalyze the conversion of epichlorohydrin (ECH) to 3-chloro-1,2-propanediol (MCP), were purified from Corynebacterium sp. strain N-1074, which catalyzes the formation of (R)-MCP from prochiral 1,3-dichloro-2-propanol via ECH. The specific activity of enzyme II_a for the formation of MCP from ECH was about 6.4-fold higher than that of enzyme II_b. Both enzymes catalyzed the conversion of 1,2-epoxides to the corresponding diol, although they differed in several enzymatic properties.     

Studies on Sugar-isomerizing Enzyme Purification,Crystallization and Some Properties of Glucose Isomerase from Streptomyces sp.

Glucose isomerase was purified by means of acetone fractionation, DEAE-cellulose column chromatography, DEAE-Sephadex column chromatography and crystallization. The purified enzyme appeared to be homogeneous on ultracentrifugation and electrophoresis. The sedimentation coefficient, s_20,w, the diffusion coefficient, D_20,w, and partial specific volume of the enzyme were 8.0S, 4 × 10^?7cm²/sec and 0.69 ml/g, respectively. The molecular weight of the enzyme was estimated to be 157,000 from the sedimentation and diffusion measurements. The crystalline glucose isomerase contained cobalt and magnesium ions. The properties of the enzyme were also studied.     

嗜碱菌(Bacillus sp.)ZBAW6的木聚糖酶的分离纯化及其性质

通过硫酸铵分级沉淀 ,阴离子交换层析 ,凝胶过滤 3步从嗜碱菌Bacillussp .ZBAW6纯化了木聚糖酶。结果表明该酶分子量为 4 5kD。N末端序列为DPFAAAVAPL。在pH5 5～ 1 0 5范围内均具有较高酶活性和稳定性 ;最适反应温度为 6 5℃ ,酶活力基本不变。该酶作用于Beech xylan的Km 为 0 1 1mg mL ,Vmax 为 2 3 89μmol (min·mg)。Hg2 + 对该酶有强的抑制作用     

海洋弧菌褐藻胶裂解酶的分离纯化及性质

从海带糜烂物中分离到一株高产胞外褐藻胶裂解酶的海洋弧菌 (Vibriosp .QY10 1) ,利用硫酸铵沉淀、离子交换层析、凝胶过滤层析等方法从发酵液中分离纯化了褐藻胶裂解酶 (alginatelyase)。SDS PAGE电泳结果表明 ,该酶分子量为 39kD。酶反应最适pH为 7.5 ,最适反应温度为 30℃。Na 、Ca2 、Mn2 对酶活性有促进作用 ,Fe2 、Ni2 以及EDTA对酶活性有抑制作用。酶的底物专一性初步分析结果表明 ,该酶具有降解多聚古罗糖醛酸[poly(G) ]及多聚甘露糖醛酸 [poly(M) ]的活性。     

Purification and some properties of chitinase produced by Vibrio sp.

Chitinase was purified from the culture filtrate of a Vibrio sp. isolated from soil and its enzymatic properties were examined. The molecular weight measured by SDS-gel electrophoresis was approximately 100,000. The chitinase hydrolyzed colloidal chitin, chitin powder, chitosan and chitin oligosaccharides more than chitotriose but did not hydrolyze glycolchitin and chitobiose.     

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.     

从海洋中分离的弧菌QY102褐藻胶裂解酶的纯化和性质研究

从马尾藻(Sargassum)表面分离到一株产生高效胞外褐藻胶裂解酶的海洋弧菌（Vibrio sp.） QY102。以褐藻胶为唯一碳源发酵培养后,发酵液上清通过0.22μm滤膜过滤、DEAESepharose离子交换和Superdex75凝胶过滤得到电泳纯的褐藻胶裂解酶。酶的性质研究表明：其分子量约为28.5kD（SDSPAGE）,反应最适温度为40℃,最适pH为7.1,Ca2+、Mg2+对酶活有促进作用,而Ni2+、Al3+、Zn2+、Ba2+对酶活有抑制作用。该酶的活性明显高于已报道的褐藻胶裂解酶,pH稳定范围广（5～10）,并且对聚甘露糖醛酸的活性高于对聚古罗糖醛酸的活性。     

Purification and Some Properties of Cyclo(Gly-Gly) Hydrolase from a Strain of Bacillus sp. No. 106

Bacillus sp. No. 106, which was isolated from soil, secreted an enzyme that hydrolyzed cyclo(Gly-Gly). The enzyme was purified to the ultracentrifugally homogeneous state and an activity more than 450-fold that of culture broth. The enzyme was activated by Na⁺, Mg²⁺, Ca²⁺, and Sr²⁺, and strongly inhibited by Ni²⁺, Cu²⁺, p-chloromercuribenzoate, and monoiodoacetic acid. The Km value for cyclo(Gly-Gly) was estimated to be 11.1 mm. The enzyme hydrolyzed only cyclo(Gly-Gly) among various diketopiperazines tested. Aslo, the enzyme was inert toward Gly-Gly, milk casein, and hemoglobin.     

Purification and Properties of a New Enzyme,Glutathione Oxidase from Penicillium sp. K-6-5

A new flavoprotein enzyme, GSH oxidase, was found in the aqueous extract of a wheat bran culture of Penicillium sp. K-6-5. The oxidase is also produced extra and intracellularly in the liquid culture, although the production is much lower than that in the wheat bran culture.

The enzyme has been purified to homogeneity. It shows absorption maxima at 270, 350 and 444 nm and a shoulder around 465 nm and contains 2 mol of FAD per mol of enzyme. The enzyme has a molecular weight of approximately 95,000 and consists of two subunits identical in molecular weight (about 47,000). Balance studies show that 2 mol of GSH are converted to 1 mol of GSSG and hydrogen peroxide with the consumption of 1 mol of oxygen. In addition to GSH, several sulfhydryl compounds are oxidized by the enzyme to a lesser extent. The Michaelis constants are as follows: 0.69 mm for GSH, 3.6 mm for l-cysteine and 6.7 mm for dithiothreitol at pH 7.4. The oxidase scarcely acts on reduced RNase A in contrast to the known sulfhydryl oxidases. The isoelectric point and the optimal pH are 4.2 and 7.4, respectively. The enzyme activity is completely inhibited by addition of 1 mm ZnSO₄.     

Purification and properties of two malate dehydrogenases from Candida sp. N-16 grown on methanol

Two malate dehydrogenases (MDH-M1 and MDH-M2) were found in a methanol-using yeast, Candida sp. N-16. MDH-M2 was induced with methanol. These enzymes were purified as electrophoretically and isoelectrophoretically homogeneous proteins. The molecular weights of MDH-M1 and MDH-M2 were estimated to be about 78,000 (homodimer) and 160,000 (homotetramer). Several kinetic properties were significantly different between the two enzymes. The value (2.07) of Vmax(oxaloacetate)/Vmax(malate) and Kcats (555 s(-1) for oxaloacetate, 481 s(-1) for NADH) of MDH-M2 were higher than the ratio (1.37) of Vmax and Kcats (241 s(-1) for oxaloacetate, 271 s(-1) for NADH) of MDH-M1, respectively. The activity of MDH-M2 was inhibited by a high concentration of NAD+ and the activity of MDH-M1 by oxaloacetate.     

Purification and Properties of Phospholipase D from Actinomadura sp. Strain No. 362

An extracellular phospholipase D from Actinomadura sp. Strain No. 362 was purified about 430-fold from the culture filtrate. The purified enzyme preparation was judged to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point of the enzyme were estimated to be about 50,000—60,000 and 6.4, respectively. The enzyme was most active at pH 5.5 and 50°C in the presence of Triton X-100, but showed the highest activity at pH 7.0 and 60 — 70°C in its absence. The enzyme was stable up to 30°C at pH 7.2 and also stable in the pH range of 4.0 to 8.0 on 2 hr incubation at 25°C. With regard to substrate specificity, this enzyme hydrolysed lecithin best among the phospholipids tested. It was activated by Fe^{3 +}, Al³⁺, Mn^{2 +}, Ca^{2 +}, diethyl ether, sodium deoxycholate and Triton X-100, but was inhibited by cetyl pyridinium chloride and dodecylsulfate.