Bacteriolytic enzymes are promising antimicrobial agents for developing new-generation drugs. Recently, we have isolated a β-lytic protease (BlpLc) from the culture liquid of Lysobacter capsici VKM B-2533^T. This BlpLc possesses a valuable property, not described for β-lytic proteases (Blps) earlier, of hydrolyzing living cells of Staphylococcus aureus 55 MRSA clinical isolate. This work phylogenetically characterized the BlpLc and investigated its properties. Analysis revealed a variability of pre-/pro-parts of Blp precursors. The mature BlpLc is the closest to the earlier annotated but not isolated Blp from Lysobacter sp. Root690. The biochemical characterization found conditions for the BlpLc general bacteriolytic activity relative to autoclaved S. aureus 209P cells to differ from that of earlier isolated Blp. Unexpected was the effect of serine (phenylmethylsulfonyl fluoride (PMSF)) and cysteine (p-chloromercuribenzoate (p-CMB)) protease inhibitors on BlpLc bacteriolytic and proteolytic activities. The specificity of BlpLc proteolytic action relative to hemoglobin, elastin, gelatin, collagen, azofibrin, myoglobin, ovalbumin, and ovamucoid was found. New types of peptide bonds—Gly-X, Ser-X, Lys-X, Ala-X, Val-X, Glu-X, and Phe-X—hydrolyzed by the enzyme in protein substrates were first revealed using MALDI-TOF. Turbidimetrically, the BlpLc was found to lyze living cells of S. aureus 209P, Micrococcus luteus B1819, and M. roseus B1236, which is important for expanding the enzyme’s applied properties.

A successful homologous expression system based on Lysobacter capsici VKM B-2533^T and the plasmid pBBR1-MCS5 was first developed for a promising bacteriolytic enzyme of this bacterium, β-lytic protease (Blp). In the expression strains, blp gene expression under the regulation of the GroEL(A) and T5 promoters increased by 247- and 667-fold, respectively, as compared with the wild-type strain. After the cultivation of the expression strains L. capsici P_GroEL(A)-blp and L. capsici P_T5-blp, the Blp yield increased by 6.7- and 8.5-fold, respectively, with respect to the wild-type strain. The cultivation of the expression strain L. capsici P_T5-blp was successfully scaled up. Under fermentation conditions the yield of the enzyme increased by 1.6-fold. The developed homologous system was used to express the gene of the bacteriolytic serine protease (Serp) of L. capsici VKM B-2533^T. The expression of the serp gene in L. capsici P_T5-serp increased by 585-fold. The developed homologous system for the gene expression of bacteriolytic Lysobacter enzymes is potentially biotechnologically valuable, and is promising for creating highly efficient expression strains.

This work investigated the antimicrobial potential of Lysobacter gummosus 10.1.1. The culture fluid of the strain was found to contain antimicrobial agents active against Staphylococcus aureus, Micrococcus luteus, and Bacillus cereus. L. gummosus was first shown to be capable of forming outer membrane vesicles, which have a bacteriolytic effect against not only Gram-positive bacteria but also against the Gram-negative pathogen Pseudomonas aeruginosa. Transcriptomic analysis revealed the genes of almost all known bacteriolytic enzymes of Lysobacter, as well as the genes of enzymes with putative bacteriolytic activity. Also identified were genes involved in the biosynthesis of a number of secondary metabolites for which antimicrobial activities are known. This research is indicative of the relevance of isolating and studying L. gummosus antimicrobial agents.

The aim of the study was to search for, isolate and characterize new bacteriolytic enzymes that show promising potential for their use in medicine, agriculture and veterinary. Using a transcriptomic analysis, we annotated in Lysobacter capsici VKM B-2533^T the genes of known bacteriolytic and antifungal enzymes, as well as of antibiotics, whose expression levels increased when cultivated on media conducive to the production of antimicrobial agents. The genes of the secreted putative bacteriolytic proteases were also annotated. Two new bacteriolytic proteases, Serp and Serp3, were isolated and characterized. The maximum bacteriolytic activities of Serp and Serp3 were exhibited at low ionic strength of 10 mM Tris-HCl, and high temperatures of, respectively, 80 °C and 70 °C. The pH optimum for Serp was 8.0; for Serp3, it was slightly acidic, at 6.0. Both enzymes hydrolyzed autoclaved cells of Micrococcus luteus Ac-2230^T, Proteus vulgaris H-19, Pseudomonas aeruginosa and Staphylococcus aureus 209P. Serp also digested cells of Bacillus cereus 217. Both enzymes hydrolyzed casein and azofibrin. The newly discovered enzymes are promising for developing proteolytic antimicrobial drugs on their basis.

The crystal structure of the Lysobacter capsici VKM B−2533^T β-lytic protease (Blp), a medicinally promising antimicrobial enzyme, was first solved. Blp was established to possess a folding characteristic of the M23 protease family. The groove of the Blp active site, as compared with that of the LasA structural homologue from Pseudomonas aeruginosa, was found to have amino acid differences. Biochemical analysis revealed no differences in the optimal reaction conditions for manifesting Blp and LasA bacteriolytic activities. At the same time, Blp had a broader range of action against living and autoclaved target cells. The results suggest that the distinction in the geometry of the active site and the charge of amino acid residues that form the active site groove can be important for the hydrolysis of different peptidoglycan types in target cells.

Secreted bacteriolytic proteases L1 and L5 of the Gram-negative bacterium Lysobacter capsici XL hydrolyze peptide bridges in bacterial peptidoglycans. Such specificity of action determines the prospects of these enzymes for medicine with the view of creating new antimicrobial drugs to combat antibiotic-resistant strains of pathogens. This research concerns the development of successful expression systems for producing active enzymes L1 and L5 in sufficient amounts for comprehensive studies. Based on L. capsici XL strains with deletions in the alpA (enzyme L1) and alpB (enzyme L5) genes and the constructed expression vectors pBBR1-MCS5 P_T5–alpA and pBBR1-MCS5 P_T5–alpB, we obtained expression strains L. capsici P_T5–alpA and L. capsici P_T5–alpB, respectively. The yields of enzymes L1 and L5 in the developed strains increased by 4 and 137 times, respectively, as compared to the wild-type strain. The cultivation of the expression strains was successfully scaled up under non-selective conditions in a 10-L bioreactor. After fermentation, the yields of enzymes L1 and L5 were 35.48 mg/L and 57.11 mg/L, respectively. The developed homologous expression systems of bacteriolytic proteases L1 and L5 have biotechnological value as compared to those obtained by us earlier based on heterologous expression systems, which have lower yields and labor-intensive purification schemes.