Purification and characterization of the antibacterial peptidase lysostaphin from Staphylococcus simulans : Adverse influence of Zn 2+ on bacteriolytic activity

Ojha, Suvash Chandra; Imtong, Chompounoot; Meetum, Kanungsuk; Sakdee, Somsri; Katzenmeier, Gerd; Angsuthanasombat, Chanan

doi:10.1016/j.pep.2018.06.013

Cited by 21 publications

(7 citation statements)

References 30 publications

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“…Mature lysostaphin requires Zn 2+ that hexacoordinates with H32, D36, H115 and three water molecules, forming the catalytic center. , This supports our observation that the protease activity of lysostaphin was compromised by EDTA and was enhanced by a low concentration of exogenous Zn 2+ . Recent research demonstrates that lysostaphin contains an extra Zn 2+ -binding site, and excessive supply of Zn 2+ leads to its binding at this site, thus inhibiting lysostaphin activity. − In line with such a theory, we observed decreasing lysostaphin activity with increasing Zn 2+ concentrations, which is also consistent with previous findings showing the inhibitory role of Zn 2+ in lysostaphin-mediated degradation of elastin …”

Section: Discussionsupporting

confidence: 91%

“…Mature lysostaphin requires Zn 2+ that hexacoordinates with H32, D36, H115 and three water molecules, forming the catalytic center. 27,38 This supports our observation that the protease activity of lysostaphin was compromised by EDTA and was enhanced by a low concentration of exogenous Zn 2+ . Recent research demonstrates that lysostaphin contains an extra Zn 2+ -binding site, and excessive supply of Zn 2+ leads to its binding at this site, thus inhibiting lysostaphin activity.…”

Section: Discussionsupporting

confidence: 85%

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Peptidoglycan-Targeting Staphylolytic Enzyme Lysostaphin as a Novel and Efficient Protease toward Glycine-Rich Flexible Peptide Linkers

Liu

Gong

et al. 2023

J. Agric. Food Chem.

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Glycine-rich flexible peptide linkers have been widely adopted in fusion protein engineering; however, they can hardly be cleaved for the separation of fusion partners unless specific protease recognition sites are introduced. Herein, we report the use of the peptidoglycan-targeting staphylolytic enzyme lysostaphin to directly digest the glycine-rich flexible linkers of various lengths including oligoglycine linkers and (G4S) x linkers, without the incorporation of extra amino acids. Using His-MBP-linker-LbCpf1 as a model substrate, we show that both types of linkers could be digested by lysostaphin, and the digestion efficiency improved with increasing linker length. The enzyme LbCpf1 retained full activity after tag removal. We further demonstrated that the proteolytic activity of lysostaphin could be well maintained under different environmental conditions and in the presence of a series of chemical reagents at various concentrations that are frequently used in protein purification and stabilization. In addition, such a digestion strategy could also be applied to remove the SUMO domain linked to LwCas13a via an octaglycine linker. This study extends the applications of lysostaphin beyond an antimicrobial reagent and demonstrates its potential as a novel, efficient, and robust protease for protein engineering.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 85%

Peptidoglycan-Targeting Staphylolytic Enzyme Lysostaphin as a Novel and Efficient Protease toward Glycine-Rich Flexible Peptide Linkers

Liu

Gong

et al. 2023

J. Agric. Food Chem.

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“…The clinical application of lysostaphin depends on the availability of large amounts of highly purified protein from a safe and nonpathogenic source. Firstly, lysostaphin was extracted from the culture of its natural host S. simulans (Chandra et al, 2018). Due to the technical difficulty, and the safety dispute, it cannot be used in actual production (Kokai-Kun, 2012).…”

Section: Introductionmentioning

confidence: 99%

Design and High Expression of Non-glycosylated Lysostaphins in Pichia pastoris and Their Pharmacodynamic Study

et al. 2021

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Lysostaphin is an effective antimicrobial agent to Staphylococcus, especially for the methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Staphylococcus aureus (MDRSA). In this study, the seven lysostaphin derived mutants (rLys) were designed to overcome the barrier of glycosylation during expression in Pichia pastoris. Among them, 127A and 127A232Q had highest antimicrobial activity (MIC values 0.07–0.3 μM) to S. aureus than others and the commercial lysostaphins (1–15.8 times). There was no glycosylation during the expression in 5-L fermenter level, with the high yield of 1315 mg/L (127A) and 1141 mg/L (127A232Q), respectively. Meanwhile, 127A and 127A232Q effectively killed 99.9% of S. aureus at low concentration (1 × MIC) within 30 min, without the regrowth of pathogen. They also showed low toxicity, high pH and temperature stability. The results of in vivo therapeutic effect of 127A and 127A232Q against high virulent S. aureus CVCC546 showed that 127A and 127A232Q increased the survival rate of infected mice up to 100% at the dose of 10 mg/kg than the untreated group, reduced the bacterial translocation by 5-7 log CFU (over 99%) in organs compared to the untreated group and alleviated multiple-organ injuries (liver, kidney and spleen). These data indicated that the non-glycosylated lysostaphin 127A and 127A232Q may be a promising therapeutic agent against MDR staphylococcal infections.

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“…The protein production was comparable to other expression systems and achieved yields of 200 mg/L. Our production in a small-scale laboratory expression system exceeded conventional laboratory expression systems (20 mg/L) and reached concentrations commonly achieved at the semi-industrial level (300 mg/L) [ 27 , 28 , 29 ]. Our final goal was to achieve a yield close to the 5 g/L medium.…”

Section: Discussionmentioning

confidence: 60%

Enzybiotics LYSSTAPH-S and LYSDERM-S as Potential Therapeutic Agents for Chronic MRSA Wound Infections

et al. 2020

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Antibacterial antibiotic therapy has played an important role in the treatment of bacterial infections for almost a century. The increasing resistance of pathogenic bacteria to antibiotics leads to an attempt to use previously neglected antibacterial therapies. Here we provide information on the two recombinantly modified antistaphylococcal enzymes derived from lysostaphin (LYSSTAPH-S) and endolysin (LYSDERM-S) derived from kayvirus 812F1 whose target sites reside in the bacterial cell wall. LYSSTAPH-S showed a stable antimicrobial effect over 24-h testing, even in concentrations lower than 1 µg/mL across a wide variety of epidemiologically important sequence types (STs) of methicillin-resistant Staphylococcus aureus (MRSA), especially in the stationary phase of growth (status comparable to chronic infections). LYSDERM-S showed a less potent antimicrobial effect that lasted only a few hours at concentrations of 15 μg/mL and higher. Our data indicate that these antimicrobial enzymes could be of substantial help in the treatment of chronic MRSA wound infections.

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Purification and characterization of the antibacterial peptidase lysostaphin from Staphylococcus simulans : Adverse influence of Zn 2+ on bacteriolytic activity

Cited by 21 publications

References 30 publications

Peptidoglycan-Targeting Staphylolytic Enzyme Lysostaphin as a Novel and Efficient Protease toward Glycine-Rich Flexible Peptide Linkers

Peptidoglycan-Targeting Staphylolytic Enzyme Lysostaphin as a Novel and Efficient Protease toward Glycine-Rich Flexible Peptide Linkers

Design and High Expression of Non-glycosylated Lysostaphins in Pichia pastoris and Their Pharmacodynamic Study

Enzybiotics LYSSTAPH-S and LYSDERM-S as Potential Therapeutic Agents for Chronic MRSA Wound Infections

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