Relationship between macrotetrolide production and specific activity of some hydrolases in a high and a low producing strain of Streptomyces griseus

1981 ◽  
Vol 11 (4) ◽  
pp. 216-221 ◽  
Author(s):  
Jochen Schindelmeiser ◽  
Hermann Pape
Keyword(s):  
Specific Activity ◽  
Streptomyces Griseus

scholarly journals Improving production of Streptomyces griseus trypsin for its application in processing insulin precursor

2019 ◽  
Author(s):  
YunFeng Zhang ◽  
Qixing Liang ◽  
Chuanzhi Zhang ◽  
Juan Zhang ◽  
Guocheng Du ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Specific Activity ◽  
Streptomyces Griseus ◽  
Dynamics Simulation ◽  
Flask Culture ◽  
Insulin Production ◽  
Chaperone Protein ◽  
Selection Approach ◽  
Pharmaceutical Manufacture ◽  
Hydrolysis Activity

Abstract Background: Trypsin has a plenty application in food and pharmaceutical manufacture. While, the commercial trypsin is usually extracted from pork pancreas, which has the risk of infectious and immunogenicity. Therefore, the microbial Streptomyces griseus trypsin (SGT) is a prior alternation because it processes efficient hydrolysis activity without the aforementioned risk. The remarkable hydrolysis efficiency of SGT caused its autolysis, and five autolysis sites R21, R32, K122, R153, and R201 were identified from its' autolysate. Results: The tbcf (K101A, R201V) mutant was screened by directed selection approach for improved activity in flask culture (60.85 ± 3.42 U·mL -1 , increased 1.5-fold). From the molecular dynamics simulation, the K101A/R201V mutation shortened the distant between catalytical residues D102 and H57 from 6.5 Å vs 7.0 Å, which afforded the improved specific activity 1527.96 ± 62.81 U·mg -1 . Further, the production of trypsin was increased 302.8% (689.47 ± 6.78 U·mL −1 ) in 3-L bio-reactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris. Conclusions: The SGT protein could be an adequate trypsin for insulin production. When working with hydrolysates analysis and direction selection, the tbcf (K101A, R201V) mutant increased 1.5-fold activity. Further, the production of trypsin was improved 3-fold by overexpressing chaperone protein in Pichia pastoris . The future study should be emphasized on the application of SGT in insulin manufacture and pharmaceutical.

Isolation of 57Co-cobalamin coenzymes at high specific activity from Streptomyces griseus

1978 ◽  
Vol 160 (1) ◽  
pp. 101-108 ◽  
Author(s):  
E.V. Quadros ◽  
A. Hamilton ◽  
D.M. Matthews ◽  
J.C. Linnell
Keyword(s):  
Specific Activity ◽  
Streptomyces Griseus ◽  
High Specific Activity

The Morphology of the Rat Kidney Following Folic Acid Administration

1970 ◽  
Vol 28 ◽  
pp. 200-201
Author(s):  
Aline Byrnes ◽  
Elsa E. Ramos ◽  
Minoru Suzuki ◽  
E.D. Mayfield
Keyword(s):  
Folic Acid ◽  
De Novo ◽  
Specific Activity ◽  
Rat Kidney ◽  
Present Report ◽  
Intracellular Localization ◽  
Male Rats ◽  
Renal Hypertrophy ◽  
Electron Microscopic ◽  
Biochemical Alterations

Renal hypertrophy was induced in 100 g male rats by the injection of 250 mg folic acid (FA) dissolved in 0.3 M NaHCO3/kg body weight (i.v.). Preliminary studies of the biochemical alterations in ribonucleic acid (RNA) metabolism of the renal tissue have been reported recently (1). They are: RNA content and concentration, orotic acid-c14 incorporation into RNA and acid soluble nucleotide pool, intracellular localization of the newly synthesized RNA, and the specific activity of enzymes of the de novo pyrimidine biosynthesis pathway. The present report describes the light and electron microscopic observations in these animals. For light microscopy, kidney slices were fixed in formalin, embedded, sectioned, and stained with H & E and PAS.

Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

1991 ◽  
Vol 66 (04) ◽  
pp. 453-458 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Specific Activity ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Clinical Importance ◽  
Potential Method ◽  
Quantitative Expression ◽  
Increased Risk ◽  
Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

Extraction of Protein-Bound ATP and ADP from Human Platelets in Plasma

1990 ◽  
Vol 63 (02) ◽  
pp. 286-290 ◽  
Author(s):  
Christina Beurling-Harbury ◽  
Pehr B Harbury
Keyword(s):  
Specific Activity ◽  
Platelet Rich Plasma ◽  
Binding Protein ◽  
Human Platelets ◽  
Luciferase Assay ◽  
First Time ◽  
Plasma Extraction

SummaryActin is the major ATP and ADP binding protein in platelets, 0.9–1.3 nmol/108 cells, 50–70% in the unpolymerized state. The goal of these experiments was to develop a method for extracting all protein-bound ATP and ADP from undisturbed platelets in plasma. Extraction of actin-bound ADP is routine while extraction of actin-bound ATP from platelets in buffer has been unsuccessful. Prior to extraction the platelets were exposed to 14-C adenine, to label the metabolic and actin pools of ATP and ADP. The specific activity was determined from the actin-bound ADP in the 43% ethanol precipitate. Sequential ethanol and perchlorate extractions of platelet rich plasma, and the derived supernatants and precipitates were performed. ATP concentrations were determined with the luciferase assay, and radioactive nucleotides separated by TLC. A total of 1.18 nmol/108 cells of protein-bound ATP and ADP was recovered, 52% ATP (0.61 nmol). The recovery of protein-bound ADP was increased from 0.3 to 0.57 nmol/108 cells. This approach for the first time successfully recovered protein bound ATP and ADP from platelets in a concentration expected for actin.

On the Preparation of Bovine α-Thrombin

1978 ◽  
Vol 39 (01) ◽  
pp. 193-200 ◽  
Author(s):  
Erwin F Workman ◽  
Roger L Lundblad
Keyword(s):  
Immobilized Enzyme ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Low Molecular Weight ◽  
Reversible Process ◽  
Gel Beads ◽  
Tissue Thromboplastin ◽  
C 50 ◽  
Hydrolysis Of

SummaryAn improved method for the preparation of bovine α-thrombin is described. The procedure involves the activation of partially purified prothrombin with tissue thromboplastin followed by chromatography on Sulfopropyl-Sephadex C-50. The purified enzyme is homogeneous on polyacrylamide discontinuous gel electrophoresis and has a specific activity toward fibrinogen of 2,200–2,700 N.I.H. U/mg. Its stability on storage in liquid media is dependent on both ionic strenght and temperature. Increasing ionic strength and decreasing temperature result in optimal stability. The denaturation of α-thrombin by guanidine hydrochloride was found to be a partially reversible process with the renatured species possessing properties similar to “aged” thrombin. In addition, the catalytic properties of a-thrombin covalently attached to agarose gel beads were also examined. The activity of the immobilized enzyme toward fibrinogen was affected to a much greater extent than was the hydrolysis of low molecular weight, synthetic substrates.

A New Method for Plasminogen Standardization

1968 ◽  
Vol 20 (03/04) ◽  
pp. 548-554
Author(s):  
J Gajewski ◽  
G Markus
Keyword(s):  
Proteolytic Activity ◽  
Specific Activity ◽  
Molar Ratio ◽  
Sharp Transition ◽  
Equivalence Point ◽  
Constant Amount ◽  
Residual Level ◽  
Activity Measurements ◽  
Complete Saturation ◽  
Human Plasminogen

SummaryA method for the standardization of human plasminogen is proposed, based on the stoichiometric interaction between plasminogen and streptokinase, resulting in inhibition of proteolytic activity. Activation of a constant amount of plasminogen with increasing amounts of streptokinase yields linearly decreasing activities, as a function of streptokinase, with a sharp transition to a constant residual level. The point of transition corresponds to complete saturation of plasmin with streptokinase in a 1:1 molar ratio, and is therefore a measure of the amount of plasminogen present initially, in terms of streptokinase equivalents. The equivalence point is independent of the kind of protein substrate used, buffer, pH, length of digestion and, within limits, temperature. The method, therefore, is not subject to the variations commonly encountered in the usual determination based on specific activity measurements.

Newborn Platelet Dysfunction: a Storage Pool and Release Defect

1976 ◽  
Vol 36 (01) ◽  
pp. 200-207 ◽  
Author(s):  
Donald G. Corby ◽  
Thomas F. Zuck
Keyword(s):  
Specific Activity ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Newborn Infants ◽  
Specific Radioactivity ◽  
High Dose ◽  
Platelet Dysfunction ◽  
Paired Samples ◽  
Normal Adults

SummaryPer cent aggregation, release and content of adenine nucleotides, and specific radioactivity were evaluated in citrated platelet-rich plasma (PRP) prepared from paired samples of maternal and cord blood. Platelets of newborn infants aggregated normally in response to high dose ADP (20 μM), strong collagen suspensions, and thrombin; however, when compared with PRP from the mothers or from normal adults, per cent aggregation in response to lower concentrations of ADP (2 μM), weak collagen, and part particularly epinephrine was markedly reduced. Nucleotide release after stimulation of the newborns’ PRP with the latter two inducers was also impaired. ATP and ADP content of the newborns’ platelets was also significantly less than that of their mothers or of normal adults, but specific activity was normal. The data suggest that the impairment of ADP release in the platelets of newborn infants is due to decreased sensitivity to external stimuli. Since metabolic ATP is necessary for the platelet release reaction, it is postulated that the platelet dysfunction results from a lack of metabolic ATP.

An International Collaborative Study to Investigate Standardisation of Hirudin Potency

1993 ◽  
Vol 69 (05) ◽  
pp. 430-435 ◽  
Author(s):  
Colin Longstaff ◽  
Man-Yu Wong ◽  
Patrick J Gaffney
Keyword(s):  
Specific Activity ◽  
Collaborative Study ◽  
Residual Activity ◽  
Quality Standard ◽  
Upper Limit ◽  
Assay Procedure ◽  
Specific Activities ◽  
International Collaborative ◽  
Range Of Values ◽  
International Collaborative Study

SummaryAn international collaborative study has been carried out to investigate the reproducibility of hirudin assays in 13 laboratories using four recombinant hirudins and one natural, sulphated product. A simple assay procedure was proposed involving the titration of α-thrombin with inhibitor and measurement of residual activity using a chromogenic substrate. A standard α-thrombin preparation was supplied to ensure that this reagent was of uniform quality throughout the study. The method appeared to present no difficulties and laboratories reported similar potencies for the 5 hirudin samples, in line with expected values. This gave 200–222 Thrombin Inhibitory Units/ampoule (TIU/ampoule) of lyophilised hirudin, with geometric coefficient of variation (gcv) values ranging from 10.15–15.97%. This corresponds to specific activities of approximately 14,300–15,900 TIU/mg protein. This is close to the upper limit of previously reported values of specific activity. We conclude that the precision of this determination compared with the wider range of values in the literature (8,000–16,000 thrombin inhibitory units [TIU]/mg) results from the use of good quality standard α-thrombin by all laboratories. This study has important implications for hirudin standardisation.

Preparation and Properties of Human Prothrombin Complex

1970 ◽  
Vol 24 (03/04) ◽  
pp. 325-333 ◽  
Author(s):  
G. H Tishkoff ◽  
L. C Williams ◽  
D. M Brown
Keyword(s):  
Molecular Weight ◽  
Proteolytic Enzyme ◽  
Enzyme Inhibitor ◽  
Specific Activity ◽  
Crystalline Form ◽  
Sedimentation Equilibrium ◽  
Crystalline Complex ◽  
Interaction Product ◽  
Proteolytic Enzyme Inhibitor ◽  
Purification Scheme

SummaryAs a corollary to our previous studies with bovine prothrombin, we have initiated a study of human prothrombin complex. This product has been isolated in crystalline form as a barium glycoprotein interaction product. Product yields were reduced compared to bovine product due to the increased solubility of the barium glycoprotein interaction product. On occasion the crystalline complex exhibited good yields. The specific activity of the crystalline complex was 1851 Iowa u/mg. Further purification of human prothrombin complex was made by removal of barium and by chromatography on Sephadex G-100 gels. The final product evidenced multiple procoagulant activities (II, VII, IX and X). The monomeric molecular weight determined by sedimentation equilibrium in a solvent of 6 M guanidine-HCl and 0.5% mercaptoethanol was 70,191 ± 3,057 and was homogeneous with respect to molecular weight. This product was characterized in regard to physical constants and chemical composition. In general, the molecular properties of human prothrombin complex are very similar to the comparable bovine product. In some preparations a reversible proteolytic enzyme inhibitor (p-aminophenylarsonic acid) was employed in the ultrafiltration step of the purification scheme to inhibit protein degradation.

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