scholarly journals Assisted refolding of recombinant prochymosin with the aid of protein disulphide isomerase

1994 ◽  
Vol 301 (1) ◽  
pp. 17-20 ◽  
Author(s):  
B Tang ◽  
S Zhang ◽  
K Yang
Keyword(s):  
Active Form ◽  
Thiol Groups ◽  
Active Protein ◽  
Protein Disulphide Isomerase ◽  
Ph Values ◽  
Free Thiol ◽  
Optimum Ph

Protein disulphide isomerase (PDI) was shown to be able to accelerate the refolding of unfolded recombinant prochymosin and to enhance the overall yield of active protein. Unlike previous reports in this study PDI was found to be active at pH values as high as 11. The coincidence of the similar apparent optimum pH values of uncatalysed and PDI-catalysed reactions suggests that conditions favourable to spontaneous refolding of proteins may help PDI to catalyse thiol/disulphide interchange. Under the conditions described here no exogenously added dithiothreitol was required for PDI-catalysed renaturation, implying that the disulphide form of PDI was reduced to its active form by the free thiol groups in prochymosin molecules.

scholarly journals Human xylosyltransferase I: functional and biochemical characterization of cysteine residues required for enzymic activity

2005 ◽  
Vol 386 (2) ◽  
pp. 227-236 ◽  
Author(s):  
Sandra MÜLLER ◽  
Manuela SCHÖTTLER ◽  
Sylvia SCHÖN ◽  
Christian PRANTE ◽  
Thomas BRINKMANN ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Biochemical Characterization ◽  
Active Form ◽  
Inhibitory Effect ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Thiol Groups ◽  
Wild Type ◽  
Free Thiol

XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure–function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561→Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.

Trans-sialidase Associated with Atherosclerosis: Defining the Identity of a Key Enzyme Involved in the Pathology

2019 ◽  
Vol 20 (9) ◽  
pp. 938-941
Author(s):  
Victor Y. Glanz ◽  
Veronika A. Myasoedova ◽  
Andrey V. Grechko ◽  
Alexander N. Orekhov
Keyword(s):  
Acid Metabolism ◽  
Research Subject ◽  
Disease Pathogenesis ◽  
Sialidase Activity ◽  
Ph Values ◽  
Ldl Particles ◽  
Optimum Ph ◽  
Key Enzyme ◽  
St6gal I

Atherosclerosis is associated with the increased trans-sialidase activity, which can be detected in the blood plasma of atherosclerosis patients. The likely involvement in the disease pathogenesis made this activity an interesting research subject and the enzyme that may perform such activity was isolated and characterized in terms of substrate specificity and enzymatic properties. It was found that the enzyme has distinct optimum pH values, and its activity was enhanced by the presence of Ca2+ ions. Most importantly, the enzyme was able to cause atherogenic modification of lowdensity lipoprotein (LDL) particles in vitro. However, the identity of the discovered enzyme remained to be defined. Currently, sialyltransferases, mainly ST6Gal I, are regarded as major contributors to sialic acid metabolism in human blood. In this mini-review, we discuss the possibility that atherosclerosis- associated trans-sialidase does, in fact, belong to the sialyltransferases family.

scholarly journals Critical importance of pH and collector type on the flotation of sphalerite and galena from a low-grade lead–zinc ore

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Abdolrahim Foroutan ◽  
Majid Abbas Zadeh Haji Abadi ◽  
Yaser Kianinia ◽  
Mahdi Ghadiri
Keyword(s):  
Iron Ore ◽  
Low Grade ◽  
Zinc Recovery ◽  
Flotation Process ◽  
Ph Values ◽  
Lead And Zinc ◽  
Optimum Ph ◽  
Iron And Zinc ◽  
Lead Zinc ◽  
Zinc Concentrate

AbstractCollector type and pulp pH play an important role in the lead–zinc ore flotation process. In the current study, the effect of pulp pH and the collector type parameters on the galena and sphalerite flotation from a complex lead–zinc–iron ore was investigated. The ethyl xanthate and Aero 3418 collectors were used for lead flotation and Aero 3477 and amyl xanthate for zinc flotation. It was found that maximum lead grade could be achieved by using Aero 3418 as collector at pH 8. Also, iron and zinc recoveries and grades were increased in the lead concentrate at lower pH which caused zinc recovery reduction in the zinc concentrate and decrease the lead grade concentrate. Furthermore, the results showed that the maximum zinc grade and recovery of 42.9% and 76.7% were achieved at pH 6 in the presence of Aero 3477 as collector. For both collectors at pH 5, Zinc recovery was increased around 2–3%; however, the iron recovery was also increased at this pH which reduced the zinc concentrate quality. Finally, pH 8 and pH 6 were selected as optimum pH values for lead and zinc flotation circuits, respectively.

scholarly journals The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase

1991 ◽  
Vol 275 (2) ◽  
pp. 335-339 ◽  
Author(s):  
H C Hawkins ◽  
R B Freedman
Keyword(s):  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Native Enzyme ◽  
Liver Protein ◽  
Thiol Groups ◽  
Protein Disulphide Isomerase ◽  
Order Rate ◽  
Reactive Groups

1. The number of reactive thiol groups in mammalian liver protein disulphide-isomerase (PDI) in various conditions was investigated by alkylation with iodo[14C]acetate. 2. Both the native enzyme, as isolated, and the urea-denatured enzyme contained negligible reactive thiol groups; the enzyme reduced with dithiothreitol contained two groups reactive towards iodoacetic acid at pH 7.5, and up to five reactive groups were detectable in the reduced denatured enzyme. 3. Modification of the two reactive groups in the reduced native enzyme led to complete inactivation, and the relationship between the loss of activity and the extent of modification was approximately linear. 4. Inactivation of PDI by alkylation of the reduced enzyme followed pseudo-first-order kinetics; a plot of the pH-dependence of the second-order rate constant for inactivation indicated that the essential reactive groups had a pK of 6.7 and a limiting second-order rate constant at high pH of 11 M-1.s-1. 5. Since sequence data on PDI show the presence within the polypeptide of two regions closely similar to thioredoxin, the data strongly indicate that these regions are chemically and functionally equivalent to thioredoxin. 6. The activity of PDI in thiol/disulphide interchange derives from the presence of vicinal dithiol groups in which one thiol group of each pair has an unusually low pK and high nucleophilic reactivity at physiological pH.

scholarly journals The Effect of pH Stress on the Survival of Rhizobium Trifolii and Sinorhizobium Meliloti in Vitro

2013 ◽  
Vol 70 (2) ◽  
pp. 449-450
Author(s):  
Monica NISTE ◽  
Roxana VIDICAN ◽  
Ioan ROTAR ◽  
Rodica POP
Keyword(s):  
Sinorhizobium Meliloti ◽  
Agar Medium ◽  
Rhizobium Trifolii ◽  
Ph Values ◽  
Optimum Ph ◽  
Yeast Extract Mannitol ◽  
Rhizobial Species ◽  
The Impact ◽  
Different Soils

Nitrogen-fixing symbiotic bacteria known as rhizobia can exist in different soils and adapt to different environmental conditions. The aim of this study was to determine the impact of pH on the growth of Rhizobium trifolii and Sinorhizobium meliloti. Rhizobial species were isolated using yeast extract mannitol agar medium) in which the pH values were adjusted to 5.0, 6.0, 8.0 and 9.0 by adding HCl and NaOH. The optimum pH for rhizobia is neutral or slightly alkaline (pH 8) and they are more sensitive to acidity. Sinorhizobium meliloti developed better in an acid medium compared to Rhizobium trifolii.

Coagulation-Direct Filtration of Soft, Low Alkalinity Humic Waters

1999 ◽  
Vol 40 (9) ◽  
pp. 55-62 ◽  
Author(s):  
Bjørnar Eikebrokk
Keyword(s):  
Natural Organic Matter ◽  
Pilot Scale ◽  
Quality Parameters ◽  
Efficient Removal ◽  
Direct Filtration ◽  
Ph Values ◽  
Optimum Ph ◽  
Calcium Sources ◽  
Ph Conditions ◽  
Removal Efficiencies

Optimisation of coagulation-direct filtration processes with respect to efficient removal of humic substances, i.e. natural organic matter (NOM) has gained a lot of focus in many countries over the last years. This paper presents experimental results from pilot scale research studies aimed at optimising the coagulation-direct filtration process applied to soft and humic raw waters with low turbidity and alkalinity levels. Comprehensive tests of 3 types of raw waters with different NOM content, 5 types of coagulants, and 3 calcium sources for the purpose of corrosion control have been conducted. Removal efficiencies with respect to relevant water quality parameters are presented, with typical relationships between raw water NOM content, coagulant dose requirements and pH. Generally, when applying metal-based coagulants, residual metal concentration was the critical parameter regarding minimum coagulant dose requirements. Typical NOM removal efficiencies were in the range of 75-90% and 40-70% with respect to colour and organic carbon, respectively. Optimum pH conditions for the removal of NOM and/or residual metals do not always coincide with that of turbidity. The experiments also showed that poly-aluminium and ferric chlorides might have some benefits over alum in terms of dose requirements and range of optimum pH values, and that chitosan may be used for colour removal with good results.

THE PYRUVIC PHOSPHOFERASE OF ERYTHROCYTES: I. PROPERTIES OF THE ENZYME AND ITS ACTIVITY IN ERYTHROCYTES OF VARIOUS SPECIES

1955 ◽  
Vol 33 (1) ◽  
pp. 38-45 ◽  
Author(s):  
P. F. Solvonuk ◽  
H. R. Collier
Keyword(s):  
Mammalian Species ◽  
Thiol Groups ◽  
Newborn Rats ◽  
Free Thiol ◽  
The Mean ◽  
Very High

Mammalian erythrocytes contain a pyruvic phosphoferase (PPFase) which is activated by K+ and Mg++ and inhibited by Na+ and Ca++. The K+ can be replaced by Rb+ or NH4+, and the Mg++ can be partially replaced by Mn++ or Co++ as activators of the enzyme. The PPFase apparently requires free thiol groups for its activity, as it is completely inhibited by 10−4 M p-chloromercuribenzoate and this inhibition is partially reversed by glutathione. The mean PPFase of the erythrocytes of six mammalian species was determined and found to be in the following order of decreasing activity: man, rat, dog, rabbit, cat, ox. The erythrocytes of chicks and of newborn rats showed a very high PPFase activity.

Study on the Kinetics of Adsorption of Cadmium by Chitosan

2010 ◽  
Vol 160-162 ◽  
pp. 1804-1809
Author(s):  
Qiang Bi ◽  
Juan Qin Xue ◽  
Ying Juan Guo ◽  
Yu Jie Wang ◽  
Yun Feng Xue
Keyword(s):  
Kinetic Equations ◽  
Adsorption Kinetic ◽  
Maximum Adsorption Capacity ◽  
Kinetics Of Adsorption ◽  
Ph Values ◽  
Optimum Ph ◽  
Pseudo Second Order ◽  
Simulated Wastewater ◽  
Isotherm Equations ◽  
Kinetics Of

The adsorption of cadmium in simulated wastewater by chitosan was investigated. The influence of temperature, contact time and pH on adsorption efficiency of cadmium was examined. Some related mathematical models were used in the fitting of experimental data. The results showed that at room temperature, the optimum pH of adsorption is between 4 and 7. At lower pH values, a strong competition existed between cadmium ions and protons for sorption sites and the sorption efficiency was decreased. After 60 minutes the adsorption equilibrium can be achieved. Chitosan is very effective at removing cadmium with the maximum adsorption capacity is 112.05mg•g-1. The adsorption kinetic curves agree with the pseudo-second-order adsorption kinetic equations and the adsorption isotherms could be well described by Langmuir isotherm equations.

scholarly journals Properties of pig synovial collagenase

1980 ◽  
Vol 189 (2) ◽  
pp. 349-357 ◽  
Author(s):  
J A Tyler ◽  
T E Cawston
Keyword(s):  
Isoelectric Focusing ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Type Ii ◽  
Polyacrylamide Gels ◽  
Optimum Ph ◽  
Detectable Difference

1. Properties of a purified chemically activated form of pig synovial collagenase were examined and compared with a spontaneously active form of the enzyme. 2. The active enzyme has a specific activity of 53 000 units (microgram/min)/mg, a mol.wt. of 44 000 (by sodium dodecyl sulphate/polyarcylamide-gel electrophoresis in 2-mercaptoethanol) and pI 5.2 (by isoelectric focusing in polyacrylamide gels). 3. The activity has the characteristics of a metalloproteinase that degrades types I and III soluble or insoluble collagens in preference to type II, at an optimum pH of 6.5-8.5. 4. There is no detectable difference in these properties between the chemically activated and spontaneously active form of collagenase.

scholarly journals Preparation of Chitosan/Magnetic Porous Biochar as Support for Cellulase Immobilization by Using Glutaraldehyde

Polymers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 2672
Author(s):  
Haodao Mo ◽  
Jianhui Qiu
Keyword(s):  
Alkali Activation ◽  
Covalent Bonds ◽  
Ph Values ◽  
Maximum Reaction ◽  
Carboxymethyl Cellulose Sodium ◽  
Optimum Ph ◽  
Potential Applications ◽  
Km Value ◽  
Immobilized Cellulase ◽  
Menten Constant

In this work, porous biochar was obtained from sugarcane bagasse by alkali activation and pyrolysis and then magnetized with γ-Fe2O3 by calcination. After functionalization with chitosan and activation with glutaraldehyde, the as-prepared chitosan/magnetic porous biochar served as a support to immobilize cellulase by covalent bonds. The immobilization amount of cellulase was 80.5 mg cellulase/g support at pH 5 and 25 °C for 12 h of immobilization. To determine the enzymatic properties, 1% carboxymethyl cellulose sodium (CMC) (dissolved in 0.1 M buffer) was considered as a substrate for hydrolysis at different pH values (3–7) and temperatures (30–70 °C) for 30 min. The results showed that the optimum pH and temperature of the free and immobilized cellulase did not change, which were pH 4 and 60 °C, respectively. The immobilized cellulase had a relatively high activity recovery of 73.0%. However, it also exhibited a higher Michaelis–Menten constant (Km) value and a slower maximum reaction velocity (Vmax) value compared to the free enzyme. In the reusability assay, the immobilized cellulase showed initial glucose productivity of 330.9 mg glucose/g CMC and remained at 86.0% after 10 uses. In conclusion, the chitosan/magnetic porous biochar has great potential applications as a support for enzyme immobilization.

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