Kotzia Georgia A, Labrou Nikolaos E
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece.
J Biotechnol. 2007 Jan 20;127(4):657-69. doi: 10.1016/j.jbiotec.2006.07.037. Epub 2006 Sep 18.
Bacterial L-asparaginases (L-ASNases) catalyze the conversion of L-asparagine to L-aspartate and ammonia. In the present work, we report the cloning and expression of L-asparaginase from Erwinia chrysanthemi 3937 (ErL-ASNase) in Escherichia coli BL21(DE3)pLysS. The enzyme was purified to homogeneity in a single-step procedure involving cation exchange chromatography on an S-Sepharose FF column. The enzymatic and structural properties of the recombinant enzyme were investigated and the kinetic parameters (K(m), k(cat)) for a number of substrates were determined. In addition, we found that the enzyme can be efficiently immobilized on epoxy-activated Sepharose CL-6B. The immobilized enzyme retains most of its activity (60%) and shows high stability at 4 degrees C. The approach offers the possibility of designing an ErL-ASNase bioreactor that can be operated over a long period of time with high efficiency, which can be used in leukaemia therapy.
细菌L-天冬酰胺酶（L-ASNases）催化L-天冬酰胺转化为L-天冬氨酸和氨。在本研究中，我们报道了来自菊欧文氏菌3937（ErL-ASNase）的L-天冬酰胺酶在大肠杆菌BL21(DE3)pLysS中的克隆与表达。通过在S-Sepharose FF柱上进行阳离子交换色谱的单步程序，将该酶纯化至同质。研究了重组酶的酶学和结构特性，并测定了多种底物的动力学参数（K(m)，k(cat)）。此外，我们发现该酶可以有效地固定在环氧活化的Sepharose CL-6B上。固定化酶保留了大部分活性（60%），并在4℃下表现出高稳定性。该方法为设计一种可长时间高效运行的ErL-ASNase生物反应器提供了可能性，该生物反应器可用于白血病治疗。
