Approaches to studying the increase in the activity and stability of the recombinant aminoacylase from Escherichia coli LGE 36

Abstract

Background: Optically active amino acids, obtained using the aminoacylase enzyme, play a crucial role in the food, pharmaceutical, and medical industries. Researchers have developed approaches to enhance the activity and stability of the recombinant intracellular aminoacylase from Escherichia coli LGE 36. Various stabilizing reagents—dithiothreitol (DTT), glutathione, mercaptoethanol, ascorbic acid, sodium cyanide and sodium sulfide—were studied, with freezing used as a control for comparison. 

Results showed that DTT at a concentration of 10-3М facilitated to preserve the activity of the studied enzyme up to 105%.  Additionally, the combined use of DTT and Co²⁺ ions tripled the enzyme's activity and improved its stability by 2.5 times. Notably, that the simultaneous action of DTT and Со+2 ions also facilitate enzyme activity regeneration. The most effective way to preserve the activity of the recombinant aminoacylase is to freeze the enzyme. 

Objective: The objective of the work is to develop approaches to studying the increase in the activity and stability of the recombinant intracellular aminoacylase from Escherichia coli LGE 36.

Methods: Recombinant strain-producer of aminoacylase from Escherichia coli LGE 36 was used in this study. E. coli cells were grown in M9 minimal medium with supplements at 370C. Aminoacylase activity was measured at 37°C in a reaction medium with a final volume of 0.2 mL, containing Buffer A (100 mM Na/K-phosphate buffer, pH 7.0), 0.2 mM CoCl₂, 40 mM N-acetyl-D,L-methionine, and the required amount of enzyme. One unit of acylase activity was defined as the amount of the enzyme catalyzing the formation of 1 µmol of L-methionie in 1min at 370C at pH 7.0.

Results: The recombinant intracellular aminoacylase from Escherichia coli LGE 36 was obtained using a previously developed method. The effect of various reagents on the stability and activity of the enzyme structure was studied. Results showed that DTT stabilized the recombinant aminoacylase up to 105% compared to the control. The combined use of Со2+ ions and DTT increases the enzyme activity threefold and increases the stability by 2.5 times. Additionally, the simultaneous action of DTT and Со+2 ions facilitated the regeneration of the enzyme activity. Among the tested methods, freezing proved to be the most effective strategy for preserving the recombinant aminoacylase activity.

Novelty: This study uniquely demonstrates the synergistic effect of dithiothreitol (DTT) and cobalt ions (Co+2) in significantly enhancing both the activity and stability of recombinant intracellular aminoacylase from Escherichia coli LGE 36. This novel approach, including the enzyme's activity regeneration, significantly advances optimizing this industrially important enzyme for producing optically active amino acids.

Conclusion: The obtained data on the properties of the recombinant intracellular aminoacylase make it possible to more fully characterize the biochemical and physiological features of the aminoacylase of Escherichia coli 36, an industrially important enzyme. Optically active amino acids obtained by biocatalysis using the recombinant intracellular aminoacylase of Escherichia coli LGE 36 can serve as a basis for creating new medicinal drugs and biologically active supplements used in the functional food industry.

Keywords: recombinant aminoacylase, Escherichia coli, enzyme, ion metal, reagents, biologically active supplements, functional food industry.