The subtilisin-like proteinase (AprBp) from Bacillus pumilus 3-19 is a promising candidate for industrial use as a food additive. However, in order to obtain a high yield of the enzyme, it is necessary to develop a very efficient expression system.
The aim of the study was to obtain stable expression of the protease gene of B. pumilus 3-19 in the Pichia pastoris expression system and to assess the correlation of enzyme activity with the choice of type. vector and signal peptides. In this study, for the stable expression of the optimized protease gene in methylotrophic yeast cells, the vectors pPINK-HC and pPINK-LC were selected. For the extracellular secretion of recombinant protease, a set of efficient signal peptides was used. The presequence of the conjugation factor repeatedly led to an increase in the secretion of recombinant proteins. On the other hand, the sequences of the killer signal peptides and lysozymes have not been used before for the expression of the bacterial protease in P. pastoris.
Colonies of transformants with constructs based on the vector pPINK-LC were detected on the 3rd day of incubation and showed a higher transformation efficiency than for colonies with constructs based on the vector pPINK-HC, which did not were detected only on the 7th day of incubation. The transformation efficiency on the 10th day of incubation for structures based on the pPINK-LC vector was on average 1.3 x 103 transformants / μg DNA, while 1.03 x 103 transformants / μg DNA was recorded for constructions with the vector pPINK-HC. Colony PCR showed that all selected transformed colonies contained the subtilisin-like protease gene integrated into the yeast genome.
The study showed that the incubation time affects the synthesis of the protease in P. pastoris, and the maximum activity of the enzyme was observed. Yeast strains with constructions based on the low copy number vector pPINK-LC showed higher protease activity (U / ml) in the hydrolysis of azocasein (2.63 ± 0.16 for the peptide signal killer (SP), 2.49 ± 0.08 for the presequence of the mating factor, 2.19 ± 0.11 for lysozyme SP) than strains with constructs based on the vector pPINK-HC (1 , 86 ± 0.09 for the killer SP, 2.21 ± 0.07 for the presequence of the -mating factor, 1.31 ± 0.11 for the lysozyme SP), whatever signal peptide was used. The maximum protease activity was obtained for the yeast strains with the constructs pPINK-LC-killer-aprBp (5.75 ± 0.08 U / ml) and pPINK-LC-α-mat.factor-aprBp (4, 33 ± 0.07 U / ml).
This study demonstrated that the subtilisin-type protease from recombinant strains of P. pastoris exhibits proteolytic activity, which depends on the incubation time and the choice of signal peptide and vector. The production of bacillary protease by the yeast-based heterologous expression system makes this system promising for the development of new feed additives for breeding.
Proteases are classified among the three largest groups of industrial enzymes and account for about 60% of total world sales of enzymes, of which 40% belong to bacterial proteases. Bacterial serine proteases have high substrate specificity and high resistance to various conditions, which justifies their use in biotechnology, in particular as bio-additives for birds and farm animals. In addition, supplementing food with proteases improves the absorption of amino acids by animals and saves funds for the purchase of synthetic amino acids. The use of proteases as food additives has also been shown to increase the bioavailability of nitrogen, which in turn reduces ammonia pollution levels in the soil. In addition, proteases facilitate the action of other enzymes and increase the profitability of feed, while promoting the growth of beneficial microflora in the intestines of broilers.
Today, a limited number of commercial proteases from foreign manufacturers are used in Russia. The main representatives on the Russian market are the imported food additives CIBENZA DP100 (NOVUS (88.1%), USA), RONOZYME® ProAct (DSM (9.4%), Switzerland), Axtra® PRO TPT (DuPont (2, 4%), Germany). The recombinant obtained comprises strains of P. pastoris with the bacillary protease gene of the subtilisin type (aprBp) and three different signal peptides (α conjugation factor, killer protein and lysozyme). Further expression and purification of the protease from the yeast culture liquid and the study of its biochemical and enzymatic properties will allow evaluation of the biotechnological prospects of the recombinant enzyme.