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.     

Oxygen-dependent Oxidation of Formaldehyde by Cell-free Extract of a Methanol-utilizing Yeast,Candida sp.

The purpose of this study was to investigate the effects of soy germ protein intake on body composition. Wistar rats were fed experimental diets for 16 weeks. These consisted of soy germ protein, soy protein, or casein. Abdominal adipose tissue weights significantly lower and hindlimb muscle weights were significantly higher in the soy germ protein group than in the casein group.     

Purification and Characterization of Formaldehyde Dehydrogenase in a Methanol-utilizing Yeast,Kloeckera sp. No. 2201

An NAD-linked formaldehyde dehydrogenating enzyme was found in the cell-extract of Kloeckera sp. No. 2201, which utilized methanol as a sole source of carbon. The enzyme was inducibly formed in methanol-grown cells. This fact suggests that the enzyme may play a significant role in the methanol metabolism of this yeast. The enzyme was purified from a cell-extract by ammonium sulfate fractionation, column chromatographies on DEAE-cellulose and on hydroxylapatite, and Sephadex G-200 gel filtration. From an experiment with the purified enzyme, it was found that the enzyme specifically required reduced glutathione for activity, and was reactive toward methylglyoxal as well as formaldehyde. The enzyme catalyzed the following reaction:

the enzyme was concluded to be a kind of formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC 1.2.1.1). Other properties of the enzyme were also investigated.     

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.     

Induction of Catalase Activity in a Methanol-utilizing Yeast,Kloeckera sp. No. 2201

The methanol-grown cells of Kloeckera sp. No. 2201 exhibit a markedly high catalase activity as compared with the glucose-grown and ethanol-grown cells. In this connection, specific organelles (“microbodies”) appear only in the methanol-grown cells. When the yeast cells harvested from a methanol medium (cells whose catalase activity had been enhanced to an appreciable extent: “partially induced cells”) were transferred into media containing glucose, ethanol or methanol as the sole carbon and energy source, further increase of catalase activity was mediated only by methanol. This induction of catalase activity was partially inhibited by cycloheximide at its high concentration, but chloramphenicol did not show any effect. Glucose inhibited strongly the induction by methanol, while galactose gave no effect. Electron microscopical observation revealed that the development of microbodies in the cells growing on methanol was hardly affected by cycloheximide. Disappearance of microbodies was observed electron microscopically after the methanol-grown cells (partially induced cells) were transferred to a methanol-glucose medium and cultivated for 8 hr. 3′,5′-Cyclic AMP or dibutyryl-3′,5′-cyclic AMP could not eliminate the inhibitory effect of glucose on the catalase induction. Addition of caffeine or theophylline did not promote the action of the cyclic nucleotides. 3-Amino-1,2,4-triazole inhibited only 40% of the hydrogen peroxide-decomposing activity in the cell homogenate of methanol-grown cells even at its concentration of as high as 10 mm, while sodium azide inhibited the enzyme activity completely at the concentration of 1 mm.     

Purification and Some Properties of Exo-type Fucoidanases from Vibrio sp. N-5

Three fucoidanases were purified from Vibrio sp. N-5 by ammonium sulfate fractionation and chromatography with DEAE-Toyopearl 650 M, Sephacryl S-300 HR, and chromatofocusing. The purified enzymes gave a single band on disc polyacrylamide gel electrophoresis. The molecular weights of the enzymes, E-1, E-2, and E-3 were 39,500, 68,000, and 68,000, respectively, by SDS polyacrylamide gel electrophoresis and 158,000, 68,500, and 67,500 by gel filtration. The enzymes hydrolyzed gagome-fucoidan to give small oligosaccharides containing sulfate as main product.     

Oxidation of Methanol and Formaldehyde by a System Containing Alcohol Oxidase and Catalase Purified from Candida sp. N-16

The oxidation of methanol and formaldehyde was investigated by using some combination systems of alcohol oxidase, catalase, which were purified from Candida N-16, and hydrogen peroxide. The activity of alcohol oxidase was irreversibly inhibited when the enzyme was incubated with 2.5 mm hydrogen peroxide for 15 min. However, the oxidation of methanol to formaldehyde by alcohol oxidase in the presence of catalase was extremely promoted by the addition of 30 mm hydrogen peroxide. Alcohol oxidase could oxidize not only methanol but also formaldehyde as follows: HCHO + 0₂ + H₂O→HCOOH + H₂O₂. The formaldehyde oxidizing activity was inhibited by hydrogen peroxide. The system containing alcohol oxidase and catalase appears to be the entity of the oxygen-dependent oxidation system of formaldehyde previously found in the cell-free extract of the yeast.     

Characterization of a biosurfactant, mannosylerythritol lipid produced from Candida sp. SY16

One yeast strain, SY16, was selected as a potential producer of a biosurfactant, and identified as a Candida species. A biosurfactant produced from Candida sp. SY16 was purified and confirmed to be a glycolipid. This glycolipid-type biosurfactant lowered the surface tension of water to 29 dyne/cm at critical micelle concentration of 10 mg/l (1.5 × 10⁻⁵ M), and the minimum interfacial tension was 0.1 dyne/cm against kerosene. Thin-layer and high-pressure liquid chromatography studies demonstrated that the glycolipid contained mannosylerythritol as a hydrophilic moiety. The hydrophilic sugar moiety of the biosurfactant was determined to be β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by nuclear magnetic resonance (NMR) and fast atom bombardment mass–spectroscopy analyses. The hydrophobic moiety, fatty acids, of the biosurfactant was determined to be hexanoic, dodecanoic, tetradecanoic, and tetradecenoic acid by gas chromatography–mass spectroscopy. The structure of the native biosurfactant was determined to be 6-O-acetyl-2,3- di-O-alkanoyl-β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by NMR analyses. We newly determined that an acetyl group was linked to the C-6 position of the d-mannose unit in the hydrophilic sugar moiety. Received: 18 December 1999 / Received last revision: 2 June 1999 / Accepted: 4 June 1999     

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 Some Properties of an inhibitor for a Yeast Pleiotropic Drug Resistant Pump from Kitasatospora sp. E-420

A strain producing an inhibitor for pleiotropic drug resistant 5 (Pdr5) was isolated using our original screening system in yeast. The strain was classified and named as Kitasatospora sp. E-420. The purified inhibitor (molecular weight=1,193 by FAB-MS) inhibited a Pdr5-mediated efflux of cycloheximide and cerulenin. The intracellular accumulation of a fluorescent dye, rhodamine 123, by the inhibitor was also confirmed. Some physicochemical data suggested that the Pdr5-specific inhibitor was different from an immunosuppressant, FK506, reported as the only inhibitor of Pdr5 in vivo.     

Expression, Isolation, Purification, and Biochemical Properties of Trehalose-6-phosphate Hydrolase from Thermoresistant Strain Bacillus sp. GP16

Here we describe cloning, expression, and purification of the enzyme trehalose-6-phosphate hydrolase from thermoresistant strain Bacillus sp. GP16. Principal biochemical properties of the enzyme at different pH and temperature values were determined. Entropy and enthalpy of activation of the enzyme for substrates trehalose-6-phosphate and p-nitrophenyl glucoside were calculated, and the dependence of the kinetic parameters from ionic strength was established.     

Purification and Properties of a Glycoprotein Acid Phosphatase from Candida albicans

An acid phosphomonoesterase was purified 87-fold with a 4% recovery from disintegrated cells of Candida albicans by four stages of column chromatography. The purified enzyme was homogeneous by ultracentrifugal, electrophoretic, and immunological analyses. The fully corrected sedimentation coefficient, s(20,w), was calculated to be 5.51s. Molecular weight estimated from ultracentrifugal data was 124.3 x 10(3), from gel chromatography was 115 x 10(3), and from acrylamide gel electrophoretic data was 131 x 10(3). Buoyant density in sucrose was 1.15 g/cm(3). The enzyme was a mannoprotein with a hexose to protein ratio of 7: 1. The Michaelis constant of the enzyme was 3.3 x 10(-4) M for p-nitrophenyl phosphate as substrate, and the pH optimum was 4.5. The enzyme was competitively inhibited by inorganic phosphate (K(i) = 10(-4) M) and by arsenate (K(i) = 0.5 x 10(-4) M). A wide range of inorganic cations and anions did not affect enzyme activity, but Hg(2+), Cd(2+), and Cu(2+) were inhibitory. F(-) was also inhibitory at low concentrations, but the effect was reversed at higher concentrations. Phosphatase activity was completely destroyed by exposure of the enzyme to 70 C for 12 min, but was destroyed only slowly by proteolytic hydrolysis. The purified glycoprotein enzyme gave a line of identity with the "b" antigen of crude C. albicans homogenates in immunodiffusion and immunoelectrophoresis tests with sera from rabbits inoculated with intact C. albicans cells and from humans with proven candidiasis. Preliminary evidence suggests that the mannan and not the protein portion of the enzyme molecule is responsible for this antigenicity.     

Purification and Properties of a Soluble Methane Monooxygenase from Methylocystis sp. M

A soluble methane monooxygenase (sMMO: EC 1.14.13.25) was purified from a type II obligate methanotroph, Methylocystis sp. M. Ion exchange chromatography elution separated the sMMO into three components, I, II, and III. Components II and III were purified to homogeneity and were essential for the sMMO activity. Components II and III had molecular masses of approximately 233,000 and 39,000, respectively. Component II consisted of three subunits with molecular masses of 55,000, 44,000, and 21,000, which appeared to be present in stoichiometric amounts, suggesting a (αβγ)₂ configuration in the native protein. Component II contained 1–4 mol of iron and was considered to be a hydroxylase. Component III was a flavoprotein, which contained 1 mol of FAD as well as 1–2mol of iron. It catalyzed the reduction of K₃Fe(CN)₆ and 2,6-dichloroindophenol by NADH. Component I, which was partially purified and not essential for sMMO activity, stimulated the activity by about 11-fold. Its stimulation could be replaced by addition of Fe²⁺. The molecular mass of the partially purified component I was estimated to be from 35,000 to 40,000 based on gel filtration, which suggested the presence of a new type of regulatory protein of sMMO.     

类芽孢杆菌环果寡糖糖基转移酶的分离纯化和酶学性质

采用硫铵沉淀、疏水层析和DEAE离子交换层析对产自类芽孢杆菌Paenibacillus sp.Lfos16的环果寡糖糖基转移酶(Cycloinulooligosaccharide Fructanotransferase,CFTase)进行分离纯化,得到电泳纯的CFTase,最终纯化倍数为13. 9,比活力为33. 3 U/mg。纯化的CFTase经SDSPAGE和Native-PAGE电泳分析得出其相对分子量大小约为120 000,且是双亚基蛋白。探究了CFTase的酶学性质,最适反应条件为:40℃～45℃,p H 7. 0;温度稳定范围为30℃～45℃,p H稳定范围为5. 0～9. 0。并对CFTase降解菊糖的催化机制进行了分析,初步确定为外切加环化作用形成环果寡糖。     

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     

A New Yeast Species,Candida aurita sp. nov., from Oligotrophic Bogs of Western Siberia

Five anamorphous yeast strains of ascomycetous affinity with a specific mode of budding were isolated from high bog soils of the Bakcharskoe Bog (Tomsk oblast). According to their morphological and physiological properties, these strains belong to the genus Candida but differ from all species described previously. The level of DNA–DNA homology with species similar in the assimilation spectrum was as low as 7%. Based on these data, the new species Candida aurita sp. nov. is described.     

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.     

Purification,Crystallization and Properties of Primary Alcohol Dehydrogenase from a Methanol-oxidizing Pseudomonas sp. No. 2941

A simple method for purification and crystallization of primary alcohol dehydrogenase (EC 1.1.99.8) is reported. The purification procedures consisted of four steps: protamine sulfate treatment, ammonium sulfate fractionation, passage through a column of DEAE-cellulose at pH 8.0 and Sephadex G-200 gel filtration. Crystallization was performed by the addition of ammonium sulfate at 65 % saturation with an overall yield of 39 %. The crystalline enzyme had an isoelectric point of pH 7.38 and a sedimentation coefficient 8.44s. A molecular weight of 128,000 was estimated, and the enzyme consisted of two subunits each having a molecular weight of 62,000. The enzyme showed an affinity toward the lower primary alcohols, methanol to n-pentanol. Formaldehyde was also oxidized by the crystalline enzyme. The Km values for methanol and formaldehyde were found to be 20 μm and 70 μm, respectively. Ammonium ions were required for enzyme activity.     

Purification,Characterization and Antibacterial Properties of Peptide from Marine Ascidian Didemnum sp.

International Journal of Peptide Research and Therapeutics - Marine ecosystems are unique and a largely diverse chest of natural resources which are still to be explored for new marine species....     

Purification and Properties of a Novel Enzyme,Maltooligosyl Trehalose Synthase,from Arthrobacter sp. Q36

Arthrobacter sp. Q36 produces a novel enzyme, maltooligosyl trehalose synthase, which catalyzes the conversion of maltooligosaccharide into the non-reducing saccharide, maltooligosyl trehalose (α-maltooligosyl α-D-glucoside) by intramolecular transglycosylation. The enzyme was purified from a cell-free extract to an electrophoretically homogeneous state by successive column chromatography on Sepabeads FP-DA13, DEAE-Sephadex A-50, Ultrogel AcA44, and Butyl-Toyopearl 650M. The enzyme was specific for maltooligosaccharides except maltose, and catalyzed the conversion to form maltooligosyl trehalose. The K_m of the enzyme for maltotetraose, maltopentaose, maltohexaose, and maltoheptaose were 22.9mM, 8.7mM, 1.4mM, and 0.9mM, respectively. The enzyme had a molecular mass of 81,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.1 by gel isoelectrofocusing. The N-terminal and C-terminal amino acids of the enzyme were methionine and serine, respectively. The enzyme showed the highest activity at pH 7.0 and 40°C, and was stable from pH 6.0 to 9.5 and up to 40°C. The enzyme activity was inhibited by Hg²⁺ and Cu2⁺.