Characterization of kinetics and thermostability of Acremonium strictum glucooligosaccharide oxidase

The kinetic and thermostability properties of a glucooligosaccharide oxidase from Acremonium strictum were determined. This enzyme produces only maltobionic acid from maltose. It is most active at pH 9 to 10.5, and is most stable at pH 6.5. Values of both K(M) and V(max) on maltose are highest at pH 10. The highest values of K(M) and V(max) occur with glucose, maltopentaose, and maltoheptaose, whereas the lowest values of K(M) are with maltotriose and of V(max) are with maltohexaose. Values of K(M) with any substrate and at any pH are always substantially above 1 mM. Activation energies for catalysis and thermoinactivation are 23 kJ/mol and 421 kJ/mol, respectively. The N-terminal sequence is not homologous with any other oxidase, but has some homology with other proteins having different functions. These unusual properties suggest that glucooligosaccharides may not be the primary substrates of this enzyme.     

A novel enzyme, L-tryptophan oxidase, from a basidiomycete, Coprinus sp. SF-1: purification and characterization

A basidiomycete, Coprinus sp. SF-1, was found to produce an L-Trp-oxidizing enzyme by screening from the culture collection of our laboratory. After solubilization by 1 M NaSCN from the particulate fraction of disrupted cells of the strain, the enzyme was purified about 76-fold to essential homogeneity. The enzyme had a molecular mass of about 420 kDa and the subunit molecular mass was 68 kDa. The enzyme contained 1 mol of non-covalently bound FAD per mol of the subunit. It catalyzed the simultaneous reactions of oxidative deamination and oxygenative decarboxylation of L-Trp to form indolepyruvic acid and indole-3-acetamide, the former of which was further oxidized to indole-3-acetic acid. The molar ratio of the respective reaction products was about 9:1. The enzyme specifically oxidized L-Trp, and slightly acted on L-Phe and L-Tyr. The Km for L-Trp was about 0.5 mM in both oxidase and oxygenase reactions. Thus, the enzyme is a novel one and was tentatively designated "L-Trp oxidase (deaminating and decarboxylating)". The optimum pHs of oxidase and oxygenase activities were 7.0 and 9.0, respectively. The optimum temperatures of both activities were 50 degrees C. The enzyme was stable at pH 6.0-10.5 and below 50 degrees C, and at 4 degrees C for 1 year.     

Intracellular glucose oxidase from Penicillium funiculosum 46.1

A method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1, using ultrafiltration membranes, was developed. Two samples of the enzyme with a specific activity of 914-956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6.0. The effective rate constant of glucose oxidase inactivation at pH 2.6 and 16 degrees C was 2.74 x 10(-6) s-1. This constant decreased significantly as pH of the medium increased (4.0-10.0). The temperature optimum for glucose oxidase-catalyzed beta-D-glucose oxidation was in the range 30-65 degrees C. At temperatures below 30 degrees C, the activation energy for beta-D-glucose oxidation was 6.42 kcal/mol; at higher temperatures, this parameter was equal to 0.61 kcal/mol. Kinetic parameters of glucose oxidase-catalyzed delta-D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of 2-deoxy-D-glucose, maltose, and galactose.     

Purification and characterization of <Emphasis Type="Italic">Chromobacterium</Emphasis> sp. DS-1 cholesterol oxidase with thermal,organic solvent,and detergent tolerance

A new screening method for 6beta-hydroperoxycholest-4-en-3-one (HCEO)-forming cholesterol oxidase was devised in this study. As the result of the screening, a novel cholesterol oxidase producer (strain DS-1) was isolated and identified as Chromobacterium sp. Extracellular cholesterol oxidase of strain DS-1 was purified from the culture supernatant. The molecular mass of the purified enzyme was 58 kDa. This enzyme showed a visible adsorption spectrum having peaks at 355 and 450 nm, like a typical flavoprotein. The enzyme oxidized cholesterol to HCEO, with the consumption of 2 mol of O2 and the formation of 1 mol of H2O2 for every 1 mol of cholesterol oxidized. The enzyme oxidized 3beta-hydroxysteroids such as cholesterol, beta-cholestanol, and pregnenolone at high rates. The Km value for cholesterol was 26 microM. The enzyme was stable at pH 3 to 11 and most active at pH 7.0-7.5, showing optimal activity at pH 7.0 and 65 degrees C. The enzyme retained about 80% of its activity after incubation for 30 min at 85 degrees C. The thermal stability of the enzyme was the highest among the cholesterol oxidases tested. Moreover, the enzyme was more stable in the presence of various organic solvents and detergents than commercially available cholesterol oxidases.     

Thermal stability of Penicillium adametzii glucose oxidase]

The thermal stability of glucose oxidase was studied at temperatures between 50 and 70 degrees C by kinetic and spectroscopic (circular dichroism) methods. The stability of glucose oxidase was shown to depend on the medium pH, protein concentration, and the presence of protectors in the solution. At low protein concentrations (< 15 micrograms/ml) and pH > 5.5, the rate constants kin (s-1) for thermal inactivation of glucose oxidase were high. Circular dichroic spectra suggested an essential role of beta structures in stabilizing the protein globule. At a concentration of 15 micrograms protein/ml, the activation energy Ea of thermal inactivation of glucose oxidase in aqueous solution was estimated at 79.1 kcal/mol. Other thermodynamic activation parameters estimated at 60 degrees C had the following values: delta H = 78.4 kcal/mol, delta G = 25.5 kcal/mol, and delta S = 161.9 entropy units. The thermal inactivation of glucose oxidase was inhibited by KCl, polyethylene glycols, and polyols. Among polyols, the best was sorbitol, which stabilized glucose oxidase without affecting its activity. Ethanol, phenol, and citrate exerted destabilizing effects.     

Thermodynamic control of D-amino acid oxidase by benzoate binding

The redox properties of D-amino acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating) EC1.4.3.3) have been measured at 18 degrees C in 20 mM sodium pyrophosphate, pH 8.5, and in 50 mM sodium phosphate, pH 7.0. Over the entire pH range, 2 eq are required per mol of FAD in D-amino acid oxidase for reduction to the anion dihydroquinone. The red anion semiquinone is thermodynamically stable as indicated by the separation of the electron potentials and the quantitative formation of the semiquinone species. The first electron potential is pH-independent at -0.098 +/- 0.004 V versus SHE while the second electron potential is pH-dependent exhibiting a 0.060 mV/pH unit slope. The redox behavior of D-amino acid oxidase is consistent with that observed for other oxidase enzymes. On the other hand, the behavior of the benzoate-bound enzyme under the same conditions is in marked contrast to the thermodynamics of free D-amino acid oxidase. Spectroelectrochemical experiments performed on inhibitor-bound (benzoate) D-amino acid oxidase show that benzoate binding regulates the redox properties of the enzyme, causing the energy levels of the benzoate-bound enzyme to be consistent with the two-electron transfer catalytic function of the enzyme. Our data are consistent with benzoate binding at the enzyme active site destroying the inductive effect of the positively charged arginine residue. Others have postulated that this positively charged group near the N(1)C(2) = O position of the flavin controls the enzyme properties. The data presented here are the clearest examples yet of enzyme regulation by substrate which may be a general characteristic of all flavoprotein oxidases.     

Purification and Properties of Alcohol Oxidase from Candida methanosorbosa M-2003

Alcohol oxidase from Candida methanosorbosa was purified about sixfold with a yield of 37.6% from the culture broth of Candida methanosorbosa M-2003. The enzyme preparation was homogeneous on slab gel electrophoresis. The purified enzyme had an optimal pH from 6.0 to 9.0 and was stable in the range 6.0–8.5. Its optimal temperature of reaction was 50°C, and it was stable below 50°C. In the presence of NaN₃, the enzyme retained its initial activity at 30°C for 35 days, indicating stability for a long term, so far. The isoelectric point was pH 4.3. Its molecular weight was 620,000 by gel filtration chromatography and 80,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. These results indicate that the enzyme consists of 8 subunits. Received: 1 October 1996 / Accepted: 12 December 1996     

颗粒状固定化青霉素酰化酶的研究

将巨大芽孢杆菌 (Bacillusmegaterium)胞外青霉素酰化酶通过共价键结合到聚合物载体EupergitC颗粒环氧基团上 ,制成的颗粒状固定化青霉素酰化酶表现活力达 1 40 0 μ/g左右。固定化酶水解青霉素的最适 pH8 0 ,最适温度为 55℃。在pH6 0～ 8 5、温度低于 40℃时固定化酶活力稳定。在 pH8 0、温度 37℃时 ,固定化酶对青霉素的表现米氏常数Ka为 2×1 0 - 2 mol/L ;苯乙酸为竞争性抑制剂 ,抑制常数Kip为 2 8× 1 0 - 2 mol/L ;6 APA为非竞争性抑制剂 ,抑制常数Kia为 0 1 2 5mol/L。固定化酶水解青霉素 ,投料浓度为 8% ,在使用 2 0 0批后 ,保留活力 80 %左右 ,6 APA收率平均达 89 48%。     

聚丙烯腈纤维固定化青霉素酰化酶性质的研究

将巨大芽孢杆菌（Bacillusmegaterium）青霉素酞化酶连接到聚丙烯腈纤维载体上,制成固定化青霉素酰化酶。其表现活力约为2000u／g。水解青霉素G的最适温度为50℃;最适PH为9．0;在PHS．5～10．3、温度50℃以下酶的活力稳定;表观米氏常数Ka为1．33×10-8mol／L;最大反应速度Vm为2．564mmol·min-1;苯乙酸为竞争性抑制剂,抑制常数为0．16mol／L。水解10％的青霉素G钾盐溶液,使用20批,保留酶活力80％。     

Immobilized enzyme system for determination of sialic acid in serum or urine.

An immobilized enzyme system has been developed and employed to determine the concentration of sialic acid (N-acetylneuraminic acid) in human serum and urine. Two enzyme pairs, neuramindiase-Neu-5-Ac lyase and pyruvate oxidase-peroxidase, have been respectively co-immobilized onto 1,12-aminododecane-agarose with glutaraldehyde. The relative specific activity of the co-immobilized neuraminidase and Neu-5-Ac lyase were 60% and 78%, and those of pyruvate oxidase and peroxidase were 50% and 95% of the corresponding soluble enzymes, respectively. The optimal reaction pH at 37 degrees C for each of the co-immobilized enzymes was about one pH unit higher than that of the corresponding soluble enzyme. The optimal reaction temperature of each enzyme was increased as a result of immobilization. The thermal stability at 45 degrees C of the immobilized neuraminidase, Neu-5-Ac lyase, pyruvate oxidase, and peroxidase were increased 80-, 83-, 115-, and 147-fold, respectively. Km and Vm of each immobilized and co-immobilized enzyme have also been determined. The system provided a convenient and rapid method to determine the concentration of total sialic acid without pretreatment of the sample. The results correlated satisfactorily with those obtained by using a soluble enzyme system. The co-immobilized enzymes were stable for at least 1 year of 500 tests when used repeatedly. The system is thus a reproducible and reliable novel assay method for sialic acid in the serum or urine sample.     

3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Properties of its acetyl derivative.

Ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) reacts with acetyl-CoA to form a complex in which the acetyl group is covalently bound to the enzyme. This acetyl group can be removed by addition of acetoacetyl-CoA or CoA. The extent of acetylation and release of CoA were found to be highly temperature-dependent. At temperatures above 20 degrees C, a maximum value of 0.85 mol of acetyl group bound/mol of enzyme dimer was observed. Below this temperature the extent of rapid acetylation was significantly lowered. Binding stoichiometries close to 1 mol/mol of enzyme dimer were also observed when the 3-hydroxy-3-methylglutaryl-CoA synthase activity was titrated with methyl methanethiosulphonate or bromoacetyl-CoA. This is taken as evidence for a 'half-of-the-sites' reaction mechanism for the formation of 3-hydroxy-3-methylglutaryl-CoA by 3-hydroxy-3-methylglutaryl-CoA synthase. The Keq. for the acetylation was about 10. Isolated acetyl-enzyme is stable for many hours at 0 degrees C and pH 7, but is hydrolysed at 30 degrees C with a half-life of 7 min. This hydrolysis is stimulated by acetyl-CoA and slightly by succinyl-CoA, but not by desulpho-CoA. The site of acetylation has been identified as the thiol group of a reactive cysteine residue by affinity-labelling with the substrate analogue bromo[1-14C]acetyl-CoA.     

聚丙烯腈纤维固定化青霉素酰化酶合成头孢氨苄的研究

将巨大芽孢杆菌胞外青霉素酰化酶通过共价键结合到聚丙烯腈纤维的衍生物上。制成的丝状固定化青霉素酰化酶表现活力达 1 5 3U g(湿重 )。固定化酶合成头孢氨苄的最适pH为 6 5 ,最适温度为 40℃。 7 ADCA的投料浓度以 4%为好 ,7 ADCA与PGME的投料量比率为1∶2 ,最佳用酶量为 1 70U g 7 ADCA。在pH6 5、温度 3 0℃时 ,固定化酶对 7 ADCA的表观米氏常数K7 ADCA为 0 1 6 2mol L ,对PGME的表观米氏常数KPGME为 0 3 6 4mol L ,最大反应速度Vmax为0 0 4 6 2mol·L- 1·min- 1,用固定化酶合成头孢氨苄 ,使用 5 0次保留酶活力 83 9%     

Cholesterol oxidase catalyzed oxidation of cholesterol in mixed lipid monolayers: effects of surface pressure and phospholipid composition on catalytic activity

The catalytic activity of cholesterol oxidase from Streptomyces sp. in mixed monolayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), N-oleoylsphingomyelin (O-SPM), and cholesterol (CHL) has been determined at lateral surface pressures between 10 and 30 mN/m. The highest cholesterol oxidase activity (determined at 37 degrees C) was observed at surface pressures around 20 mN/m in a POPC/CHL monolayer (50:50 mol %). Above and below this surface pressure, the enzyme activity decreased markedly. A similar optimal activity vs surface pressure relationship was observed also for an O-SPM/CHL monolayer (50:50 mol %). The activity of cholesterol oxidase toward cholesterol in the O-SPM/CHL monolayer was, however, less than in the corresponding POPC mixed monolayer. The surface activity of cholesterol oxidase decreased markedly when the temperature was lowered to 20 degrees C, and hardly any enzyme activity was observed in an O-SPM/CHL monolayer at 25 mN/m or above. With a monolayer containing POPC/O-SPM/CHL (42:18:40 mol %), maximal cholesterol oxidase activity was observed at the lowest surface pressure tested (i.e., 10 mN/m), and the catalytic activity decreased markedly with increasing lateral surface pressures in the monolayer. The results of this study show (i) that the activity of cholesterol oxidase in general is highly dependent on the lateral surface pressure in the substrate membranes and (ii) that sphingomyelin, by interacting tightly with cholesterol, can prevent or restrain the accessibility of cholesterol for oxidation by cholesterol oxidase.     

Polyvinylferrocenium modified Pt electrode for anaerobic glucose monitoring

An amperometric enzyme electrode for the determination of glucose under anaerobic solution conditions was developed by immobilizing glucose oxidase and then by adsorbing ferrocene in polyvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of ferrocene that the reduced flavin adenine dinucleotide centers of glucose oxidase was measured at a constant potential. The response characteristics of the enzyme electrode were investigated. The effects of the thickness of the polymeric film, the amount of the enzyme immobilized, the amount of the mediator, the glucose concentration, the applied potential, operating pH and temperature on the response of the enzyme electrode were studied. The response time and the optimum pH were found to be 30-40 s and pH 7.4 at 25 degrees C, respectively. The linear response was observed up to 5.0 mM glucose concentration that the produced detectable current was 0.0075 mM glucose concentration. The activation energy (E(a)) of immobilized enzyme reaction was calculated to be 41.3 kJ mol(-1) from the Arrhenius plot. The apparent Michaelis-Menten constant (K(Mapp)) was found to be 6.05 mM glucose according to the Lineweaver-Burk graph of the Michaelis-Menten equation under the optimum conditions. The interference signal due to the most common electrochemical interfering species was also evaluated.     

球孢白僵菌Bb174固态发酵产几丁质酶产酶及酶学特征研究

对球孢白僵菌(Beauveria bassiana)Bb174产几丁质酶进行了固态发酵条件及酶学特征的研究．结果表明,以4：1麸皮：蚕蛹粉、蛋白胨1g·L^-1作为产酶最适培养基,在7．5g培养基中接种3ml液态种子,自然pH下28℃培养2d,酶活可达最高,为126U·g^-1(干培养基)．粗酶液的最适反应温度为40℃,最适反应pH5．0,在30-70℃保温1h,得半失活温度48℃．在30--40℃、pH4～6范围内,酶的性质最稳定．根据Lineweaver-Burk作图法,得到该酶的动力学参数Km为0．52mg·ml^-1,Vm为0．7△E680·h^-1．     

Degradation of ribonucleic acid by immobilized ribonuclease.

An immobilized enzyme (pancreatic ribonuclease bound to porous titania) was investigated for the degradation of purified yeast ribonucleic acid as a substrate. The immobilized enzyme is active and stable in the pH range 4--8. Dependence of enzymatic activity on ionic strength, pH, temperature, fluid flow rate, and substrate concentration were investigated. A cumulative fluid residence time of 6 sec is sufficient for 50% substrate conversion at 25 degrees C and pH 7.0. The critical flow rate (i.e., the fluid flow rate necessary to remove film diffusion resistance) approximately doubles with each 10 degree C rise in reaction temperature. The critical flow rates obtained in this study are about 40 times greater than those obtained for a similar study on immobilized glucose oxidase. Arrhenius plots gave activation energies of -9.6 and -7.1 kcal/g mol at pH 4.6 and 7.0, respectively. The work reported herein is a bench-scale investigation of an immobilized enzyme with primary emphasis on the mass transfer and kinetic characteristics of the system. The rapid reaction rates obtainable at relatively low temperatures offer a potential alternative method of purifying yeast single cell protein (SCP) with miminum loss of desired protein. The key questions are how such a system would react in a yeast homogenate, what conditions in such a system must be controlled, and what type of immobilized reactor should be utilized, if such further work continued to show promise.     

Production of novel oligosaccharide oxidase by wheat bran solid-state fermentation

A search for oxidases that catalyze the oxidation of oligosaccharides has resulted in the isolation of several soil-derived fungus strains which produced novel oligosaccharide oxidases with different substrate specificity on wheat bran solid culture. One of these oxidases produced by Acremonium strictum T1 strain has been characterized. This enzyme showed high reactivity toward maltose, lactose, cellobiose and maltooligosaccharides composed of up to seven glucose units, and was named as glucooligosaccharide oxidase based on its substrate specificity. Strain T1 was subjected to a strain improvement program, and an enzyme hyper-producing mutant strain T1-38 was selected. This mutant strain produced glucooligosaccharide oxidase 75 times higher than the wild type strain T1. When cultivated in a solid medium comprised of 1 part of wheat bran and 1 part of water (w/w), enzyme activity reached a maximum level of 6 units per g of culture medium after 4 days cultivation. Characteristics of the enzyme including the substrate specificity were compared with two other novel oligosaccharide oxidases isolated in this laboratory. Batch type conversion of lactose to lactobionic acid using crude enzyme was also discussed.     

芽孢杆菌M50产生β—甘露聚糖酶的条件研究

从土壤中分离到９株产生β－甘露聚糖酶的芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｐ．）。Ｂａｃｉｌｌｕｓ ｓｐ．Ｍ５０２５０ｍＬ三角瓶摇瓶培养试验,以４％的魔芋粉为碳源,１．０％（ＮＨ４）２ＳＯ４为氮源,０．３５％Ｎａ２ＣＯ３,３０～３４℃培养６０ｈ产酶达到高峰。酶活力为１８０～２２０ｕ／ｍＬ。１００Ｌ罐发酵,在３０～３２℃,１：０．７５ｖｖｍ通气量,２００ｒ／ｍｉｎ条件下,发酵液酶活力高达３３０ｕ／ｍＬ。     

2-Chloro-2-phenylethylamine as a mechanistic probe and active site-directed inhibitor of monoamine oxidase from bovine liver mitochondria

The reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B was investigated to study the mechanism of this enzyme and its inactivation by this compound. 2-Chloro-2-phenylethylamine is a substrate with a Km of 30 microM and a turnover number of 80 min-1 at pH 6.5 at 30 degrees C. Incubation of 2-chloro-2-phenylethylamine with the enzyme led to the normal oxidation product, 2-chloro-2-phenylacetaldehyde, but only traces (0.25 mol%) of 2-phenylacetaldehyde, the product anticipated if the oxidation of substrate involved a stabilized carbanion at C-1 and elimination of chloride ion. These data suggest that a carbanion is not a likely intermediate in the oxidation of amines by monoamine oxidase. During the mechanistic studies we noted time-dependent inactivation of monoamine oxidase B by 2-chloro-2-phenylethylamine under both aerobic and anaerobic conditions. Inactivation was not reversible. Aerobically 2-chloro-2-phenylethylamine is oxidized to 2-chloro-2-phenylacetaldehyde which covalently modifies the enzyme (tau 1/2 = 40 min). Benzyl alcohol, a substrate analog, gives substantial protection against inactivation under aerobic conditions (tau 1/2 = 320 min), suggesting that an active site residue is modified. Anaerobic reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B probably proceeds by direct alkylation of an enzyme residue (tau 1/2 = 140 min). Reduction with [3H]NaBH4 of the inactivated enzyme gave from 0 to 0.7 and from 4.5 to 5.6 mol of hydride incorporation for enzyme inactivated anaerobically and aerobically, respectively. The latter results are in agreement with inactivation by unmodified inhibitor and inactivation by oxidized inhibitor for the anaerobic and aerobic reactions, respectively. It is suggested that 2-chloro-2-phenylethylamine or its oxidation product 2-chloro-2-phenylacetaldehyde may serve as an active site affinity reagent for monoamine oxidase.     

Trapping choline oxidase in a nonfunctional conformation by freezing at low pH

Choline oxidase is a flavin-dependent enzyme that catalyzes the oxidation of choline to glycine-betaine, with oxygen as electron acceptor. Storage at pH 6 and -20 degrees C resulted in a change in the conformation of choline oxidase, which was associated with complete loss of catalytic activity when the enzyme was assayed at pH 6. Incubation of the inactive enzyme at pH values > or = 6.5 and 25 degrees C resulted in a fast and partial reactivation of the enzyme, which occurred with slow onset of steady state during enzymatic turnover. The rate of approaching steady state was independent of the concentrations of choline and enzyme, but increased to a limiting value with increasing pH, defining a pKa value of approximately 7.3 for an unprotonated group required for enzyme activation. Prolonged incubation of the inactive enzyme at pH 6 and temperatures > or = 20 degrees C, at which no hysteretic behavior was observed, resulted in the slow and full recovery of activity over 3 h, associated with a conformational change that reverted the enzyme to the native form. Activation of the enzyme at pH 6 was enthalpy-driven with deltaH(double dagger) and TdeltaS(double dagger) values of approximately 112 kJ mol(-1) and approximately 20 kJ mol(-1) determined at 25 degrees C. These data suggest that freezing the enzyme at low pH induces a localized and reversible conformational change that is associated with the complete and reversible loss of catalytic activity.