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Frontiers of Chemical Science and Engineering

ISSN 2095-0179

ISSN 2095-0187(Online)

CN 11-5981/TQ

邮发代号 80-969

2019 Impact Factor: 3.552

Frontiers of Chemical Science and Engineering  2013, Vol. 7 Issue (1): 37-42   https://doi.org/10.1007/s11705-013-1303-z
  RESEARCH ARTICLE 本期目录
Purification of L-asparaginase II by crystallization
Purification of L-asparaginase II by crystallization
Y. LIU1(), M. PIETZSCH2, J. ULRICH1()
1. Thermal Process Engineering, Center for Engineering Science, Martin Luther University, Halle-Wittenberg, D-06099 Halle (Saale), Germany; 2. Department of Downstream Processing, Institute of Pharmacy, Faculty of Natural Sciences I, Martin Luther University, Halle-Wittenberg, D-06099 Halle (Saale), Germany
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Abstract

Here a case study of L-asparaginase II out of a recombinant Escherichia coli is presented. The target protein was obtained by simple cell disintegration and acetone precipitation. The L-asparaginase II has been crystallized in three different forms in the following microbatch crystallization. The rod-shaped crystals (~400 μm edge length) were obtained at either 8°C or 22°C after 17 h by addition of PEG6000. The rectangular-shaped crystals were obtained after further recrystallization of the rod-shaped crystals. The rhombic-shaped crystals formed at 8°C after 12 days when cold ethanol was used instead of PEG6000. All crystallizations were performed in tris-acetate buffer (50 mmol·L-1, pH 5.1). By crystallization, the specific activity of L-asparaginase II has increased 5-fold. The protein content and the purity of the crystals were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The more concentrated L-asparaginase II out of an extract mixture and the presence of only less minor proteins after crystallization demonstrates that crystallization is an effective and mild method to purify the target protein. The single crystal X-ray diffraction pattern reveals that the crystals are proteins and the X-ray powder diffraction (XRPD) pattern shows clearly that the crystals forming in PEG6000 and ethanol have different crystal structures.

Key wordsprotein crystallization    L-asparaginase II    purification
收稿日期: 2012-09-27      出版日期: 2013-03-05
Corresponding Author(s): LIU Y.,Email:yi.liu@iw.uni-halle.de; ULRICH J.,Email:ulrich@iw.uni-halle.de   
 引用本文:   
. Purification of L-asparaginase II by crystallization[J]. Frontiers of Chemical Science and Engineering, 2013, 7(1): 37-42.
Y. LIU, M. PIETZSCH, J. ULRICH. Purification of L-asparaginase II by crystallization. Front Chem Sci Eng, 2013, 7(1): 37-42.
 链接本文:  
https://academic.hep.com.cn/fcse/CN/10.1007/s11705-013-1303-z
https://academic.hep.com.cn/fcse/CN/Y2013/V7/I1/37
Protein solutionPurification stepTotal activity /USpecific activity /(U·mg-1)
Crude extract1st acetone precipitation74176.7 (100%)11.1
pH 5.1 extractpH 5.1 tris-acetate buffer46261.7 (55.6%)26.9
CrystalsCrystallization with PEG600025799.3 (34.8%)57.2
Tab.1  
Fig.1  
Fig.1  
Fig.2  
Fig.2  
Fig.3  
Fig.3  
Crystals morphologyPrecipitantGrowth time/hAverage growth rate a) /m·s-1Final size b) /μm
Rod-shaped crystals(Fig. 1(b))PEG6000170.27 × 10-9400 × 23 × 2
Rectangular-shaped crystals (Fig. 1(c))PEG6000170.23 × 10-9179 × 34 × 2
Rhombic-shaped crystals (Fig. 2)Cold ethanol2880.04 × 10-9241 × 132 × 10
Tab.2  
Fig.4  
Fig.4  
Fig.5  
Fig.5  
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