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One‐step purification and kinetic properties of the recombinant l ‐asparaginase from Erwinia carotovora
Author(s) -
Krasotkina Julya,
Borisova Anna A.,
Gervaziev Yuri V.,
Sokolov Nikolay N.
Publication year - 2004
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20030138
Subject(s) - recombinant dna , erwinia , enzyme , asparaginase , enterobacteriaceae , microbiology and biotechnology , specific activity , escherichia coli , glutaminase , biology , biochemistry , chemistry , immunology , lymphoblastic leukemia , gene , leukemia , amino acid , glutamine
ECAR‐LANS, the recombinant l ‐asparaginase from Erwinia carotovora , is a prospective therapeutic enzyme for leukaemia treatment. An efficient and economical scheme was developed for the purification, cloning and expression in Eschericha coli of ECAR‐LANS. More than 90% purity, complemented with 72% active enzyme recovery, was achieved with a single chromatographic purification step. The activity of purified l ‐asparaginase was 630 i.u./mg. The ECAR‐LANS K m value was 98×10 −6 M for the main physiological substrate l ‐Asn and 3400×10 −6 M for l ‐Gln. ECAR‐LANS was found to have low relative glutaminase activity (1.2%) at physiological concentrations of l ‐Asn and l ‐Gln in blood. Kinetic studies of ECAR‐LANS showed that the recombinant asparaginase combined the main advantages of Erw. chrysanthemi and E. coli l ‐asparaginases II, currently used in the treatment of acute lymphoblastic leukaemia.