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Heterologous Expression of Bacillus pumilus 3–19 Protease in Pichia pastoris and Its Potential Use as a Feed Additive in Poultry Farming


Proteases are one of the most innovative products used to improve the efficiency of feed additives. Due to their hydrolytic properties, they enhance the absorption of amino acids, which can reduce the protein content and the cost of feeds. The subtilisin-like proteinase of Bacillus pumilus 3–19 is a promising candidate for industrial use as a feed additive. However, in order to obtain a high yield of the enzyme, it is necessary to develop a highly efficient expression system. The aim of the study was to obtain stable expression of the optimized B. pumilus 3–19 protease gene in the Pichia pastoris expression system and evaluate the correlation of enzyme activity with the choice of vector type and signal peptides. The efficient secretion of subtilisin-like protease into the culture fluid of the recombinant yeast strains was confirmed. The study showed that the incubation time affects the synthesis of protease in P. pastoris, and the maximum activity of the enzyme was observed at 72 h of growth of the yeast culture. Yeast strains with constructs based on the low-copy vector pPINK-LC showed higher protease activity (U/mL) in the hydrolysis of azocasein (2.63 ± 0.16 for killer signal peptide (SP), 2.49 ± 0.08 for α-mating factor presequence, 2.19 ± 0.11 for lysozyme SP) than strains with constructs based on the pPINK-HC vector (1.86 ± 0.09 for killer SP, 2.21 ± 0.07 for α-mating factor presequence, 1.31 ± 0.11 for lysozyme SP), regardless of which signal peptide was used. The ability of the recombinant protease to hydrolyze a specific substrate confirms that the enzyme is a member of the subtilisin family. The maximum protease activity was obtained for yeast strains with pPINK-LC-killer-aprBp (5.75 ± 0.08 U/mL) and pPINK-LC-α-mat.factor-aprBp (4.33 ± 0.07 U/mL) constructs. This study demonstrated that the subtilisin-like protease from recombinant P. pastoris strains exhibits proteolytic activity, which depends on the incubation time and the choice of signal peptide and vector. The production of bacillary protease by the heterologous yeast-based expression system makes this system promising for the development of new feed additives for animal husbandry.

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This work was performed in accordance with the Russian Government Program of Competitive Growth of Kazan Federal University.


This work was supported by the Russian Science Foundation (Project No. 16–16-04062).

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DP and YV conducted cloning of the gene encoding a B. pumilus 3–19 protease and performed an analysis of protease expression. DP prepared the manuscript. MS developed the study concept and contributed materials for analyses. All authors were involved in editing the manuscript and read and approved the submitted version.

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Correspondence to D. S. Pudova.

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Pudova, D.S., Vasilyeva, Y.A. & Sharipova, M.R. Heterologous Expression of Bacillus pumilus 3–19 Protease in Pichia pastoris and Its Potential Use as a Feed Additive in Poultry Farming. BioNanoSci. 11, 989–997 (2021).

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  • Protease
  • Bacillus pumilus
  • Feed additive
  • Heterological expression system
  • Pichia pastoris