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.     

Fabrication of butyrylcholinesterase sensor using polyurethane-based ion-selective membranes.

A simple method of enzyme immobilization was investigated which is useful for fabrication of enzyme sensors based on polymeric ion-selective membranes. The enzyme membrane was built by coating a thin hydrophilic polyurethane (HPU) film directly mixed with an enzyme over an underlying polyurethane (PU)-based ion-selective membrane. This highly simple method of enzyme immobilization was applied to the fabrication of a potentiometric butyrylcholinesterase-based biosensor for the determination of organophosphorus pesticides. The enzyme was well entrapped within the HPU film and the intrinsic potentiometric response of the underlying ion-selective PU membrane was not influenced significantly by the outer HPU/enzyme membrane. The enzyme electrode was optimized by changing systematically the composition of the enzyme membrane to evaluate the effect of the changes on sensor response. The sensor was successfully applied to the analysis of paraoxon, an organophosphorus pesticide.     

Comparative studies on dihydrofolate reductases from Plasmodium falciparum and Aotus trivirgatus.

Dihydrofolate reductase (E.C. 1.5.1.3) from Plasmodium falciparum and from its host, the owl monkey (Aotus trivirgatus), were partially purified and characterized. The molecular weight of the parasite enzyme was estimated to be over 10 times as high as that of the host enzyme. The host enzyme had 2 pH optima whereas the parasite enzyme only one. The activity of the host enzyme was greatly stimulated by KCl and urea, while that of the parasite enzyme was inhibited at high concentrations of such chaotropic agents. Km of the parasite enzyme was significantly higher than that of the host enzyme. The parasite enzyme had much lower Ki for pyrimethamine than the host enzyme. Dihydrofolate reductases isolated from pyrimethamine-resistant and pyrimethamine sensitive strains of P. falciparum were found to be similar.     

Purification and characterization of dipeptidyl peptidase IV from Streptococcus salivarius HHT.

Dipeptidyl peptidase IV (DAP IV) was purified from Streptococcus salivarius HHT by anion-exchange chromatography, gel filtration and affinity chromatography after lysis of cell walls with N-acetylmuramidase. DAP IV was purified 114-fold with a yield of 16.6% from total activity of the crude extract. The purified enzyme was shown to be homogeneous by disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 109,000 by gel filtration and 47,000 by sodium dodecylsulphate SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a dimeric form. The optimum pH for the reaction was 8.7 in Gly-NaOH buffer, and the isoelectric point of the enzyme was pH 4.2. The enzyme hydrolysed specifically N-terminal X-Pro from X-Pro-p-nitroanilides. The enzyme activity was hardly affected by various cations, sulphydryl-blocking reagents and metal chelators. The enzyme activity was markedly inhibited by 1 mM diisopropylfluoride, and the desialysed enzyme was attacked by proteinases.     

Characterization of rat muscle fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphatase has been purified from rat muscle. Although the specific activity of the enzyme in the crude extract of rat muscle was extremely low, purification by the present procedure is highly reproducible. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The subunit molecular weight of the muscle enzyme was 37,500 in contrast to 43,000 in the case of the liver enzyme. Immunoreactivity of the muscle enzyme to anti-muscle and anti-liver fructose 1,6-bisphosphatase sera was clearly distinct from that of the liver enzyme. All one-dimensional peptide mappings of the muscle enzyme with staphylococcal V8 protease, chymotrypsin, and papain showed different patterns from those of the liver enzyme. When incubated with subtilisin, the extent of activation of muscle fructose 1,6-bisphosphatase at pH 9.1 was smaller than that of the liver enzyme. The subtilisin digestion pattern of the muscle enzyme on SDS-polyacrylamide gel electrophoresis was distinct from that of the liver enzyme. The AMP-concentration giving 50% inhibition of the muscle enzyme was 0.54 microM, whereas that of the liver enzyme was 85 microM. The concentrations of fructose 2,6-bisphosphate that gave 50% inhibition of rat muscle and liver enzymes were 6.3 and 1.5 microM, respectively. Fructose 1,6-bisphosphatase protein was not detected in soleus muscle by immunoelectroblotting with anti-muscle fructose 1,6-bisphosphatase serum.     

Purification of inactivated photoresponsive nitrile hydratase

Photoresponsive nitrile hydratase from Rhodococcus sp. N-771 was purified in its inactivated form. The enzyme had a molecular weight of approximately 60 kDa and consisted of 2 subunits each having molecular weight of 27.5 and 28 kDa. The enzyme also contained 2 iron atoms/enzyme as a cofactor. The enzyme was more stable in its inactivated form, rather than the activated during storage in the dark. The enzyme was most stable in the temperature region of 0-35 degrees C, and lost its activity above 40 degrees C. The enzyme was most stable in the pH region of 6-8. The optimum temperature and pH for the enzyme activity was 30 degrees C and 7.8, respectively. The enzyme showed wide substrate specificity, and most of the metal ions did not affect enzyme activity significantly. The absorption spectrum revealed the presence of some cofactor which changed form after photoirradiation.     

Immobilization of Bacillus licheniformis 5A1 milk-clotting enzyme and characterization of its enzyme properties

Milk-clotting enzyme from Bacillus licheniformis 5A1 was immobilized on Amberlite IR-120 by ionic binding. Almost all the enzyme activity was retained on the support. The immobilized milk-clotting enzyme was repeatedly used to produce cheese in a batch reactor. The production of cheese was repeated 5 times with no loss of activity. The specific activity calculated on a bound-protein basis was slightly higher than that of free enzyme. The free and immobilized enzyme were highly tolerant to repeated freezing and thawing. The optimum temperature for milk-clotting activity was 70 °C with the free enzyme whereas, it was ranged from 70 to 80 °C with the immobilized milk-clotting enzyme. The activation energy (E _A) of the immobilized milk-clotting enzyme was lower than the free enzyme (E _A = 1.59 and 1.99 Kcal mol⁻¹ respectively). The immobilized milk-clotting enzyme exhibited great thermal stability. The milk-clotting optimum pH was 7.0 for both free and immobilized enzyme. The Michaelis constant K _m of the immobilized milk-clotting enzyme was slightly lower than the free enzyme.     

Potent fibrinolytic enzyme from a thermophilic Streptomyces megasporus strain SD5

As a therapeutic agent in thrombosis the fibrinolytic enzymes are of interest and the search for a new enzyme continues. A strong fibrin-specific fibrinolytic enzyme was purified from the cell-free spent broth of thermophilic Streptomyces megasporus strain SD5. The crude enzyme was concentrated using ammonium sulphate, dialysis and lyophilization. Approximately 0.11 mg ml(-1) crude enzyme with a specific activity of 4.2 U microg(-1) was obtained. Post-electrophoretic reactivity revealed a monomeric form of the enzyme with a molecular weight of 35 kDa. The optimum pH and temperature for production of the enzyme were 8 and 55 degrees C, respectively. The enzyme was resistant to a broad range of pH ranging from 6 to 9 and temperature ranging from 37 to 60 degrees C. The enzyme was a chymotrypsin-like serine peptidase and the activity of the enzyme was N-terminal-dependent. The in vitro clot lysis by the enzyme at 37 degrees C was encouraging.     

Isolation and properties of rat plasma lecithin-cholesterol acyltransferase

Lecithin-cholesterol acyltransferase was purified from rat plasma and the properties of this enzyme during the purification procedures and those of the purified enzyme were investigated in comparison with the human enzyme. The rat enzyme was not adsorbed on hydroxyapatite, which was employed for the purification of the human enzyme. When purified human enzyme was incubated at 37 degrees C in 0.1 mM phosphate buffer (pH 7.4; ionic strength, 0.00025), no alteration of enzyme activity was observed for up to 6 h. In the case of the rat enzyme, however, approximately 40% of the enzyme activity was lost under the same conditions. The human enzyme and rat enzyme were both retained on a Sepharose 4B column to which HDL3 was covalently linked, in 39 mM phosphate buffer, pH 7.4. Although the human enzyme was eluted from the column in 1 mM phosphate buffer, the rat enzyme was dissociated from the column at a lower buffer concentration (0.1 mM phosphate buffer). These findings indicate that the rat enzyme effectively associated with HDL3 in 39 mM phosphate buffer, pH 7.4, but the association was more sensitive to increase of ionic strength compared with that of the human enzyme.     

Immobilization–stabilization of a new recombinant glutamate dehydrogenase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.     

Effects of phospholipids on L-lactate dehydrogenase from membranes of Escherichia coli. Activation and stabilization of the enzyme with phospholipids.

Membrane-bound L-lactate dehydrogenase was freed from the detergent used during purification. The detergent-free enzyme had about one-half the specific activity of the enzyme in 1.0% Tween 80, and was only partially sensitive to the specific antibody. This enzyme was activated about 3-fold with phosphatidylglycerol, cardiolipin, or a mixture of phospholipids. The phospholipid-activated enzyme had a similar Km value for L-lactate to that of the membrane enzyme and was completely inhibited by the specific antibody. On heat treatment, the phospholipid-activated enzyme was more stable than detergent-free enzyme and was as stable as membrane-bound enzyme. The alpha helical content of the enzyme increased 1.7-fold during preincubation with these lipids and the alpha helix became more stable during heat treatment than that of the detergent-free enzyme. These results suggest that the enzyme showed monomolecular dispersion in the lipid bilayer and that its conformation, including its active site and secondary structure, was different from that of the detergent-free enzyme. Phosphatidylethanolamine, dilauroyl lecithin and lecithin from egg yolk had none of the above effects on the activity or the secondary structure of the enzyme. On the other hand, mixtures of each of these lipids and cholate had essentially similar effects to phosphatidylglycerol.     

Purification and properties of human placental aminopeptidase B.

Aminopeptidase B (EC 3.4.11.6; L-arginyl-beta-naphthylamidase) was purified 1,800-fold from human placental cytoplasm and characterized. The enzyme was subjected to ammonium sulfate fractionation and a series of chromatographies on DE-52, hydroxylapatite, Bio-gel A 0.5 m and L-arginine-Sepharose. The native molecular mass of the enzyme was estimated to be 220,000 by gel filtration. The molecular mass was estimated to be about 83,000 by SDS/PAGE in the absence of 2-mercaptoethanol, suggesting that the enzyme exists in a polymeric form. The isoelectric point of the enzyme was 5.4. The purified enzyme was most active at pH 7.2 with L-arginyl-beta-naphthylamide as substrate and the Km value for this enzyme was 0.3 mmol/l. Human placental aminopeptidase B was markedly activity by Cl-. Bestatin and arphamenin, low molecular weight peptides, showed appreciable inhibition of this enzyme. However, amastatin and puromycin did not inhibit the enzyme. Bacitracin markedly activated this enzyme.     

Change in Hatching Enzyme Activity during Development of the Sea Urchin, Hemicentrotus Pulcherrimus

The time course of change in hatching enzyme activity during development of embryos of the sea urchin Hemicentrotus pulcherrimus was observed. The enzyme was present in the particulate fraction in embryos until the time of hatching and was maximal at the time of hatching. Cell fractionation studies suggested the existence of an inhibitor of the hatching enzyme. This possibility was subsequently substantiated by experiments in mixtures of fractions: the activity of hatching enzyme in the particulate fraction was inhibited by the supernatant of embryos. This inhibitory factor was heat-stable and non-dialyzable, but it was not characterized further. The activity of secreted hatching enzyme was not inhibited by this factor, suggesting that the molecular forms of hatching enzyme in embryos and in the culture supernatant are different. After hatching, the amount of increase in the hatching enzyme activity in the culture supernatant was 3.5 times the amount of decrease in enzyme activity in the embryos, suggesting that the enzyme was activated during its secretion.     

Findings of trigonelline demethylating enzyme activity in various organisms and some properties of the enzyme from hog liver

Trigonelline demethylating enzyme activity was found widely in animals, plants and microorganisms. Very high enzyme activity of this enzyme was detected in hog liver. Properties of the hog liver enzyme were investigated. Optimum pH for the enzymic reaction was observed at 8.5. The Km value for trigonelline was calculated at 2.77 mM. Addition of any cofactor is not required for the reaction The enzyme activity was inhibited by heavy metal ions. The reaction product was identified as nicotinic acid. Proposed enzyme reaction mechanism and the role of this enzyme in biosynthesis and metabolism of NAD are discussed.     

Synthesis and degradation of phenylalanine ammonia-lyase of Rhodosporidium toruloides.

The regulation of the enzyme phenylalanine ammonia-lyase (PAL), which is of potential use in oral treatment of phenylketonuria, was investigated. Antiserum against PAL was prepared and was shown to be monospecific for the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme and two inactive mutant forms of the enzyme were purified to homogeneity by immunoaffinity chromatography, using anti-PAL immunoglobulin G-Sepharose 4B. Both mutant enzymes contained intact prosthetic groups. The formation of PAL catalytic activity after phenylalanine was added to yeast cultures was paralleled by the appearance of enzyme antigen. During induction, uptake of [3H]leucine into the enzyme was higher than uptake into total protein. Our results are consistent with de novo synthesis of an enzyme induced by phenylalanine, rather than activation of a proenzyme. The half-lives of PAL and total protein were similar in both exponential and stationary phase cultures. No metabolite tested affected the rate of enzyme degradation. Glucose repressed enzyme synthesis, whereas ammonia reduced phenylalanine uptake and pool size and so may repress enzyme synthesis through inducer exclusion. The synthesis of enzyme antigen by a mutant unable to metabolize phenylalanine indicated that this amino acid is the physiological inducer of the enzyme.     

Purification and some properties of chlorogenic acid oxidase from apple (Malus pumila).

Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malus pumila cv. Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65,000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6-8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (-)-epicatechin and 4-methylcatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The Km value for the enzyme was found to be 122 microM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyl-dithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity.     

多聚半乳糖醛酸内切酶的固定化及其性质研究

棉花枯萎病菌多聚半乳糖醛酸内切酶在pH大于7时不稳定,故对它进行多种化学修饰而又不影响其活性,必须在pHd小于7的体系中进行。本文报道将PGAUase在还原剂存在下,与稀酸处理的Sepharose 4B交联,获得较高活力的固定化酶。固定化酶催化动力学表明,最适pH为4,4,最适温度为55℃,在pH1至8.0范围内稳定。和溶液酶比较,对热稳定性提高,但对碱稳定性下降。以多聚半乳糖醛酸为底物,Km为0.27mmol/L,Vmax为66.67nmol/L·min,均大于溶液酶(Km=0.07mmol/L,Vmax=28.00nmol/L·min)。在pH4.8,30℃,聚半乳糖醛酸在固相酶的柱中循环水解不同的时间降解产物经圆盘电泳和等电聚焦测定,得到不同大小的寡糖片段混合物,证明固相酶和溶液酶的作用方式相同,同时使以酶解法制备一定大小的有生物活性的寡糖分子成为可能。     

DISTRIBUTION AND PROPERTIES OF ANGIOTENSIN CONVERTING ENZYME OF RAT BRAIN

Abstract— Angiotensin converting enzyme of rat brain was studied using Hip-His-Leu as substrate. The highest specific activity of the enzyme was associated with the microsomal fraction. The specific activity of the microsomal enzyme in several regions of the rat brain varied significantly. For example, the specific activities of the striatal and pituitary enzymes were about 10-fold greater than that of the cerebral cortical enzyme. The enzyme required chloride ion; moreover, activity was inhibited in the presence of disodium EDTA or O-phenanthroline, effects suggesting that the converting enzyme of brain is a metalloprotein. SQ-20881, a nonapeptide that inhibits converting enzyme in peripheral tissue, was a potent inhibitor of the enzyme of brain. In addition to Hip-His-Leu, the microsomal fraction was capable of liberating C terminal dipeptides from angiotensin I, Hip-Gly-Gly and Z-Gly- Gly-Val. The broad substrate specificity of the enzyme suggests that, in addition to the possible contribution of the enzyme to the brain renin-angiotensin system, other naturally occurring peptides might also be substrates for the enzyme.     

Purification and characterization of a thermostable carboxypeptidase (carboxypeptidase Taq) from Thermus aquaticus YT-1.

A thermostable carboxypeptidase, which we named carboxypeptidase Taq, was purified from Thermus aquaticus YT-1 and characterized. The molecular weight of the enzyme was estimated to be about 56,000 and 58,000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme has a monomeric structure. The optimum pH of the enzyme was 8.0, and the optimum temperature for the reaction was 80 degrees C. The enzyme activity was dependent on cobalt ion and was inhibited by metal-chelating reagents, indicating that the enzyme is a metalloenzyme. The enzyme had high thermostability independent of cobalt ion; about 90% of its activity remained even after treatment at 80 degrees C for 5 h. The enzyme showed broad substrate specificity, although proline at the C-terminus of peptides was not cleaved. The enzyme released amino acids sequentially from the C-terminus.     

Amine Oxidases of Microorganisms

There was an increase in copper content proportional to the increase in specific activity of the enzyme during the purification of amine oxidase of Aspergillus niger. The recrystallized enzyme preparation contained 1 g atom of copper per 83,000 g of protein. This indicated that the enzyme contained 3 g atoms of copper per mole of enzyme. The copper in the enzyme was removed by dialysis against sodium diethyldithiocarbamate with concomitant loss of activity. The dialyzed enzyme was reactivated by the addition of cupric copper.

The copper in the enzyme was present in cupric state. No valency change of the copper was observed in the catalytic activity.

The enzyme was inhibited by various chelating agents, and potently inhibited by various carbonyl reagents.