scholarly journals Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede,Chamberlinius hualienensis

2015 ◽  
Vol 112 (34) ◽  
pp. 10605-10610 ◽  
Author(s):  
Mohammad Dadashipour ◽  
Yuko Ishida ◽  
Kazunori Yamamoto ◽  
Yasuhisa Asano
Keyword(s):  
Specific Activity ◽  
Enantiomeric Excess ◽  
High Specific Activity ◽  
Potassium Cyanide ◽  
Aromatic Aldehydes ◽  
Building Blocks ◽  
Industrial Biotechnology ◽  
Hydroxynitrile Lyase ◽  
Wide Range ◽  
Very High

Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipedeChamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

The labeling of brassinosteroids by tritium

RSC Advances ◽  
2015 ◽  
Vol 5 (80) ◽  
pp. 65214-65220 ◽  
Author(s):  
Aleš Marek ◽  
Mahadeo R. Patil ◽  
Tomáš Elbert
Keyword(s):  
Convenient Method ◽  
Specific Activity ◽  
High Specific Activity ◽  
Very High

A convenient method for the synthesis of tritium-labeled brassinosteroids with very high specific activity is reported.

scholarly journals Production of Sm-153 With Very High Specific Activity for Targeted Radionuclide Therapy

2021 ◽  
Vol 8 ◽  
Author(s):  
Michiel Van de Voorde ◽  
Charlotte Duchemin ◽  
Reinhard Heinke ◽  
Laura Lambert ◽  
Eric Chevallay ◽  
...  
Keyword(s):  
Neutron Irradiation ◽  
Radionuclide Therapy ◽  
High Efficiency ◽  
Separation Efficiency ◽  
Specific Activity ◽  
High Specific Activity ◽  
Mass Separation ◽  
Full Potential ◽  
Targeted Radionuclide Therapy ◽  
Very High

Samarium-153 (153Sm) is a highly interesting radionuclide within the field of targeted radionuclide therapy because of its favorable decay characteristics. 153Sm has a half-life of 1.93 d and decays into a stable daughter nuclide (153Eu) whereupon β− particles [E = 705 keV (30%), 635 keV (50%)] are emitted which are suitable for therapy. 153Sm also emits γ photons [103 keV (28%)] allowing for SPECT imaging, which is of value in theranostics. However, the full potential of 153Sm in nuclear medicine is currently not being exploited because of the radionuclide's limited specific activity due to its carrier added production route. In this work a new production method was developed to produce 153Sm with higher specific activity, allowing for its potential use in targeted radionuclide therapy. 153Sm was efficiently produced via neutron irradiation of a highly enriched 152Sm target (98.7% enriched, σth = 206 b) in the BR2 reactor at SCK CEN. Irradiated target materials were shipped to CERN-MEDICIS, where 153Sm was isolated from the 152Sm target via mass separation (MS) in combination with laser resonance enhanced ionization to drastically increase the specific activity. The specific activity obtained was 1.87 TBq/mg (≈ 265 times higher after the end of irradiation in BR2 + cooling). An overall mass separation efficiency of 4.5% was reached on average for all mass separations. Further radiochemical purification steps were developed at SCK CEN to recover the 153Sm from the MS target to yield a solution ready for radiolabeling. Each step of the radiochemical process was fully analyzed and characterized for further optimization resulting in a high efficiency (overall recovery: 84%). The obtained high specific activity (HSA) 153Sm was then used in radiolabeling experiments with different concentrations of 4-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA). Even at low concentrations of p-SCN-Bn-DOTA, radiolabeling of 0.5 MBq of HSA 153Sm was found to be efficient. In this proof-of-concept study, we demonstrated the potential to combine neutron irradiation with mass separation to supply high specific activity 153Sm. Using this process, 153SmCl3 suitable for radiolabeling, was produced with a very high specific activity allowing application of 153Sm in targeted radionuclide therapy. Further studies to incorporate 153Sm in radiopharmaceuticals for targeted radionuclide therapy are ongoing.

Eine Methode zur qualitativen und quantitativen Bestimmung von drei Juvenilhormonen von Insekten

1974 ◽  
Vol 29 (3-4) ◽  
pp. 161-168 ◽  
Author(s):  
K. H. Trautmann ◽  
A. Schuler ◽  
M. Suchý ◽  
H.-K. Wipf
Keyword(s):  
Quantitative Determination ◽  
Specific Activity ◽  
High Specific Activity ◽  
Ether Extract ◽  
Qualitative And Quantitative ◽  
Work Up ◽  
First Time ◽  
Very High ◽  
Tenebrio Molitor Larvae

Abstract A method is presented permitting the qualitative and quantitative determination of all three presently known hormones (JH1-3). The determination is based on the method of radioactive isotope dilution, whereby a very small known amount of tritium-labelled JH-1 is added to the ether extract of the particular species. The addition of radioactive JH-1 permits the isolation of all three hormones, because of their similar behaviour during the chosen work up. The quantitative determination was carried out by gas chromatography and the identification was confirmed with the help of retention-times and GC-MS combination. The method was checked by using an extract of Hyalophora cecropia. For the first time methyl 10,11-epoxy-3,7,11-trimethyl-2-trans-6-trans-dodecadienoate (JH-3) could also be identified as the juvenile hormone of Melo­lontha melolontha. In Vanessa io larvae, Tenebrio molitor larvae and adults and in Musca domestica larvae none of the three known hormones could be detected. The preparation of JH-1 labelled with tritium in the methyl group of the ester was accomplished with very high specific activity (4.34 Ci/mmol) of the tritiated acid with diazomethane.

Factor VIII:E113A Represents a High Specific Activity Factor VIII Arising from a Single Point Mutation within a Ca2+ Binding Site.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1725-1725 ◽  
Author(s):  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Wild Type ◽  
Activity Increase ◽  
Wide Range

Abstract We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca2+, an ion necessary for cofactor activity (Wakabayashi et al., J. Biol. Chem.279:12677–12684, 2004). Using Ala-scanning mutagenesis, it was determined that replacement of residue E113 with Ala yielded a factor VIII point mutant that possessed an ~2-fold increased affinity for Ca2+ as compared with wild type, suggesting that this residue did not directly contribute to Ca2+ coordination but rather modulated the affinity of the ion at this site. Furthermore, the E113A factor VIII possessed twice the specific activity of wild type as determined by a one-stage clotting assay. This increased activity was not likely a result of increased affinity for Ca2+, since assays were performed at saturating Ca2+ levels. Saturation mutagenesis at position 113 revealed that substitution at this position with relatively small, nonpolar residues were well-tolerated, whereas replacement with a number of polar or charged residues was detrimental to activity. Ala-substitution yielded the greatest activity increase of ~2-fold and this level was observed over a wide range of factor VIII concentrations. Time course experiments of factor VIII activation following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Interestingly, results from factor Xa generation assays using purified reactants showed the mutant possessed <10% greater specific activity than wild type and yielded similar values for Km for substrate factor X, kcat for factor Xa generation and Kd for factor IXa. Thus the single amino acid substitution minimally altered cofactor structure or inter-molecular interactions relating to its participation in factor Xase. These results indicate that mutations within this Ca2+ coordination site may selectively enhance cofactor specific activity as measured in a plasma-based assay compared to activity determined in a purified system. The enhanced activity observed for E113A factor VIII may derive from a subtle alteration in conformation affecting a yet to be identified functional parameter.

scholarly journals Identification, Cloning, and Characterization of a Novel Ketoreductase from the Cyanobacterium Synechococcus sp. Strain PCC 7942

2008 ◽  
Vol 74 (21) ◽  
pp. 6697-6702 ◽  
Author(s):  
Kathrin H�lsch ◽  
Jan Havel ◽  
Martin Haslbeck ◽  
Dirk Weuster-Botz
Keyword(s):  
Specific Activity ◽  
Acyl Carrier Protein ◽  
Asymmetric Reduction ◽  
Enantiomeric Excess ◽  
High Specific Activity ◽  
Lactobacillus Brevis ◽  
Ph Optimum ◽  
Synechococcus Sp ◽  
Prochiral Ketones ◽  
Pcc 7942

ABSTRACT A new ketoreductase useful for asymmetric synthesis of chiral alcohols was identified in the cyanobacterium Synechococcus sp. strain PCC 7942. Mass spectrometry of trypsin-digested peptides identified the protein as 3-ketoacyl-[acyl-carrier-protein] reductase (KR) (EC 1.1.1.100). The gene, referred to as fabG, was cloned, functionally expressed in Escherichia coli, and subsequently purified to homogeneity. The enzyme displayed a temperature optimum at 44�C and a broad pH optimum between pH 7 and pH 9. The NADPH-dependent KR was able to asymmetrically reduce a variety of prochiral ketones with good to excellent enantioselectivities (>99.8%). The KR showed particular high specific activity for asymmetric reduction of ethyl 4-chloroacetoacetate (38.29 � 2.15 U mg−1) and 2′,3′,4′,5′,6′-pentafluoroacetophenone (8.57 � 0.49 U mg−1) to the corresponding (S)-alcohols. In comparison with an established industrial enzyme like the alcohol dehydrogenase from Lactobacillus brevis, the KR showed seven-times-higher activity toward 2′,3′,4′,5′,6′-pentafluoroacetophenone, with a remarkably higher enantiomeric excess (>99.8% [S] versus 43.3% [S]).

Modified Method for the Preparation of Purified Bovine Prothrombin of High Specific Activity

1968 ◽  
Vol 19 (01/02) ◽  
pp. 178-185 ◽  
Author(s):  
O. P Malhotra ◽  
J. R Carter
Keyword(s):  
Moving Boundary ◽  
Specific Activity ◽  
High Specific Activity ◽  
Disc Electrophoresis ◽  
Factor Vii ◽  
Voltage Gradient ◽  
Factor V ◽  
Isolation And Purification ◽  
Modified Method ◽  
Very High

SummaryA modified method for the isolation and purification of bovine prothrombin is described. The preparations have a very high specific activity, viz. 3,200 ± 200 u/mg of protein, show a single symmetrical peak in the analytical ultracentrifuge and by moving boundary electrophoresis at both pH 6.86 and 8.6. They do not undergo inactivation or dissociation during electrophoresis at high voltage gradient (7.5) at alkaline pH. Disc electrophoresis also shows essentially a single component. For optimal activation, prothrombin requires the presence of factor VII-X complex in addition to factor V.

Properties and Genesis of Hot Particles from the Chernobyl Reactor Accident

1996 ◽  
Vol 465 ◽  
Author(s):  
Peter Schubert-Bischoff ◽  
Werner Lutze ◽  
Boris E. Burakov
Keyword(s):  
Fission Product ◽  
Specific Activity ◽  
High Specific Activity ◽  
Chemical Compositions ◽  
Fuel Particle ◽  
Reactor Unit ◽  
Hot Particles ◽  
Hot Particle ◽  
Dust Generation ◽  
Wide Range

ABSTRACTOn April 25, 1986, the nuclear reactor Unit 4 (RBMK) at Chernobyl, Ukraine, exploded. Besides molecular species, the fallout contained particles of relatively high specific activity (hot particles) with a wide range of chemical compositions. The composition of a hot particle bears information about its genesis. Particle sizes ranged from a few to 100s of micrometers. Data on a hot particle, found in Berlin, Germany, is presented and discussed in context with earlier measurements on other particles to understand their genesis. The chemical composition was determined by electron probe micro analysis. Our particles are either reactor fuel (one) or fission product alloys (nine). The alloys were formed during normal reactor operation. Strongly varying concentrations of Fe and Ni suggest that at least some of our particles reacted with molten structural material of the reactor. The particles were mobilized by fuel oxidation or fuel dust generation during the accident. The fission product composition can only be explained if we assume that the alloys remained in the solid state in the course of the accident. Some particles may have been ejected during the explosion, others later while the reactor was burning. Activities (103Ru and 106Ru, originally up to 160,000 Bq) of our ten year old particles were re-measured but were no longer detectable. No long-lived γ-emitters were found. The 99Tc activity was calculated and found to only lBq. The γ -spectrum of the fuel particle still shows 137Cs (1 Bq) and 60Co (<1 Bq).

ULTRAFILTRATION STUDIES OF STEROID-PROTEIN BINDING

1961 ◽  
Vol 23 (2) ◽  
pp. 129-137 ◽  
Author(s):  
P. S. CHEN ◽  
I. H. MILLS ◽  
F. C. BARTTER
Keyword(s):  
Protein Binding ◽  
Specific Activity ◽  
Binding Protein ◽  
High Specific Activity ◽  
Wide Range

SUMMARY A method is described for determination of the extent of protein binding of steroids by ultrafiltration after addition of radioactively-labelled steroid tracers of high specific activity. Results obtained with specifically labelled 14C-steroids did not differ significantly from those with steroids randomly labelled with tritium. A number of steroids have been studied with this technique. The 'S' shaped curve obtained with some corticosteroids by plotting percentage ultrafilterable against steroid added to the plasma confirmed the presence of the corticosteroid-binding protein which has greater affinity for steroid than albumin. This method of plotting the ultrafiltration data afforded a method of assessing the amount of binding protein (or the number of sites). Cortisone, 17-hydroxy-11-deoxycorticosterone, cortisol, and Δ1 cortisol showed this type of curve. Binding of aldosterone, progesterone and 17-ketosteroids occurred to the same extent in plasma as in 5% albumin. The ultrafilterable fraction of these steroids in plasma was constant over a wide range of total steroid concentration. Binding of testosterone was greater with plasma than with albumin, and did not decrease upon addition of large amounts of testosterone.

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