Staphylococcus simulans Recombinant Lysostaphin: Production, Purification, and Determination of Antistaphylococcal Activity.
Boksha I S,Lavrova N V,Grishin A V,Demidenko A V,Lyashchuk A M,Galushkina Z M,Ovchinnikov R S,Umyarov A M,Avetisian L R,Chernukha M Iu,Shaginian I A,Lunin V G,Karyagina A S
Biochemistry. Biokhimiia
Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by Sigma-Aldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.
10.1134/S0006297916050072
Synergistic Anti-Staphylococcal Activity Of Niosomal Recombinant Lysostaphin-LL-37.
International journal of nanomedicine
PURPOSE: is the most common persistent pathogen in humans, so development of new formulations to combat pathogen invasion is quite necessary. METHODS:In the current study, for the first time, the synergistic activity of recombinant lysostaphin and LL-37 peptide was studied against . Moreover, different niosomal formulations of the peptide and protein were prepared and analyzed in terms of size, shape, zeta potential, and entrapment efficiency. Also, a long-term antibacterial activity of the best niosomal formulation and free forms was measured against in vitro. RESULTS:The optimal niosomal formulation was obtained by mixing the surfactants (span60 and tween60; 2:1 w/w), cholesterol, and dicetylphosphate at a ratio of 47:47:6, respectively. They showed uniform spherical shapes with the size of 565 and 325 nm for lysostaphin and LL-37, respectively. This formulation showed high entrapment efficiency for the peptide, protein, and a slow-release profile over time. Release kinetic was best fitted by Higuchi model indicating a diffusion-based release of the drugs. The lysostaphin/LL-37 niosomal formulation synergistically inhibited growth of for up to 72 hours. However, the same amounts of free forms of both anti-microbial agents could not hold the anti-microbial effect and growth was seen in the following 72 hours. Cytotoxicity assay specified that lysostaphin/LL-37 niosomal combination had no deleterious effect on normal fibroblast cells at effective antimicrobial concentrations. CONCLUSION:This study indicated that the use of lysostaphin in combination with LL-37, either in niosomal or free forms, synergistically inhibited growth of in vitro. In addition, niosomal preparation of antimicrobial agents could provide a long-term protection against bacterial infections.
10.2147/IJN.S230269
Enhanced production of recombinant lysostaphin using medium engineering.
Duman Zeynep Efsun,Ünlü Aişe,Çakar Mehmet Mervan,Ünal Hayriye,Binay Barış
Preparative biochemistry & biotechnology
, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in . A new effective inducible promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 °C. The production cost of 1000 U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.
10.1080/10826068.2019.1599393
Self-cleaved expression of recombinant lysostaphin from its cellulose binding domain fusion.
Applied microbiology and biotechnology
Mature lysostaphin (mLst) is a glycineglycine endopeptidase, capable of specifically cleaving penta-glycine crosslinker in the peptidoglycan of Staphylococcus aureus cell wall. It is a very effective therapeutic enzyme to kill the multidrug-resistant S. aureus often encountered in hospital acquired infections. Fusing cellulose binding domain (CBD) to mLst significantly reduced the insoluble expression of mLst in E. coli. Employing mLst-cleavable peptides as fusion linkers leaded to an effective self-cleavage expression that CBD and mLst could be completely cleaved off from the fusions during the expression process. The presence of residue linker fragment at N-terminus of the cleaved-off mLst strongly inhibited the cell lytic activity of the recovered recombinant mLst, and only ~ 50% of the wild-type mLst activity could be retained. Intact CBD-Lst fusions were obtained when uncleavable peptide linkers were employed. With CBD at N-terminus of mLst, the intact fusion completely lost its cell lytic activity but the dipeptidase activity still remained. In contrast, approximately 10% cell lytic activity of mLst still could be maintained for the fusion with CBD at C-terminus of mLst. KEY POINTS: • CBD fusion enhanced soluble expression of recombinant lysostaphin. • In vivo self-cleavage of fusion linkers by the expressed lysostaphin fusions. • Self-cleaved lysostaphin fusions retain most of dipeptidase but lose 50% cell lytic activity.
10.1007/s00253-022-12047-1
Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation.
Biotechnology and bioengineering
We describe a facile strategy to identify sites for the incorporation of noncanonical amino acids into lysostaphin-an enzyme that degrades the cell wall of Staphylococcus aureus-while retaining stapholytic activity. We used this strategy to generate active variants of lysostaphin incorporating para-azidophenylalanine. The incorporation of this "reactive handle" enabled the orthogonal site-specific modification of the enzyme variants with polyethylene glycol (PEG) using copper-free click cycloaddition. PEGylated lysostaphin variants could retain their stapholytic activity, with the extent of retention depending on the site of modification and the PEG molecular weight. The site-specific modification of lysostaphin could be useful not only for PEGylation to improve biocompatibility but also for the incorporation of the enzyme into hydrogels and other biomaterials and for studies of protein structure and dynamics. Moreover, the approach described herein could be readily applied to identify suitable sites for the incorporation of reactive handles into other proteins of interest.
10.1002/bit.28360
Lysostaphin: Engineering and Potentiation toward Better Applications.
Journal of agricultural and food chemistry
Lysostaphin is a potent bacteriolytic enzyme with endopeptidase activity against the common pathogen . By digesting the pentaglycine crossbridge in the cell wall peptidoglycan of including the methicillin-resistant strains, lysostaphin initiates rapid lysis of planktonic and sessile cells (biofilms) and has great potential for use in agriculture, food industries, and pharmaceutical industries. In the past few decades, there have been tremendous efforts in potentiating lysostaphin for better applications in these fields, including engineering of the enzyme for higher potency and lower immunogenicity with longer-lasting effects, formulation and immobilization of the enzyme for higher stability and better durability, and recombinant expression for low-cost industrial production and biocontrol. These achievements are extensively reviewed in this article focusing on applications in disease control, food preservation, surface decontamination, and pathogen detection. In addition, some basic properties of lysostaphin that have been controversial and only elucidated recently are summarized, including the substrate-binding properties, the number of zinc-binding sites, the substrate range, and the cleavage site in the pentaglycine crossbridge. Resistance to lysostaphin is also highlighted with a focus on various mechanisms. This article is concluded with a discussion on the limitations and future perspectives for the actual applications of lysostaphin.
10.1021/acs.jafc.2c03459