从胡萝卜软腐果胶杆菌 MTCC 1428 中纯化和表征不含谷氨酰胺酶的 L-天冬酰胺酶。
Purification and characterization of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428.
机构信息
Biochemical Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.
出版信息
Bioresour Technol. 2011 Jan;102(2):2077-82. doi: 10.1016/j.biortech.2010.07.114. Epub 2010 Aug 1.
An intracellular glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428 was isolated to apparent homogeneity. The homotetramer enzyme has a molecular mass of 144.4 kDa (MALDI-TOF MS) and an isoelectric point of approximately 8.4. The enzyme is very specific for its natural substrate, L-asparagine. The activity of L-asparaginase is activated by mono cations and various effectors including Na+, K+, L-cystine, L-histidine, glutathione and 2-mercaptoethanol whereas it is moderately inhibited by various divalent cations and thiol group blocking reagents. Kinetic parameters, Km, Vmax and kcat of purified L-asparaginase from P. carotovorum MTCC 1428 were found to be 0.657 mM, 4.45 U μg(-1) and 2.751×10(3) s(-1), respectively. Optimum pH of purified L-asparaginase for the hydrolysis of L-asparagine was in the range of 8.0-10.0, and its optimum temperature was found to be 40 °C. The purified L-asparaginase has no partial glutaminase activity, which can reduce the possibility of side effects during the course of anti-cancer therapy.
从 Pectobacterium carotovorum MTCC 1428 中分离出一种不含细胞内谷氨酰胺酶的 L-天冬酰胺酶，达到明显的均一性。该同四聚体酶的分子量为 144.4 kDa（MALDI-TOF MS），等电点约为 8.4。该酶对其天然底物 L-天冬酰胺具有很高的特异性。L-天冬酰胺酶的活性被单价阳离子和各种效应物激活，包括 Na+、K+、L-胱氨酸、L-组氨酸、谷胱甘肽和 2-巯基乙醇，而各种二价阳离子和巯基组阻断试剂则中度抑制其活性。从 P. carotovorum MTCC 1428 中分离出的纯化 L-天冬酰胺酶的动力学参数 Km、Vmax 和 kcat 分别为 0.657 mM、4.45 U μg(-1) 和 2.751×10(3) s(-1)。纯化 L-天冬酰胺酶水解 L-天冬酰胺的最适 pH 值范围为 8.0-10.0，最适温度为 40°C。纯化的 L-天冬酰胺酶没有部分谷氨酰胺酶活性，这可以降低在抗癌治疗过程中产生副作用的可能性。
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