Anastasia O. Koryagina

A comparative study of the expression of B. pumilus extracellular serine proteinases (subtilisin-like proteinase and glutamyl endopeptidase) under the control of different promoters and signal peptides based on the LIKE expression system in various B. subtilis recipient strains was carried out using mass spectral analysis in the monitoring of multiple reactions (MRM) mode. The maximum expression of the subtilisin-like proteinase was established under the control of a simulated signal peptide (SP_Asp) and an inducible promoter of the P_LiaI—LIKE expression system in the protease-deficient strain B. subtilis 20-36; the amount of secreted protein was 1.6 µg/µL of the culture liquid. The optimal expression of glutamyl endopeptidase was established under the control of the signal peptide of the Bacillus megaterium glycoside hydrolase gene (SP_Yngk) and the inducible promoter P_LiaI in the protease-deficient strain B. subtilis 20-36, the maximum amount of protein was 0.06 µg/µL. It was concluded that selection of all components of the expression system for individual secreted proteins is of importance, including the selection of the optimal signal peptide.

We evaluated the effect of adding subtilisin-like Bacillus pumilus proteinase to the diet of Hubboard broiler chickens on growth, digestibility of feed nutrients, blood characteristics, meat quality and histological parameters. One hundred and eighty broiler chickens were divided into 6 groups and received 2 feed rations from 0 to 35 days. The diet was either a basic diet without additives (BD) or a basic diet supplemented with B. pumilus proteinase at a concentration of 10 units/kg feed. We found that the addition of proteinase did not adversely (P < 0.05) affect the blood counts of broilers throughout the experiment. In the balanced experiment, we showed that with the addition of the microbial enzyme, the digestibility of the organic matter of the broilers diet increases considerably (P < 0.05). Veterinary sanitary examination and histological studies led to the conclusion that the broiler meat of the experimental group complies with Russian state standards (GOST) for fresh meat of good quality. It was established that the addition of proteinase to the main diet for broilers enhances the digestibility of the organic and mineral components of the broiler feed ration and reduces the amount of feed consumed.

Bacillus subtilis produces eight industrially important exo-proteases. For the detection of proteases, the activity- and antibody-based assays are normally used. Current activity-based assays require expensive multiplex chemical substrates which allow specificity determination of each enzyme. In this study, we provide evidences pertaining to the usefulness of the label-free multiple reaction monitoring (MRM) assay for a rapid identification and quantitation of specific proteins in bacteria. We used wild-type B. pumilus cells producing at least two serine proteases, subtilisin-like protease (AprBp) and glutamyl endopeptidase (GseBp), as well as optimized recombinant B. subtilis cells containing the same protease genes under control of the LIKE expression system. The Skyline software was used for the selection of three specific proteotypic peptides and their fragment ions for quantification and confirmation of AprBp and GseBp in complex mixtures. MRM indicated that the production of AprBp and GseBp exo-enzymes were respectively 0.9- and 26.6-fold higher in the culture medium of B. pumilus strain in comparison to the recombinant B. subtilis strains carrying optimized LIKE expression systems under identical conditions. The developed procedure in this study is fast, easy to perform and dependable. Additionally, it achieves accurate proteins identification and quantification in complex mixture.