Expression of the vanadium-dependent bromoperoxidase gene from a marine macro-alga Corallina pilulifera in Saccharomyces cerevisiae and characterization of the recombinant enzyme

The vanadium-dependent bromoperoxidase from the marine macro-alga Corallina pilulifera was heterologously expressed in Saccharomyces cerevisiae. The enzyme was purified and crystals in "tear drop" form were obtained. The catalytic properties of the recombinant enzyme were studied and compared with those of the native enzyme purified from C. pilulifera. Differences in thermal stability and chloroperoxidase activity were observed. The recombinant enzyme retained full activity after preincubation at 65 degrees C for 20 min, but the native enzyme was completely inactivated under the same conditions. The chlorinating activity of the native enzyme was more than ten times higher than that of the recombinant enzyme. Other properties, such as K(m) values for KBr and H(2)O(2), and optimal temperature and pH, were similar for each source of C. pilulifera bromoperoxidase.     

Increased thermostability and phenol removal efficiency by chemical modified horseradish peroxidase

Horseradish peroxidase was modified by phthalic anhydride and glucosamine hydrochloride. The thermostabilities and removal efficiencies of phenolics by native and modified HRP were assayed. The chemical modification of horseradish peroxidase increased their thermostability (about 10- and 9-fold, respectively) and in turn also increased the removal efficiency of phenolics. The quantitative relationships between removal efficiency of phenol and reaction conditions were also investigated using modified enzyme. The optimum pH for phenol removal is 9.0 for both native and modified forms of the enzyme. Both modified enzyme could suffer from higher temperature than native enzyme in phenol removal reaction. The optimum molar ratio of hydrogen peroxide to phenol was 2.0. The phthalic anhydride modified enzyme required lower dose of enzyme than native horseradish peroxidase to obtain the same removal efficiency. Both modified horseradish peroxidase show greater affinity and specificity of phenol.     

Purification and biochemical characterization of recombinant rat liver phenylalanine hydroxylase produced in Escherichia coli.

Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to rat phenylalanine hydroxylase. Recombinant rat phenylalanine hydroxylase was placed into an expression vector in Escherichia coli. The enzyme has been purified to homogeneity and its physical and catalytic properties have been characterized. The molecular weight and the fluorescence emission spectrum of the recombinant enzyme were identical to those of the native enzyme. The recombinant enzyme could be activated by incubation with phenylalanine or lysolecithin or by phosphorylation, as is the rat liver enzyme. The extent of activation is the same as that for the native enzyme in each case except for phenylalanine, which activates the recombinant enzyme only 5- to 10-fold rather than the 15- to 30-fold activation observed with the native enzyme. The kinetic constants determined for the recombinant enzyme are also essentially the same as those reported for the native enzyme. We conclude that this enzyme is essentially identical to the native enzyme and should be very useful in the future study of this important hydroxylase.     

Mutants of d-aminopeptidase with increased thermal stability

Mutant d-aminopeptidases from Ochrobactrum anthropi with increased thermal stability were obtained by random mutagenesis. One of the mutants, no. 65, was derived from E. coli cells transformed with DNA treated with sodium nitrite. The remaining activity of the purified mutant ezyme no. 65 after heat treatment at 52°C for 10 min was 20% that of the untreated mutant enzyme no. 65, whereas the native enzyme showed 5% of the untreated native enzyme activity after the same treatment. The gene for the mutant enzyme no. 65 was sequenced and it was found that Gly155 and Gly279 in the native enzyme were replaced by Ser and Asp, respectively. Five mutants carrying one or two mutations were generated from the native gene by site-specific mutagenesis. The enhancement of the thermal stability of mutant enzyme no. 65 was attributed to the substitution of Gly155 to Ser.     

棕色固氮菌含铁超氧化物歧化酶重组研究

将棕色固氮菌230含铁超氧化物歧化酶对8mol/L脲,10mmol/L EDTA透析制备无活性缺辅基蛋白;将其在8mol/L脲中对10mmol/L硫酸亚铁铵透析得到重组超氧化物歧化酶。重组酶含有与天然酶相近的铁含量,活性为天然酶的89.1％。缺辅基蛋白,重组酶与天然酶都是由二个相同的亚基组成;重组酶的吸收光谱与荧光光谱与天然酶几乎一样,而缺辅基蛋白则有较大的差异;从园二色谱的分析得知,缺辅基蛋白不含有α—螺旋,而天然酶和重组酶中α螺旋的含量分别为21％和20％;缺辅基蛋白比天然酶或重组酶具有更大的巯基反应性。     

In vitro and in vivo activity of soluble cross-linked uricase-albumin polymers: A model for enzyme therapy

The possibility of using soluble cross-linked enzyme-albumin polymers as a means of enzyme therapy for the treatment of certain enzyme deficiency diseases is investigated. The hyperuricemic Dalmatian coach hound is used as an experimental animal and the enzyme uricase (urate oxidase) as the administered enzyme. Chemically cross-linking uricase with an excess of canine albumin yields a soluble enzyme polymer that is significantly more heat stable and resistant to proteolytic activity than the native enzyme. Intravenous administration of similar amounts of enzyme in the native or polymeric form indicated that the “solubilized” enzyme survived in the circulation for a longer period of time (clearance half-time of 26 hours as opposed to 4 hours for the native enzyme) and was more effective in lowering plasma uric acid levels for longer periods. In vivo administration of the native enzyme lowered uric acid levels by about 35% with a return to normal levels with a half-time of about 24 hours. Subsequent injections of native uricase proved less effective and produced a severe hypersensitivity reaction following the third injection. No such adverse reactions or decreased activity of the administered “solubilized” uricase-albumin polymers were observed. The plasma uric acid levels were decreased by about 40% and only after 48 hours did the substrate levels begin to rise towards their resting levels.     

Purine nucleoside phosphorylase of the malarial parasite, Plasmodium lophurae

Purine nucleoside phosphorylase (EC 2.4.2.1, purine nucleoside:orthophosphate ribosyltransferase) was purified and characterized from the malarial parasite, Plasmodium lophurae, using a chromatofocusing (Pharmacia) column and a formycin B affinity column. The apparent isoelectric point of the native protein, as determined by chromatofocusing, was 6.80. By gel filtration and both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the native enzyme appeared to be a pentamer with a native molecular weight of 125,300 and a subunit molecular weight of 23,900. The enzyme had a broad pH optimum, pH 5.5-7.5, with maximum activity at pH 6.0-6.5. The enzyme reaction was readily reversible with a Km for inosine of 33 microM and a Km for hypoxanthine of 82 microM. Thioinosine, guanosine, and guanine were also substrates for the plasmodial enzyme, but allopurinol and adenine were not. The parasite enzyme was competitively inhibited by formycin B (Ki = 0.39 microM). Formycin A, azaguanine, and 8-aminoguanosine were not inhibitors of the enzyme.     

Immobilized flounder muscle glyceraldehyde 3-phosphate dehydrogenase

Summary Partially purified flounder muscle (Pseudopleuronectus americanus) glyceraldehyde 3-phosphate dehydrogenase was immobilized on cyanogen bromide-activated Sepharose. The catalytic properties of the immobilized preparation were studied to determine if immobilization alters the kinetic properties of the native holoenzyme. The results indicate that the pH activity profile of immobilized glyceraldehyde 3-phosphate dehydrogenase did not differ from that of the native enzyme. The Michaelis constants (Km) for NAD and glyceraldehyde 3-phosphate were somewhat altered. The enzyme stability toward various inactivation treatments in the presence and absence of NAD was characterized and compared to that of he native enzyme. When either form of the enzyme was incubated with urea at concentrations greater than 2m, inactivation occurred very rapidly. Incubation in 0.1% trypsin for 60 minutes decreased the activity of immobilized glyceraldehyde 3-phosphate dehydrogenase by 45% and of the native soluble enzyme by 70%. The immobilized enzyme also exhibited considerably more stability than the native soluble enzyme when exposed to a temperature of 50° or to 20 mm ATP. In all cases NAD either greatly reduced the rate of inactivation or completely protected the enzyme from inactivation.     

Production of levan using recombinant levansucrase immobilized on hydroxyapatite

Levansucrase of Zymomonas mobilis was immobilized onto the surface of hydroxyapatite by ionic binding. Optimum conditions for the immobilization were: pH 6.0, 4 h of immobilization reaction time, and 20 U of enzyme/g of matrix. The enzymatic and biochemical properties of the immobilized enzyme were similar to those of the native enzyme, especially towards the effect of salts and detergents. The immobilized enzyme showed sucrose hydrolysis activity higher as that of the native enzyme, but levan formation activity was 70% of the native enzyme. HPLC analysis of levan produced by immobilized enzyme showed the presence of two different types of levan: high-molecular-weight levan and low-molecular-weight levan. The proportion of low-molecular-weight levan to total levan produced by the immobilized enzyme was much higher than that with the native enzyme, indicating that immobilized levansucrase could be applied to produce low-molecular-weight levan. Immobilized levansucrase retained 65% of the original activity after 6 times of repeated uses and 67% of the initial activity after 40 d when stored at 4 °C.     

Structural specificity of the allosteric inhibitor of phosphoenolpyruvate carboxylase of Escherichia coli.

The structural specificity of the allosteric inhibitor of phosphoenolpyruvate carboxylas [EC 4.1.1.31] of Escherichia coli W was investigated using native enzyme and photooxidized enzyme which was desensitized to L-aspartate. Inhibitory activity was expressed in terms of the concentration of the compound required for 50% inhibition (I0.5). For the native enzyme, L-aspartate and L-malate were the strongest inhibitors with I0.5 values of about 0.10-0.15 mM among about 20 componds tested. For the photooxidized enzyme, oxaloacetate and L-malate were relatively strong inhibitors wiht I0.5 values of about 11-16 mM. The results obtained suggest that the inhibition of the native enzyme mainly reflects allosteric inhibition.     

Purification and characterisation of chicken brain hypoxanthine-guanine phosphoribosyltransferase

Hypoxanthine-guanine phosphoribosyltransferase enzyme (EC 2.4.2.8) from chicken brain has been purified 10 000-fold to homogeneity. The molecular mass of the native enzyme is 85 kDa, with four subunits, each of 26 kDa, and exerts its maximum activity at pH 10.0. The Km values for hypoxanthine and guanine are 5.2 and 1.8 microM, respectively. The half-life of the enzyme is 30 min at 85 degrees C. Monoclonal antibodies were raised against the native purified enzyme and were used for purification of enzyme to homogeneity. The monoclonal antibody did not bind to the active centre of the enzyme.     

尼龙网固定化果胶酶的制备及其性质研究

用尼龙网作载体,经3-二甲氨基丙胺活化,用戊二醛将果胶酶固定化。所得固定化酶Km值与自然酶接近;对温度的稳定性有较大的提高,100℃保温30min才能使其失活。固定化酶在较宽的pH范围内能保持其正常活力,它对金属离子抑制剂的耐受性有较显著的提高,用0.5％果胶溶液作底物,重复使用10次后酶活力保留44％。固定化果胶酶与自然酶相比较,对不同果汁的澄清效果不同。固定化果胶酶在无保护剂存在的条件下,室温放置四个月活力不减少。     

Function of Mannan Chains of Yeast Repressible Acid Phosphatase on Its Enzymatic Properties

To find the function of the mannan chains covalently attached to yeast repressible acid phosphatase, the N-glycosidic carbohydrate chains were removed by endo-β-N-acetyl-glucosaminidase H under native conditions. Almost all of the N-glycosidic mannan chains were cleaved off by the glycosidase. The deglycosylated enzyme was shown to be a dimer structure as is the native enzyme. The deglycosylated enzyme retained enzyme activity, the same Km, and the same circular dichroism spectra as the native enzyme. These results indicate that the carbohydrate chains are not essential for maintaining the active enzyme structure, but the deglycosylated enzyme was shown to be more sensitive to acidic pH and high temperature.     

Action of crystalline acid carboxypeptidase from Penicillium janthinellum.

Acid carboxypeptidase (EC 3.4.12.-) crystallized from culture filtrate of Penicillium janthinellum has been investigated for its use in carboxy-terminal sequence determination of Z-Gly-Pro-Leu-Gly, Z-Gly-Pro-Leu-Gly-Pro, angiotensin I, native lysozyme, native ribonuclease T1, and reduced S-carboxy-methyl-lysozyme. The examination indicated that proline and glycine were liberated from Z-Gly-Pro-Leu-Gly-Pro. At high enzyme concentration, the enzyme catalyzed complete sequential release of amino acids from the carboxy-terminal leucine to the amino-terminal aspartic acid of angiotensin I. The enzyme released the carboxy-terminal leucine from native lysozyme, however, no release of the threonine from native ribonuclease T1 was observed after a prolonged period of incubation with the enzyme. The sequence of the first nine carboxy-terminal residues of denatured lysozyme, leucine, arginine, S-carboxymethyl-cysteine, glycine, arginine, isoleucine, tryptophane, alanine, and glutamine, could be deduced unequivocally from a time release plot of an incubation mixture with the enzyme.     

The role of metal in liver alcohol dehydrogenase catalysis. Spectral and kinetic studies with cobalt-substituted enzyme.

The kinetic and spectral properties of native and totally cobalt-substituted liver alcohol dehydrogenase have been compared. Based on titrimetric determinations of enzyme active site concentration, the turnover number at pH 7.0 for cobalt enzyme was the same as for native enzyme. At pH 10, however, the turnover number was slower for cobalt-substituted enzyme, 3.14 s-1 as compared with 4.05 s-1 for native enzyme. A comparison between native and totally cobalt-substituted enzyme showed a blue-shifted enzyme-NADH double difference spectrum and a splitting and red-shifted enzyme-NAD+-pyrazole double difference spectrum in the near-ultraviolet. The 655-nm peak of the cobalt-substituted enzyme was perturbed by the formation of enzyme-NADH binary complex, enzyme-NAD+-trifloroethanol ternary complex, and enzyme-NAD+ binary complex formation. At pH 7.0, the only observable step in the reaction sequence with a significantly different rate constant for cobalt enzyme was the catalytic hydrogen-transferring step. The rate constant for this step is 92 s-1 for totally cobalt-substituted enzyme as compared with 138 s-1 for native liver alcohol dehydrogenase. The results of this study indicate that zinc is involved in catalysis alcohol and NADH.     

Purification and characterization of Mycobacterium tuberculosis KatG, KatG(S315T), and Mycobacterium bovis KatG(R463L)

Isoniazid, a first-line antibiotic used for the treatment of tuberculosis, is a prodrug that requires activation by the Mycobacterium tuberculosis enzyme KatG. The KatG(S315T) mutation causes isoniazid resistance while the KatG(R463L) variation is thought to be a polymorphism. Much of the work to date focused on isoniazid activation by KatG has utilized recombinant enzyme overexpressed in Escherichia coli. In this work, native KatG and KatG(S315T) were purified from M. tuberculosis, and KatG(R463L) was purified from Mycobacterium bovis. The native molecular weight, enzymatic activity, optical, resonance Raman, and EPR spectra, K(D) for isoniazid binding, and isoniazid oxidation rates were measured and compared for each native enzyme. Further, the properties of the native enzymes were compared and contrasted with those reported for recombinant KatG, KatG(S315T), and KatG(R463L) in order to assess the ability of the recombinant enzymes to act as good models for the native enzymes.     

Biochemical and genetic characterization of the structure of yeast ornithine decarboxylase

The ornithine decarboxylase gene of S. cerevisiae encodes a predicted protein of approximately 53 kD highly homologous with the ornithine decarboxylase of other species. However, the native enzyme has been reported as an 86 kD protein. Our molecular sieve analysis indicated a Mr = 110,000 for the native enzyme. SDS-PAGE analysis of [H3]-alpha-difluoromethylornithine labelled enzyme demonstrated a subunit Mr of approximately 50 kD and suggested the native enzyme is a dimer. Genetic analyses support this conclusion. The complementary, ornithine decarboxylase deficient mutations spe 1A and spe 1B were mapped to the enzyme structural gene by linkage analysis and gene conversion mapping. This demonstrated that the mutations exhibit intragenic complementation which suggests protein-protein interactions and an oligomeric structure for the yeast enzyme. We conclude that yeast ornithine decarboxylase is a dimeric enzyme of 53 kD subunits.     

Monovalent cation-induced conformational change in glucose oxidase leading to stabilization of the enzyme

Glucose oxidase (GOD) from Aspergillus niger is an acidic dimeric enzyme having a high degree of localization of negative charges on the enzyme surface and dimer interface. We have studied the effect of monovalent cations on the structure and stability of GOD using various optical spectroscopic techniques, limited proteolysis, size exclusion chromatography, differential scanning calorimetry, and enzymic activity measurements. The monovalent cations were found to influence the enzymic activity and tertiary structure of GOD, but no effect on the secondary structure of the enzyme was observed. The monovalent cation-stabilized GOD was found to have a more compact dimeric structure but lower enzymic activity than the native enzyme. The enzyme's K(m) for D-glucose was found to be slightly enhanced for the monovalent cation-stabilized enzyme (maximum enhancement of about 35% for LiCl) as compared to native GOD. Comparative denaturation studies on the native and monovalent cation-stabilized enzyme demonstrated a significant resistance of cation-stabilized GOD to urea (about 50% residual activity at 6.5 M urea) and thermal denaturation (Delta T(m) maximum of 10 degrees C compared to native enzyme). However, pH-induced denaturation showed a destabilization of monovalent cation-stabilized GOD as compared to the native enzyme. The effectiveness of monovalent cations in stabilizing GOD structure against urea and thermal denaturation was found to follow the Hofmeister series: K(+) > Na(+) > Li(+).     

Immobilization of the exo-maltohexaohydrolase by the irradiation method

Exomaltohexaohydrolase (E.C.3.2.1.98) was immobilized by radiocopolymerization of some synthetic monomers which were mixed in various combinations. Irradiation was carried out while the mixture of monomers and enzymes was frozen in petroleum ether-dry-ice bath. Recovery of the immobilized enzyme was 44-75%.The optimum pH of the enzyme slightly shifted to the acidic side. The pH stability was improved remarkably by immobilization. The enzyme was stable retaining more than 90% of its original activity in the range pH 4-11. The optimum reaction temperature of the enzyme increased about 2 degrees C. Heat stability was also improved by immobilization, and that the enzyme retained about 40% of its original activity after treatment at 75 degrees C for 15 min. The immobilized enzyme was stable to the repeated use of 20 cycles. The K(m) value of the enzyme for short-chain amylose was almost the same as that of native enzyme. When soluble starch was used as the substrate, the K(m), value of the enzyme was three times as large as that of native enzyme. Effects of various metal ions and inhibitors on the immobilized enzyme were also studied compared to the native enzyme.     

Preparation of immobilized enzymes by N-vinylpyrrolidone and the general properties of the glucoamylase gel

Immobilized glucoamylase, invertase, and β-galactosidase were prepared by using N-vinylpyrrolidone monomer (VP) under γ-ray irradiation. The enzyme-VP solutions were gelled by irradiation with 2.9 Mrad and the added enzymes were almost completely entrapped. Activity losses on entrapping were 55% for the VP-glucoamylase gel, and more than 90% in the case of VP-invertase and VP-β-galactosidase gels. No leakage of enzyme from these gels could be detected within 1 hr. The VP-glucoamylase gel was capable of hydrolyzing dextrin (mol wt 10,400) to glucose and the glucose equivalent was equal to that obtain able with native enzyme. The optimum temperature, heat stability, pH activity curve, and pH stability of VP-glucoamylase gel were slightly inferior to those of native enzyme, while Km was a little larger than that of native enzyme.