Polyäthylenglykol zur Anreicherung und Kristallisation von L‐Asparaginase

Wagner, Otto; Bauer, Klaus; Irion, E; Rauenbusch, Erich; Kaufmann, W.; Arens, A

doi:10.1002/ange.19690812214

Cited by 9 publications

(5 citation statements)

References 2 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An opposite dependency on protein concentration can be recognized which means that each additional PEG fractionation step will lower the protein concentration in the remaining L-asparaginase B solution. If 50% PEG 6000 fractionation is carried out according to Wagner et al [12] and Born and Bauer [15], the decrease in protein concentration rises from the separation by centrifugation of solidified L-asparaginase B after each fractionation step. In this work, the presented specific activities measured after each fractionation step (see Tab. 3) are lower than the ones published in the patent of Wagner et al [12].…”

Section: Resultsmentioning

confidence: 99%

“…If 50% PEG 6000 fractionation is carried out according to Wagner et al [12] and Born and Bauer [15], the decrease in protein concentration rises from the separation by centrifugation of solidified L-asparaginase B after each fractionation step. In this work, the presented specific activities measured after each fractionation step (see Tab. 3) are lower than the ones published in the patent of Wagner et al [12]. Possible reasons for these obvious differences could be the different centrifugation parameters, different working temperatures, changes in buffer type and pH, or different molecular weights of the PEG.…”

Section: Resultsmentioning

confidence: 99%

“…Activity measurements and shape analysis were done after 24 h at 8°C. 1) First crystallization trials were carried out, according to Wagner et al [12] and Born and Bauer [16], by adding 50% PEG 6000 in several fractionation steps to the crude L-asparaginase B solution, starting from an addition of 17 % up to 29 %. Tab.…”

Section: Resultsmentioning

confidence: 99%

“…According to the patent of Wagner et al [12], an experimental design for acetone precipitation followed by crystallization was developed. Tab.…”

Section: Acetone Precipitation/crystallizationmentioning

confidence: 99%

See 3 more Smart Citations

Recombinant L‐Asparaginase B and its Crystallization – What is the Nature of Protein Crystals?

Müller¹,

Liu²,

Migge³

et al. 2011

Chem Eng & Technol

View full text Add to dashboard Cite

L-Asparaginase is a protein produced by Escherichia coli and exists in two types, A and B. Only type B shows a specific antitumor activity to acute lymphatic leukemia. A new recombinant E. coli BL21Gold (DE3) pET11a-ansB was used to produce L-asparaginase B in higher quantities. To enhance the purity and preserve the activity of L-asparaginase B, crystallization is a promising process. Based on available patents, first experiments on L-asparaginase B crystallization were performed. Recombinant L-asparaginase B was successfully and reproducibly crystallized in two modifications. Up to now, it is not clear in which respect these two modifications of crystalline L-asparaginase B differ. It is part of the current study to investigate the nature of protein crystals in detail. The investigations on lysozyme crystals are well in agreement with the literature.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…According to the patent of Wagner et al [12], an experimental design for acetone precipitation followed by crystallization was developed. Tab.…”

Section: Acetone Precipitation/crystallizationmentioning

confidence: 99%

See 2 more Smart Citations

Recombinant L‐Asparaginase B and its Crystallization – What is the Nature of Protein Crystals?

Müller¹,

Liu²,

Migge³

et al. 2011

Chem Eng & Technol

View full text Add to dashboard Cite

show abstract

“…E. coli strains produce two L-asparaginases, one with and the other without tumor inhibitory activity (5,34); the two enzymes can be separated by chromatography. The properties of crystalline E. coli asparaginase were studied in some detail (1, 10,13,35,37). More recently, two isozymes of E. coli L-asparaginase with tumor inhibitory activity were crystallized (1).…”

mentioning

confidence: 99%

Purification and Properties of l -Asparaginase from Serratia marcescens

Boyd

Phillips

1971

J Bacteriol

View full text Add to dashboard Cite

The purification and properties of a tumor inhibitory L-asparaginase from SeOratia marcescens are described. The following properties of the enzyme were examined: kinetics of the enzyme reaction, catalytic activity as a function of pH, boundary sedimentation velocity, electrophoresis on polyacrylamide gel, immunoelectrophoresis against homologous and heterologous antisera, immunodiffusion, blood clearance rate in mice, and inhibition of the 6C3HED lymphoma in C3H mice. Complete regression of this tumor was obtained with a smaller dose of the enzyme from S. marcescens than with enzyme from Escherichia coli. The reason for this difference was not evident from a comparison of several properties of the two enzymes.Eazyme assays. Routine L-asparaginase assays were conducted by a modified method based on that of Meister et al. (20). Portions (1 to 100 Mliters) of enzyme solution were added to 0.05 M tris(hydroxmethyl)aminomethane (Tris)-hydrochloride buffer (pH 7.4) to give a final volume of 1.5 ml. The reaction was initiated by the addition of 0.5 ml of 0.04 M L-asparagine in the same buffer and conducted at 37 C in a reciprocal water bath shaker. The reaction was stopped by the addition of 0.1 ml of 1.5 M trichloroacetic acid. If necessary, the mixture was centrifuged to remove precipitated proteins. Ammonia released in the reaction was determined by the addition of Nessler's reagent to the diluted supernatant fluid and, after 15 min, observing the absorbancy at 500 nm. Our studies on enzyme kinetics utilized a reduced nicotinamide adenine dinucleotide (NADH)-dependent coupled assay for ammonia producing systems (15). NADH and ammonia are required in equimolar amounts for the synthesis of glutamate from a-ketoglutarate by glutamic dehydrogenase. The rate of ammonia production from L-asparaginase may be calculated from the rate of NADH oxidation 578 on July 31, 2020 by guest http://jb.asm.org/ Downloaded from on July 31, 2020 by guest

show abstract