L‐Asparaginase from Lachancea Thermotolerans: Effect of Lys99Ala on Enzyme Performance and in vitro Antileukemic Efficacy

Abstract

L‐asparaginases (EC 3.5.1.1) are amidohydrolase enzymes that predominantly catalyze conversion of L‐asparagine to L‐aspartic acid and ammonia. In addition, some exhibit secondary L‐glutaminase activity. Escherichia coli and Erwinia chrysanthemi L‐asparaginases are widely used in the pharmaceutical industry to produce therapeutically important compounds. In the therapeutic use of enzymes, bacterial L‐asparaginases can trigger immune responses, leading to a high rate of adverse effects that diminish the effectiveness of the treatment. This situation has forced scientists to search for promising L‐asparaginases from new sources. Yeast L‐asparaginases could be useful in reducing toxicity and enhancing efficacy but they have been poorly studied to date. Here, we characterized the yeast Lachancea thermotolerans L‐asparaginase (LtASNase) purified by affinity chromatography. It has a specific activity of 313.8 U/mg and a high kcat value (312.4 s). We demonstrated through a semi‐rational design that the mutations of Lys99 show varying effects on catalytic activity, with the Lys99Ala mutant increasing specific activity 3.3‐fold. Furthermore, the in vitro antileukemic activity of the non‐formulated form of Lys99Ala LtASNase was evaluated against SUP‐B15 and REH cell lines. The results demonstrated that LtASNase exhibits significant antileukemic potential, comparable to commercial type II bacterial enzymes. The understanding of the mutant L‐asparaginases examined in this study will significantly contribute to the development of new and more effective yeast‐derived asparaginases.