Abstract
L-asparaginase (L-ASNase) is a biotechnologically relevant enzyme for the pharmaceutical, biosensor and food industries. Efforts to discover new promising L-ASNases for different fields of biotechnology have turned this group of enzymes into a growing family with amazing diversity. Here, we report that thermophile Melioribacter roseus from Ignavibacteriae of the Bacteroidetes/Chlorobi group possesses two L-ASNases—bacterial type II (MrAII) and plant-type (MrAIII). The current study is focused on a novel L-ASNase MrAII that was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 70 °C and pH 9.3, with a high L-asparaginase activity of 1530 U/mg and L-glutaminase activity ~19% of the activity compared with L-asparagine. The kinetic parameters KM and Vmax for the enzyme were 1.4 mM and 5573 µM/min, respectively. The change in MrAII activity was not significant in the presence of 10 mM Ni2+, Mg2+ or EDTA, but increased with the addition of Cu2+ and Ca2+ by 56% and 77%, respectively, and was completely inhibited by Zn2+, Fe3+ or urea solutions 2–8 M. MrAII displays differential cytotoxic activity: cancer cell lines K562, Jurkat, LnCap, and SCOV-3 were more sensitive to MrAII treatment, compared with normal cells. MrAII represents the first described enzyme of a large group of uncharacterized counterparts from the Chlorobi-Ignavibacteriae-Bacteroidetes clade.
Highlights
L-asparaginase (EC 3.5.1.1; L-asparagine amidohydrolase) (L-ASNase) is a biotechnologically relevant enzyme used in the pharmaceutical, biosensor and food industries [1,2,3].It catalyzes the conversion of the amino acid L-asparagine (L-Asn) to L-aspartic acid and ammonia [4]
Homology search demonstrated that putative isoaspartyl peptidase/L-asparaginase
WP_014855981.1 shared the highest identity with the previously characterized isoaspartyl peptidase/L-asparaginase from E. coli—WP_146849425.1—with 55.2% classified as planttype L-ASNase EcAIII
Summary
L-asparaginase (EC 3.5.1.1; L-asparagine amidohydrolase) (L-ASNase) is a biotechnologically relevant enzyme used in the pharmaceutical, biosensor and food industries [1,2,3]. It catalyzes the conversion of the amino acid L-asparagine (L-Asn) to L-aspartic acid and ammonia [4]. L-ASNase selectively targets the metabolism of susceptible cancer cells by exploiting deficiencies in metabolic pathways. A diminished level of L-Asn and the inability of susceptible tumor cells to synthesize their own L-Asn leads to the inhibition of protein synthesis, cell cycle arrest in the G1 phase, and apoptosis in leukemic cells [5,6]
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