The Induction of L-lysine-α-Oxidase from Trichoderma Harzianum Rifai by  Metabolic Products of Brevibacterium sp. and the Improvement of Its Isolation and  Purification Techniques

Irina  Smirnova; Ekaterina  Neborak; Valeriy Shkinev; Victor  Larichev; Yuri  Shneyder; Ida  Bashkirova; Elena  Karimova; Lyudmila  Gavrilyuk; Daria  Baranova; Maria  Ploskonos

doi:10.18502/ajmb.v17i1.17676

Irina Smirnova T.T. Berezov Department of Biochemistry, Institute of Medicine, Peoples’ Friendship University of Russia (RUDN University), 117198 Moscow, Russian Federation
Ekaterina Neborak T.T. Berezov Department of Biochemistry, Institute of Medicine, Peoples’ Friendship University of Russia (RUDN University), 117198 Moscow, Russian Federation
Valeriy Shkinev Vernadsky Institute of Geochemistry and Analytical Chemistry, 119991 Moscow, Russian Federation
Victor Larichev The Gamaleya National Research Center of Epidemiology and Microbiology, Moscow 123098, Russian Federation
Yuri Shneyder Federal State Budgetary Institution All-Russian Plant Quarantine Center, 140150 Moscow, Russian Federation
Ida Bashkirova Federal State Budgetary Institution All-Russian Plant Quarantine Center, 140150 Moscow, Russian Federation
Elena Karimova Federal State Budgetary Institution All-Russian Plant Quarantine Center, 140150 Moscow, Russian Federation
Lyudmila Gavrilyuk T.T. Berezov Department of Biochemistry, Institute of Medicine, Peoples’ Friendship University of Russia (RUDN University), 117198 Moscow, Russian Federation
Daria Baranova Free University of Bozen-Bolzano, 39100, Bolzano, Italy
Maria Ploskonos Department of Fundamental Chemistry, Astrakhan State Medical University, Astrakhan 414000, Russian Federation

DOI: https://doi.org/10.18502/ajmb.v17i1.17676

Keywords: Anti-bacterial agents, Antifungal, Antineoplastic, Brevibacterium, L-lysine oxidase, Trichoderma, Trichoderma harzianum

Abstract

Background: The enzyme L-lysine-α-oxidase from Trichoderma harzianum Rifai is a promising anticancer, antifungal and antibacterial agent. Intensive exploring of its physico-chemical properties and possible ways of application requires sufficient amounts of the protein which in turn depends on good techniques of cultivation of the micro-organism producer, enzyme soft isolation and purification, and storage.

Methods: An improved method has been suggested for isolation and purification of the enzyme. A specific combination of column sorbents was adapted and gradient elution with sodium chloride was applied to elevate the yield of the enzyme. The inductive influence of Metabolic Products (MP) of the Brevibacterium species, along with fungal metabolites of Ulocladium sp. and Trichoderma sp. was tested. The enzyme activity assay was based on the detection of oxidized dimethylbenzidine in a peroxidase reaction coupled with an L-lysine-α-oxidase reaction. Some enzyme properties were additionally explored.

Results: The upgraded technique of isolation and purification resulted in a yield of enzyme of about 79%. All strains of Brevibacterium sp. proved to be potent enhancers of L-lysine-α-oxidase activity and concomitant activities. The induced enzyme appeared to be less specific but more thermostable. Possible application scopes for the enzyme with modified properties are discussed. Phosphate buffer solution (pH=5.6) appeared to be the best one for long-term storage of the enzyme.

Conclusion: A significant inducing effect of MP of Brevibacterium sp. on L-lysine-α-oxidase has been detected, and its isolation and purification techniques have been improved.