Mahajan Richi V, Kumar Vinod, Rajendran Vinoth, Saran Saurabh, Ghosh Prahlad C, Saxena Rajendra Kumar
Department of Microbiology, University of Delhi South Campus, New Delhi, Delhi, India.
Department of Biochemistry, University of Delhi South Campus, New Delhi, Delhi, India.
PLoS One. 2014 Jun 6;9(6):e99037. doi: 10.1371/journal.pone.0099037. eCollection 2014.
L-asparaginase having low glutaminase has been a key therapeutic agent in the treatment of acute lymphpoblastic leukemia (A.L.L). In the present study, an extracellular L-asparaginase with low glutaminase activity, produced by Bacillus licheniformis was purified to homogeneity. Protein was found to be a homotetramer of 134.8 KDa with monomeric size of 33.7 KDa and very specific for its natural substrate i.e. L-asparagine. The activity of purified L-asparaginase enhanced in presence of cations including Na+ and K+, whereas it was moderately inhibited in the presence of divalent cations and thiol group blocking reagents. The purified enzyme was maximally active over the range of pH 6.0 to 10.0 and temperature of 40°C and enzyme was stable maximum at pH 9.0 and -20°C. CD spectra of L-asparaginase predicted the enzyme to consist of 63.05% α-helix and 3.29% β-sheets in its native form with T222 of 58°C. Fluorescent spectroscopy showed the protein to be stable even in the presence of more than 3 M GdHCl. Kinetic parameters Km, Vmax and kcat of purified enzyme were found as 1.4×10(-5) M, 4.03 IU and 2.68×10(3) s(-1), respectively. The purified L-asparaginase had cytotoxic activity against various cancerous cell lines viz. Jurkat clone E6-1, MCF-7 and K-562 with IC50 of 0.22 IU, 0.78 IU and 0.153 IU respectively. However the enzyme had no toxic effect on human erythrocytes and CHO cell lines hence should be considered potential candidate for further pharmaceutical use as an anticancer drug.
谷氨酰胺酶活性低的L-天冬酰胺酶一直是治疗急性淋巴细胞白血病（A.L.L）的关键治疗药物。在本研究中，对由地衣芽孢杆菌产生的具有低谷氨酰胺酶活性的细胞外L-天冬酰胺酶进行了纯化，直至达到同质。发现该蛋白质是一种134.8 KDa的同四聚体，单体大小为33.7 KDa，对其天然底物即L-天冬酰胺具有高度特异性。纯化的L-天冬酰胺酶在包括Na+和K+在内的阳离子存在下活性增强，而在二价阳离子和巯基阻断试剂存在下受到中度抑制。纯化的酶在pH 6.0至10.0的范围内以及40°C的温度下具有最大活性，并且在pH 9.0和-20°C时酶的稳定性最高。L-天冬酰胺酶的圆二色光谱预测该酶在天然形式下由63.05%的α-螺旋和3.29%的β-折叠组成，T222为58°C。荧光光谱表明，即使在存在超过3 M GdHCl的情况下，该蛋白质也是稳定的。纯化酶的动力学参数Km、Vmax和kcat分别为1.4×10(-5) M、4.03 IU和2.68×10(3) s(-1)。纯化的L-天冬酰胺酶对各种癌细胞系具有细胞毒性，即Jurkat克隆E6-1、MCF-7和K-562，IC50分别为0.22 IU、0.78 IU和0.153 IU。然而，该酶对人红细胞和CHO细胞系没有毒性作用，因此应被视为进一步作为抗癌药物用于制药的潜在候选物。
