Recombinant intracellular Rhodospirillum rubrum L-asparaginase with low L-glutaminase activity and antiproliferative effect

摘要

The recombinant producer strain expressing Rhodospirillum rubrum L-asparaginase (RrA) has been obtained and a purification procedure of RrA has been developed. The purified enzyme, RrA, has the following biochemical and catalytic characteristics: K _m for L-Asn of 0.22 mM, pH optimum at 9.2; temperature optimum at 54°C, pI = 5.1. RrA exhibited a significant cytotoxic effect towards the following cell lines: K562 (IC _(50) = 1.80 U/mL), DU145 (IC _(50) = 9.19 U/mL), and MDA-MB-231 (IC _(50) = 34.62 U/mL). Comparative analysis employing E. coli L-asparaginase II type (EcA) and Erwinia carotovora L-asparaginase (EwA) has shown that the enzyme cytotoxicity towards these cell lines decreased in the following order: EcA > RrA > EwA. Daily administration of RrA (4000 U/kg) to L5178y bearing mice for 10 days (total dose of 40000 U/kg) showed T/C = 172. Data obtained suggest that RrA may be referred to intracellular L-asparaginases with low L-glutaminase activity and marked antiproliferative effect.