Highly Active Thermophilic L-Asparaginase from Melioribacter roseus Represents a Novel Large Group of Type II Bacterial L-Asparaginases from Chlorobi-Ignavibacteriae-Bacteroidetes Clade

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 <i>Melioribacter roseus</i> from <i>Ignavibacteriae</i> 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 <i>Escherichia coli</i>, 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 K_M and V_max 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.