Manna S, Sinha A, Sadhukhan R, Chakrabarty S L
Department of Microbiology, Bose Institute, Calcutta, India.
Curr Microbiol. 1995 May;30(5):291-8. doi: 10.1007/BF00295504.
An L-asparaginase produced by Pseudomonas stutzeri MB-405 was isolated and characterized. After initial ammonium sulfate fractionation, the enzyme was purified by consecutive column chromatography on Sephadex G-100, Ca-hydroxylapatite, and DEAE-Sephadex A-50. The 665.5-fold purified enzyme thus obtained has the specific activity of 732.3 units mg protein-1 with an overall recovery of 27.2%. The apparent M(r) of the enzyme under nondenaturing and denaturing conditions was 34 kDa and 33 kDa respectively, and the isoelectric point was 6.38 +/- 0.02. It displayed optimum activity at pH 9.0 and 37 degrees C. The enzyme was very specific for L-asparagine and did not hydrolyze L-glutaminate. The Km of the L-asparaginase was found to be 1.45 x 10(-4) M towards L-asparagine and was competitively inhibited by 5-diazo-4-oxo-L- norvaline (DONV) with a Ki of 0.03 mM. Metal ions such as Mn2+, Zn2+, Hg2+, Fe3+, Ni2+, and Cd2+ potentially inhibited the enzyme activity. The activity was enhanced in the presence of thiol-protecting reagents such as DTT, 2-ME, and glutathione (reduced), but inhibited by PCMB and iodoacetamide. The tumor inhibition study with Dalton's lymphoma tumor cells in vivo indicated that this enzyme possesses antitumor properties.
对斯氏假单胞菌MB-405产生的一种L-天冬酰胺酶进行了分离和表征。经过初步硫酸铵分级分离后，该酶通过在Sephadex G-100、羟基磷灰石钙和DEAE-Sephadex A-50上连续柱层析进行纯化。由此获得的纯化了665.5倍的酶具有732.3单位mg蛋白-1的比活性，总回收率为27.2%。该酶在非变性和变性条件下的表观分子量分别为34 kDa和33 kDa，等电点为6.38±0.02。它在pH 9.0和37℃时表现出最佳活性。该酶对L-天冬酰胺具有高度特异性，不水解L-谷氨酰胺。发现该L-天冬酰胺酶对L-天冬酰胺的Km为1.45×10(-4)M，并被5-重氮-4-氧代-L-正缬氨酸（DONV）竞争性抑制，Ki为0.03 mM。金属离子如Mn2+、Zn2+、Hg2+、Fe3+、Ni2+和Cd2+可能抑制该酶的活性。在存在硫醇保护试剂如DTT、2-ME和还原型谷胱甘肽时，活性增强，但被对氯汞苯甲酸和碘乙酰胺抑制。用道尔顿淋巴瘤肿瘤细胞进行的体内肿瘤抑制研究表明该酶具有抗肿瘤特性。
