Sudhir Ankit P, Dave Bhaumik R, Prajapati Anil S, Panchal Ketankumar, Patel Darshan, Subramanian R B
BRD School of Biosciences, Sardar Patel Maidan, Sardar Patel University, Vadtal Road, Satellite Campus, PO Box 39, Vallabh Vidyanagar, 388 120, Gujarat, India.
Appl Biochem Biotechnol. 2014 Dec;174(7):2504-15. doi: 10.1007/s12010-014-1200-z. Epub 2014 Sep 16.
L-Asparaginase (3.5.1.1) is an enzyme widely used to treat the acute lymphoblastic leukemia. Two genes coding for L-asparaginase (ansA1 and ansA3) from Bacillus licheniformis MTCC 429 were cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were purified to homogeneity by one-step purification process and further characterized for various biochemical parameters. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that both the enzymes are monomers of ∼37 kDa. Recombinant ansA1 was found to be highly unstable, and recombinant ansA3 was catalytically active and stable, which showed an optimum activity of 407.65 IU/mg at 37 °C and pH 8. Recombinant ansA3 showed higher substrate specificity for L-asparagine with negligible glutaminase activity. Kinetic parameters like K m , V max, k cat, and k cat/K m were calculated for recombinant ansA3.
L-天冬酰胺酶（3.5.1.1）是一种广泛用于治疗急性淋巴细胞白血病的酶。从地衣芽孢杆菌MTCC 429中克隆出两个编码L-天冬酰胺酶的基因（ansA1和ansA3），并在大肠杆菌BL21（DE3）细胞中进行了过表达。通过一步纯化过程将重组蛋白纯化至同质，并进一步对各种生化参数进行了表征。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，这两种酶均为约37 kDa的单体。发现重组ansA1高度不稳定，而重组ansA3具有催化活性且稳定，在37°C和pH 8时显示出407.65 IU/mg的最佳活性。重组ansA3对L-天冬酰胺表现出更高的底物特异性，谷氨酰胺酶活性可忽略不计。计算了重组ansA3的动力学参数，如K m、V max、k cat和k cat/K m。
