Sindhu R, Manonmani H K
Food Protectants and Infestation Control Department, CSIR- Central Food Technological Research Institute, Mysuru 570 020, Karnataka, India.
Protein Expr Purif. 2018 Mar;143:83-91. doi: 10.1016/j.pep.2017.09.009. Epub 2017 Oct 25.
l-asparaginase (E.C. 3.5.1.1), an anti-cancer drug has been used in the treatment of acute lymphoblastic leukemia. A novel source of l-asparaginase from Pseudomonas fluorescens was addressed in the present studies. The ANS gene in Pseudomonas fluorescens MTCC 8127 which produces l-asparaginase was cloned and expressed in E. coli BL21 (DE3). The expressed recombinant protein (PfAns) which was purified, showed l-asparaginase activity. The enzyme was further characterized. The pH and temperature optima were found to be 7.5 and 37 °C. The recombinant enzyme was stable to temperature and pH. The enzyme was homotetramer with the molecular weight of the monomer being 35 kDa and a whole protein molecular weight of 140 kDa. The purified l-asparaginase had a specific activity of 26 U/mg with a K and V of 0.050 M and 4.032 molmin. The enzyme exhibited a high affinity towards l-asparagine and showed a very minimal activity with glutamine as a substrate. The enzyme activity was inhibited by PMSF, suggesting the presence of serine at the active site. The presence of Mg enhanced PfAns activity by 49%, and SDS strongly inhibited the enzyme activity. The in vitro half-life of the recombinant enzyme was ∼40 h. The enzyme demonstrated deglycosylation activity which could exhibit an additional barrier for proliferating cancer cells.
L-天冬酰胺酶（E.C. 3.5.1.1）是一种抗癌药物，已用于治疗急性淋巴细胞白血病。本研究探讨了荧光假单胞菌中L-天冬酰胺酶的一种新来源。在荧光假单胞菌MTCC 8127中产生L-天冬酰胺酶的ANS基因被克隆并在大肠杆菌BL21（DE3）中表达。纯化后的表达重组蛋白（PfAns）显示出L-天冬酰胺酶活性。该酶进一步得到了表征。发现最适pH和温度分别为7.5和37℃。重组酶对温度和pH稳定。该酶为同四聚体，单体分子量为35 kDa，全蛋白分子量为140 kDa。纯化的L-天冬酰胺酶比活性为26 U/mg，K和V分别为0.050 M和4.032 μmol/min。该酶对L-天冬酰胺表现出高亲和力，以谷氨酰胺为底物时活性极低。该酶活性受到苯甲基磺酰氟（PMSF）的抑制，表明活性位点存在丝氨酸。镁离子的存在使PfAns活性提高了49%，而十二烷基硫酸钠（SDS）强烈抑制该酶活性。重组酶的体外半衰期约为40小时。该酶表现出脱糖基化活性，这可能对增殖的癌细胞构成额外的屏障。
