米曲霉F-81产中性蛋白酶的固定化条件及其特性

以海藻酸钠为载体,戊二醛为交联剂固定化米曲霉F-81产中性蛋白酶,研究了固定化条件及固定化酶的性质。结果表明,固定化的最佳条件为:固定化时间1 h、海澡酸钠浓度4%、戊二醛浓度9%、CaCl2浓度0.7 mol/L。在此条件下固定化的中性蛋白酶活力为游离酶活力的68%。固定化酶的最适作用温度为65℃,最适作用pH值为7.0。60℃下酶稳定性较好,80℃下处理60 min,粗酶中几乎检测不到酶活力;中性蛋白酶pH稳定范围为6.5-9.5。Km值为24.83 mg/mL,最大反应速率Vmax为0.043 12 mg/min。     

Dynamics of the heart minute volume and rheological properties of the blood in the early post-resuscitation period after preagonal and agonal conditions

Fifteen experiments on heparinized (500 IU/kg) dogs (both male and female) under slight nembutal anaesthesia with promedol have revealed that polyglucin hemodilution (60% of blood; 40% of polyglucin) during resuscitation following 4 hours of hypovolemic hypotension prevented the onset of ischemic hyperperfusion syndrome for 5 minutes of post-resuscitation period. Moreover, delayed hyperperfusion syndrome was not observed 3 hours after resuscitation, as it was in animals with blood reinfusion only. Hyperperfusion syndrome was more expressed in animals, recovered after a 4-hour hypovolemic hypotension, than in animals whose agony was caused by a 2-hour arterial hypotension. The correlation was established between high blood viscosity and post-ischemic hyperperfusion (reactive hyperemia).     

Vermiculite as an economic support for immobilization of neutral protease

A new and cheap support, vermiculite was successfully used to immobilize neutral protease by adsorption and hexamethylene diamine mediated coupling using glutaraldehyde as a bifunctional agent. Neutral protease immobilized on vermiculite by adsorption showed maximum retained activity than HMD mediated coupling. The optimum temperature for both free and immobilized neutral protease was found to be 45°C. However, the pH and thermal stabilities of immobilized neutral protease was observed to be better than that of the free enzyme. The storage stability of the immobilized enzyme was also studied.     

羊毛防毡缩用蛋白酶的化学修饰

为减少防毡缩整理中蛋白酶对羊毛纤维主体结构的破坏作用,分别研究了戊二醛、微生物谷氨酰胺转氨酶(MTG)和水溶性碳二亚胺(EDC)对蛋白酶Savinase 16L的化学修饰,以期达到增大蛋白酶分子量,从而将水解作用限制在纤维表面的目的。主要通过体积排阻色谱、SDS-PAGE谱图以及荧光光谱研究修饰酶的分子量和结构变化。结果表明,戊二醛不能对蛋白酶分子进行有效修饰;MTG会被蛋白酶水解,无法催化酶分子间发生共价交联;而碳二亚胺既可以使蛋白酶分子间发生交联,又能将含有伯胺基的大分子修饰剂偶联到酶分子上。     

Serotonin participation in the mechanism of the cytoprotective action of polyglucin on the stomach

Experiments on rats with the use of ultrastructural analysis and fluorescent-histochemical and biochemical methods have shown serotonin to participate in the mechanism of the cytoprotective effect of polyglucin on the stomach. In the presence of experimental hyperthyrosis, intravenous injection of polyglucin coupled with hydrocortisone reduces the percentage of gastric mucosa lesions. This is a consequence of the restriction of serotonin release from serotonin-producing gastric cells.     

Mechanisms of Responses of Systemic Circulation: Role of the Endothelial Factor of Vascular Tone Control

Experimental data on the effect of NO synthase inhibition on hemodynamic changes (blood pressure, cardiac output, and peripheral resistance) induced by an increased (polyglucin infusion) or decreased (orthostasis) cardiac output are presented. Under conditions of NO synthase inhibition, the pressor effects of polyglucin and orthostatic hypotension increased by 70 and 72%, respectively. The response of peripheral resistance had a similar trend. Significance of NO secretion by vascular endothelium for the development of systemic hemodynamic responses is proposed.     

Immobilization of the serine protease from Thermomonospora fusca YX on porous glass

As much as 84% of the thermostable serine protease from Thermomonospora fusca strain YX was covalently attached to silanized glass using glutaraldehyde. The immobilized protease exhibited a higher temperature optimum (86 degrees C) and pH optimum (9.4) for activity compared to soluble YX-protease (80 degrees C and pH 9.0, respectively). Immobilization improved enzyme thermo-stability above 90 degrees C and reduced inactivation during prolonged storage (9% loss of activity after 90 days at 12 degrees C). A continuous-flow column reactor packed with immobilized protease readily hydrolyzed casein over broad ranges of temperature and pH.     

Enhanced stabilization of mungbean thiol protease immobilized on glutaraldehyde-activated chitosan beads

A thiol protease purified from mungbean seedlings was immobilized on chitosan beads cross-linked with glutaraldehyde. The yield of the immobilized enzyme was maximum (～99%) at 1% concentration each of chitosan and glutaraldehyde. The immobilized enzyme showed reusability for 15 batch reactions. Immobilization shifted the optimum pH of the enzyme to a more acidic range and enhanced its stability both at acidic as well as alkaline pH values compared to the free enzyme. The stability of the enzyme to temperature and in aqueous non-conventional medium (ethanol and DMSO) was significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation reflected by a higher apparent K_m value. This study produced an immobilized biocatalyst having improved characteristics and better operational stability than the soluble enzyme. The increase in stability in the presence of high concentrations of ethanol and DMSO may make it useful for catalyzing organic reactions such as trans-esterification and trans-amidation similar to other cysteine proteinases.     

Continuous proteolysis with a stabilized protease. I. Chemical stabilization of an alkaline protease

Due to the loss of enzymatic activity as a function of time, an alkaline protease, selected for the continuous preparation of protein hydrolysates (J. Boudrant and C. Cheftel, Biotechnol. Bioeng., 18 , 1735, 1976), was chemically stabilized by a simple treatment with glutaraldehyde. Two fractions, soluble and insoluble, were obtained. The activities of these two fractions were measured with casein and N-benzoyl-L -arginine ethyl ester (BAEE) as a function of glutaraldehyde concentration used. It was noted that the insoluble fraction was practically inactive with the first substrate and that the heat stability of the soluble form was likewise enhanced. Molecular weights of these two forms were unchanged, but the uv-spectrum of the soluble form was modified. From amino acid analysis, it appears that this treatment mainly provokes a decrease in lysine content.     

Study of chemically modified hemoglobin as an artificial oxygen carrier in the model of hemorrhagic shock in dogs]

Hemodynamic and gas-transporting properties of the chemically modified hemoglobin solution have been studied on the model of hemorrhagic shock in dog. It has been shown that the polymerized hemoglobin solution exerts the hemodynamic action just as the plasma substitute "polyglucin" does. However, in contrast to the latter, polyhemoglobin circulating in the vascular bed for a prolonged period of time increases the blood oxygen capacity and oxygen delivery to tissues with the resultant increase in body total oxygen taking-up.     

Immobilization of alkaline protease on nylon

The parameters involved in immobilization of alkaline protease on nylon using glutaraldehyde as coupling agent and the characteristics of the immobilized enzyme were investigated. Optimum temperature and pH of both free and immobilized enzyme for the degradation of protein was found. Immobilized enzyme showed better thermal stability than the free enzyme. The reusability and storage stability of the immobilized enzyme was also studied.     

Preparation and properties of proteases immobilized on anion exchange resin with glutaraldehyde

High activity alkaline protease was obtained when the enzyme was immobilized on Dowex MWA-1 (mesh 20–50) with 10% glutaraldehyde in chilled phosphate buffer (M/15, pH 6.5). Activity yields of the protease and rennet were 27 and 29, respectively. The highest activities appeared at 60°C, pH 10 for alkaline protease and 50°C, pH 4.0 for rennet. The properties of both proteases were not essentially changed by the immobilization except that the K_m values of both enzymes were increased about tenfold as a result of immobilization. Both proteases in the immobilized state were more stable than those in the free state at 60°C. Other peptide hydrolases, β-galactosidase, invertase, and glucoamylase, were successfully immobilized with high activities, but lipase, hexokinase, glucose-6-phosphate dehydrogenase, and xanthine oxidase became inactive.     

Hydrolysis of milk lactose by immobilized beta-galactosidase-hen egg white powder

Immobilized beta-galactosidase was obtained by crosslinking the enzyme with hen egg white using 2% glutaraldehyde. The gel obtained could be lyophilized to give a dry enzyme powder. The pH optimum of both the soluble and immobilized enzyme was found to be 6.8. The immobilized enzyme showed a higher K(m) for the substrates. The extent of enzyme inhibition by galactose was reduced upon immobilization. The stability towards inactivation by heat, urea, gamma irradiation, and protease treatment were enhanced. The bound enzyme as tested in a batch reactor could be used repeatedly for the hydrolysis of milk lactose. The possible application of this system for small-scale domestic use has been suggested.     

Cellulose and glass fiber affinity membranes for the chromatographic separation of biomolecules

Macroporous cellulose and glass membranes were prepared from filter paper and glass fiber filter, respectively. To enhance their stability, the cellulose membranes were crosslinked with epichlorohydrin, and the glass membranes were crosslinked with glutaraldehyde or organic bifunctional silanes. Several pathways for the modification, activation, and ligand immobilization were used and compared. For cellulose membranes, the diazotization method provided the best results, whereas the glutaraldehyde method provided the best performance for glass membranes, regarding both their stability and ligand immobilization capacity. The characterization of the membranes was made by using a triazine dye, bovine serum albumin, and trypsin as test ligands. The membrane morphologies and the uniformities of ligand distribution across the membrane cartridges were investigated. Numerous affinity ligands were immobilized onto the membranes, and the prepared affinity membranes have been used to separate or purify concanavalin A, peroxidase, protease inhibitors, globulin, fibronectin, and other biomolecules.     

The use of proteases for improved presentation of DNA in chromosomes and chloroplasts of Prorocentrum micans (Dinophyceae)

Summary Chromosomes and chloroplasts of the marine dinoflagellate Prorocentrum micans were investigated electron-microscopically, using standard glutaraldehyde fixation, followed by protease treatment and post-osmication. The DNA-fibrils of chromosomes and chloroplasts which usually appear coagulated after standard aldehyde fixation, recover their natural arrangement within the organelle after protease-digestion. Also the DNA-fibrils of the chloroplast appear in a comparatively high degree of structural order of a kind previously reported only from dinoflagellate chromosomes and bacterial nucleoids.The specific retention of DNA-structures after protease treatment is accompanied by enhanced contrast of the single DNA-fibrils, probably caused by adsorption of proteinaceous digestion products which are again removable by prolonged enzyme treatment.     

Reduction of active elastase concentration by means of immobilized inhibitors: a novel therapeutic approach

The inhibitory power of three different active Nylon membranes, separately loaded with three different protease inhibitors, was studied with the aim of reducing the increased elastase concentration occurring during hemodialysis or extracorporeal blood circulation in patients undergoing cardiopulmonary bypass. Chemical grafting was carried out to make the inert Nylon membrane suitable for the immobilization of the inhibitors. The behavior of immobilized alpha(1)-antitrypsin, bovine pancreatic trypsin inhibitor (BPTI), or elastatinal was separately studied. alpha(1)-Antitrypsin and BPTI were covalently immobilized by means of a diazotization process, whereas elastatinal was covalently attached via a condensation process mediated by glutaraldehyde. The inhibitory power of each membrane type was studied as a function of the amount of immobilized inhibitor and temperature. All active membranes have shown good inhibitory power. The most efficient membrane was that loaded with alpha(1)-antitrypsin, the less efficient that with BPTI.     

Membrane dynamics in human leukemia and lymphoma cells: pH dependency of diphenylhexatriene fluorescence polarization

Membrane dynamics of human leukemia and lymphoma cell lines were analyzed by investigating the effect of pH on fluorescence polarization (P) of the lipophilic probe diphenylhexatriene (DPH). The degree of P varied as a function of pH, depending on the cell lines. These variations were not detected in phospholipid vesicles. In addition, they were prevented by treatments with glutaraldehyde, sodium azide or phenylmethylsulfanyl fluoride, a specific protease inhibitor. Therefore, these P value changes might be influenced by protein modification.     

Intermolecular cross-linking of a protein crystal--acid protease from Endothia parasitica--in 2.7 M ammonium sulfate solution

A workable procedure for cross-linking of a protein crystal - acid protease from Endothia parasitica - in 2.7 M ammonium sulfate solution with bifunctional reagent glutaraldehyde has been found. Using the cross-linking technique, we have made one good quality heavy atom derivative of uranyl into three or even more isomorphous derivatives for this particular protein.     

Effect of hypervolemia on the adrenergic reactivity of the arterial system

In anaesthetised rats, dependence of haemodynamics upon increase in the blood volume due to infusion of polyglucin in amounts 1.2 and 2.4 ml (8% and 16% of the blood volume in rats, respectively) < was studied. In the former case the initial blood pressure increased by 30%, in the latter case--by 42%; cardiac output--by 16% and 40%. The metasone pressor effects, at the were reliably decreased by 27% and 65%, and those of general peripheral resistance--by 40% and 80, respectively. The cardiac output changes did not differ significantly. The data obtained suggest an effect of the blood volume increase upon a drop of the arterial system's adrenoreactivity as a result of increase in initial blood pressure.