Purification of amylase and protease of mutant strain of bacillus subtilis and their characteristics

УДК 577.112;579.64
DOI 10.33861/2071-8020-2025-5-25-29

Original Empirical Research

Valiullin L. R., Mukhammadiev Rish. S., Mukhammadiev Rin. S., Galeeva A. G., Zainullin L. R., Nesterova I. A., Samsonov A. I., Fazulzyanov I. R.

Abstract. Among the diversity of biologically active compounds of bacilli, extracellular hydrolytic enzymes are of particular interest. The main subject of research in recent years has been directed at amylases and proteases, which constitute two of the three largest groups of industrial enzymes. In this work, protease and amylase were isolated and purified from the culture fluid of mutant of Bacillus subtilis (strain 9) by ammonium sulfate fractionation, ion exchange and gel filtration chromatography with a purification degree of 10.06 and 13.65, a yield of 28.2 and 32.4%, and a specific activity of 17.9 and 113.3 U/mg protein, respectively. Physicochemical properties of purified enzymes of mutant bacterial strain were established. The molecular weight of purified native protease and amylase molecules, determined by gel chromatography, was 44.0±2.0 and 156.0±2.5 kDa, respectively. According to the results of electrophoresis and gel chromatography, the protease was a monomer consisting of one subunit, and amylase was a homodimer with two identical subunits. Both hydrolases of mutant bacterial strain were characterized by pH and temperature stability. The protease retained the highest level of hydrolase activity when incubated for 60 min at pH values from 7.0 to 9.0 units, amylase – from 5.9 to 6.1 units. The activity of both hydrolases was almost at the maximum level when the enzymes were incubated for 60 min within the temperature range from 47.0 to 57.0°C. The obtained results open up prospects for further industrial use of purified hydrolytic enzymes in the development of feed additives for farm animals.

Keywords: Bacillus subtilis, mutant strain, amylase, protease, isolation, purification, characterization, molecular weight, physicochemical properties, feed additive, animal husbandry.

Author affiliation:

Valiullin Lenar R., Ph.D. in Biology, Senior Research Fellow, Laboratory of Applied and Experimental Microbiology, All-Russian Scientific Research Institute of Phytopathology; Head of the Department of Biotechnology, Federal Center for Toxicological, Radiation and Biological Safety; Nauchnyy gorodok-2 st., Kazan, 420075; phone: 8-950-9698469; e-mail: valiullin27@mail.ru.

Mukhammadiev Rinat S., Ph.D. in Biology, Senior Research Fellow, Laboratory of Applied and Experimental Microbiology, All-Russian Scientific Research Institute of Phytopathology; Department of Virological and Ultrastructural Research, Federal Center for Toxicological, Radiation and Biological Safety; st. Scientific town-2, Kazan, 420075; phone: 8-987-4214127; e-mail: tanirtashir@mail.ru.

Galeeva Antonina G., Ph.D. in Veterinary Sciences, Leading Research Fellow, Department of Virological and Ultrastructural Research, Federal Center for Toxicological, Radiation and Biological Safety; Nauchnyy gorodok-2 st., Kazan, 420075; phone: 8-987-2338616; e-mail: antonina-95@yandex.ru.

Zaynullin Lenar Ilgizarovich, Ph.D. in Biology, Leading Researcher Fellow, Department of Biochemistry and Molecular Genetic Analysis, Federal Center for Toxicological, Radiation and Biological Safety; Nauchnyy gorodok-2 st., Kazan, 420075; phone: 8-843-2395320; e-mail: lenarilgizayn@mail.ru.

Nesterova Irina Aleksandrovna, Ph.D. candidate, Junior Researcher, Department of Virology and Ultrastructural Research, Federal Center for Toxicological, Radiation and Biological Safety; Nauchnyy gorodok-2 st., Kazan, 420075; phone: 8-962-5501761; e-mail: ircha-@mail.ru.

Samsonov Andrey Ivanovich, Ph.D. in Biology, Leading Researcher Fellow, Department of Virology and Ultrastructural Research, Federal Center for Toxicological, Radiation and Biological Safety; Nauchnyy gorodok-2 st., Kazan, 420075; phone: 8-950-3167638; e-mail: andreykaz82@yandex.ru.

Fazulzyanov Ilshat Rafailevich, Ph.D. in Biology, Researcher, Department of Virology and Ultrastructural Research, Federal Center for Toxicological, Radiation and Biological Safety; Nauchnyy gorodok-2 st., Kazan, 420075; phone: 8-950-316-5677; e-mail: fazulrif@mail.ru.

Responsible for correspondence with the editorial board: Mukhammadiev Rishat S., Ph.D. in Biology, Senior Research Fellow, Laboratory of Applied and Experimental Microbiology, All-Russian Scientific Research Institute of Phytopathology; Department of Virological and Ultrastructural Research, Federal Center for Toxicological, Radiation and Biological Safety; st. Scientific town-2, Kazan, 420075; phone: 8-939-3728789; e-mail: tashir9891@mail.ru

Authors’ Contribution:

Valiullin L.R.: conceptualization, supervision, methodology, resources.

Mukhammadiev Rish.S.: work administration, writing – original draft preparation, investigation.

Mukhammadiev Rin.S.: writing – editing, investigation.

Galeeva A.G.: data curation.

Zainullin L.R.: formal analysis.

Nesterova I.A.: visualization.

Samsonov A.I.: validation

Fazulzyanov I.R.: investigation.

Conflict of Interest Statement: the authors declare no conflict of interest.