Antibacterial activity of lysozyme after association with carboxymethyl konjac glucomannan.

The unique high isoelectric point of lysozyme (LYZ) restricts its application in composite antibacterial coating due to the unfavorable liability to electrostatic interaction with other components. In this work, the antibacterial activity of a dispersible LYZ-carboxymethyl konjac glucomannan (CMKGM) polyelectrolyte complex was evaluated. Kinetic analysis revealed that, compared with free LYZ, the complexed enzyme exhibited decreased affinity (Km) but markedly increased Vmax against Micrococcus lysodeikticus, and QCM and dynamic light scattering analysis confirmed that the complex could bind with the substrate but in a much lower ratio. The complexation with CMKGM did not alter the antibacterial spectrum of LYZ, and the complex exerted antibacterial function by delaying the logarithmic growth phase and impairing the cell integrity of Staphylococcus aureus. Since the LYZ-CMKGM complex is dispersible in water and could be assembled easily, it has great potential as an edible coating in food preservation.

Acute toxicity and anti-enterotoxigenic activity of pigment extracted from Micrococcus roseus.

Microbial pigments are considered as one of the main sources of natural types, and the attention to them is increasing in the food and pharmaceutical industries. This study aimed to investigate the effects of pigments extracted from Micrococcus roseus (PEM) on the gene expression of a and b staphylococcal enterotoxins (sea and seb) and their acute toxicity. Real-time PCR was used to study the anti-enterotoxigenic activity of PEM against Staphylococcus aureus at sub-inhibitory concentrations. In addition, the acute toxicity of PEM was evaluated on albino mice through alkaline phosphatase (ALP), aspartate aminotransferas (AST), and alanine aminotransferase (ALT) of liver and its histopathological changes. Based on the results, the expression of sea and seb was decreased in the presence of PEM at sub-inhibitory concentrations. The 2-∆∆CT was measured 0.02 and 0.01 for the expression of sea and seb of S. aureus grown in the MHB containing 16 mg/ml PEM. The results showed that the expression of seb is more sensitive to PEM compared to the expression of sea. After treatment of mice with PEM for two weeks, the condition of mice was normal, and the results of liver enzymatic activities and histopathological changes showed insignificant difference compared to the control sample.

The molecular modification, expression, and the antibacterial effects studies of human lysozyme.

Human lysozyme (hLYZ) has attracted considerable research attention due to its natural and efficient antibacterial abilities and widespread uses. In this study, hLYZ was modified to enhance its enzyme activity and expressed in a Pichia pastoris expression system. A combination mutant HZM(2R-K)-N88D/V110S demonstrated the highest enzyme activity (6213 ± 164 U/mL) in shake flasks, which was 4.07-fold higher when compared with the original strain. Moreover, the recombinant P. pastoris was inducted in a 3 L bioreactor plus methanol/sorbitol co-feeding. After 120 h induction, the antibacterial activity of hLYZ reached 2.23 ± 0.12 × 105 U/mL, with the specific activity increasing to 1.89 × 105 U/mg, which is currently the highest specific activity obtained through recombinant expression of hLYZ. Also, hLYZ supernatants showed 2-fold inhibitory effects toward Staphylococcus aureus and Micrococcus lysodeikticus when compared with HZM(2R-K). Our research generated a hLYZ mutant with high antibacterial capabilities and provided a method for screening of high-quality enzymes.

Antibacterial activity of four recombinant carbohydrate recognition domain proteins identified from the kuruma shrimp Marsupenaeus japonicus.

The carbohydrate recognition domain (CRD) is the key component of C-type lectins (CTLs) with the capacity to recognize and eliminate invading pathogens. Herein, the recombinant proteins of four CRDs identified from the kuruma shrimp, Marsupenaeus japonicus, were produced and purified by an Escherichia coli expression system and affinity chromatography. Bacterial binding and antibacterial assays showed that the four CRDs displayed various bacterial binding and antibacterial activities against different bacteria. Among the four recombinant CRDs, His-CRD2-3 exhibited the broadest spectrum of bacterial binding and antibacterial activities against gram-negative bacteria (Vibrio parahaemolyticus, V. alginolyticus and V. harveyi) and gram-positive bacteria (Staphylococcus aureus and Micrococcus lysodeikticus). Moreover, the four recombinant CRDs showed different capacities to regulate the expression of several immune effector genes (MjCTL3, MjCTL4, MjCTL, Mjily and Mjsty), among which His-CRD2-3 displayed broader and stronger inductive effects on these immune effector genes. This study indicated that the four CRDs participated in immune defense by binding and killing bacteria and regulating the transcription of other immune effector genes. In addition, our results suggested that His-CRD2-3 might be a promising agent for the prevention and treatment of bacteriosis.

Heterologous Expression of Mersacidin in Escherichia coli Elucidates the Mode of Leader Processing.

The lanthipeptide mersacidin is a ribosomally synthesized and post-translationally modified peptide (RiPP) produced by Bacillus amyloliquefaciens. It has antimicrobial activity against a range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, giving it potential therapeutic relevance. The structure and bioactivity of mersacidin are derived from a unique combination of lanthionine ring structures, which makes mersacidin also interesting from a lantibiotic-engineering point of view. Until now, mersacidin and its derivatives have exclusively been produced in Bacillus strains and purified from the supernatant in their bioactive form. However, to fully exploit its potential in lanthipeptide-engineering, mersacidin would have to be expressed in a standardized expression system and obtained in its inactive prepeptide form. In such a system, the mersacidin biosynthetic enzymes could be employed to create novel peptides, enhanced by the recent advancements in RiPP engineering, while the leader peptide prevents activity against the expression host. This system would however need a means of postpurification in vitro leader processing to activate the obtained precursor peptides. While mersacidin's native leader processing mechanism has not been confirmed, the bifunctional transporter MrsT and extracellular Bacillus proteases have been suggested to be responsible. Here, a modular system is presented for the heterologous expression of mersacidin in Escherichia coli, which was successfully used to produce and purify inactive premersacidin. The purified product was used to determine the cleavage site of MrsT. Additionally, it was concluded from antimicrobial activity tests that in a second processing step mersacidin is activated by specific extracellular proteases from Bacillus amyloliquefaciens.

Ultrasonic microencapsulation of oil-soluble vitamins by hen egg white and green tea for fortification of food.

We report the microencapsulation of oil soluble vitamins (A, D and E) using a one pot ultrasonic process and raw egg white proteins as a shell material. Green tea catechin/iron complex coating method was further developed to impart UV filtering property to the microcapsules in order to protect the encapsulated nutrients from photodegradation. The microcapsules showed antibacterial properties and long shelf-life. The encapsulated vitamins were protected from degradation upon heating, UV irradiation, simulated storage/transit and cooking processes. The in-vitro digestion study showed that functional vitamin D can be potentially released in the gastrointestinal tract improving vitamin D availability by more than 2-fold compared to the free vitamin. The vitamin D microcapsules were highly stable and maintained their microstructures once incorporated into staple food products. The low-cost egg white shell encapsulated vitamins can improve the nutritional value of staple food products to combat maternal and child malnutrition.

Polyoxometalate-Ionic Liquids (ILs) and Polyvinyl Alcohol/Chitosan/ILs Hydrogels for Inhibiting Bacteria Colonising Wall Paintings.

Historical monuments are increasingly being threatened by unexpected microbial colonizers, leading to their subsequent deterioration. Here, two tetraalkylphosphonium polyoxometalate ionic liquids (Q14-IL and Q16-IL) were successfully synthesized, which showed excellent antibacterial activity against four bacteria colonising wall paintings. Notably, Q14-IL exhibited superior antibacterial efficacy compared to longer alkyl Q16-IL. Additionally, polyvinyl alcohol/chitosan (PVA-CS) hydrogels containing two ILs were prepared, and the morphology, thermal stability, swelling ratio and antibacterial activity were systematically evaluated. The results suggest that higher CS content resulted in more uniform micropores and increased the swelling ratio. However, fewer antibacterial ILs were released and diffused over time from the matrix. Hydrogels with 5% CS content exhibited the highest antibacterial activity, which was mainly attributed to the synergetic antibacterial activity of positively charged ammonium (-NH3+) groups of CS and quaternary phosphonium cation of ILs. This study may provide an alternative strategy for fighting against bacterial communities colonising ancient artworks.

Asperorydines N-P, three new cyclopiazonic acid alkaloids from the marine-derived fungus Aspergillus flavus SCSIO F025.

Three new tricyclic cyclopiazonic acid (CPA) related alkaloids asperorydines N-P (1-3), together with six known compounds (4-9) were isolated and characterized from the fungus Aspergillus flavus SCSIO F025 derived from the deep-sea sediments of South China Sea. The structures including absolute configurations of 1-3 were deduced from spectroscopic data, X-ray diffraction analysis, and electronic circular dichroism (ECD). All compounds were evaluated for the antioxidative activities against DPPH, cytotoxic activities against four tumor cell lines (SF-268, HepG-2, MCF-7, and A549), and antimicrobial activities. Compound 9 showed significant radical scavenging activities against DPPH with an IC50 value of 62.23 µM and broad-spectrum cytotoxicities against four tumor cell lines with IC50 values ranging from 24.38 to 48.28 µM. Furthermore, compounds 4-9 exhibited weak antimicrobial activities against E scherichia coli, and compound 9 also showed antibacterial activity against Bacillus thuringiensis, Micrococcus lutea, Staphylococcus aureus, Bacillus subtilis, Methicillin resistant Staphylococcus aureus.

Modular Approaches to Lankacidin Antibiotics.

Lankacidins are a class of polyketide natural products isolated from Streptomyces spp. that show promising antimicrobial activity. Owing to their complex molecular architectures and chemical instability, structural assignment and derivatization of lankacidins are challenging tasks. Herein we describe three fully synthetic approaches to lankacidins that enable access to new structural variability within the class. We use these routes to systematically generate stereochemical derivatives of both cyclic and acyclic lankacidins. Additionally, we access a new series of lankacidins bearing a methyl group at the C4 position, a modification intended to increase chemical stability. In the course of this work, we discovered that the reported structures for two natural products of the lankacidin class were incorrect, and we determine the correct structures of 2,18-seco-lankacidinol B and iso-lankacidinol. We also evaluate the ability of several iso- and seco-lankacidins to inhibit the growth of bacteria and to inhibit translation in vitro. This work grants insight into the rich chemical complexity of this class of antibiotics and provides an avenue for further structural derivatization.

Heterologous Expression Leads to Discovery of Diversified Lobophorin Analogues and a Flexible Glycosyltransferase.

The heterologous expression of the lobophorin biosynthetic gene cluster from marine-derived Streptomyces pactum SCSIO 02999 has led to the discovery of new analogues that carry d-kijanose (or its variants with a 3-amino or hydroxyl group) at C-9. The identification of intermediates resulting from the inactivation of lobG1 (encoding C-17 kijanosyltransferase) and lobG3 (encoding C-9 digitoxosyltransferase) demonstrates the substrate flexibility of LobG3 to also accept d-kijanose and its variants. Notably, two lobophorins with d-kijanose at C-9 show improved bioactivities.

Half of 'Micrococcus spp.' cases identified by conventional methods are revealed as other life-threatening bacteria with different drug susceptibility patterns by 16S ribosomal RNA gene sequencing.

Bacterial infection during chemotherapy is a fatal complication, therefore precise identification of the pathogenic microorganism is required for treatment. We report that 2 of 4 pediatric patients with malignancy who were diagnosed with Micrococcus spp. infection by conventional methods were finally revealed to have Kytococcus schroeteri and Kocuria marina infection by 16S ribosomal RNA gene sequence analysis (16S rRNA analysis). Although K. schroeteri is morphologically similar to Micrococcus spp., its drug susceptibility profile is quite different from that of Micrococcus spp. K. schroeteri is resistant to penicillin and cephalosporin, which are effective for Micrococcus spp. In fact, penicillin-resistant lethal pneumonia caused by K. schroeteri has been reported in compromised hosts. Based on our results, Micrococcus spp. determined by conventional methods could contain other life-threatening bacteria with different drug susceptibility patterns from Micrococcus spp. To develop an effective empirical treatment for immunocompromised hosts, accumulation of pathogen data by 16S rRNA analysis is required.

Effects of biochar-immobilized bacteria on phytoremediation of cadmium-polluted soil.

This work is the first report of the ability of biochar-immobilized cadmium-resistant bacteria (CRB) on promoting the efficiency of cadmium phytoextraction by Chlorophytum laxum R.Br. The survival of CRB immobilized on biochar in cadmium-contaminated soil at a concentration of 75.45 mg kg-1 was studied. The results found that both CRB, namely Arthrobacter sp. TM6 and Micrococcus sp. MU1, can survive and grow in cadmium-contaminated soil. To study phytoextraction in the pot experiments, 2-month-old C. laxum was individually planted in cadmium-contaminated soil and divided into four treatments, including (i) untreated control, (ii) biochar, (iii) biochar-immobilized (BC) Arthrobacter sp., and (iv) BC-Micrococcus sp. The results found that biochar-immobilized CRB did not cause any effect to the root lengths and shoot heights of plants compared to the untreated control. Interestingly, inoculation of biochar-immobilized CRB significantly increased cadmium accumulation in the shoots and roots compared to the untreated control. In addition, the highest cadmium content in a whole plant, best phytoextraction performance, and greatest bioaccumulation factor was found in plant inoculated with BC-Micrococcus sp., followed by BC-Arthrobacter sp. In conclusion, inoculation of biochar-immobilized CRB enhanced cadmium accumulation and translocation of cadmium from the roots to shoots, suggesting further applying biochar-immobilized CRB in cadmium-polluted soil for promoting cadmium phytoextraction efficiency of ornamental plants. Graphical abstract.

GC-MS analysis of volatile organic compounds from Bambara groundnut rhizobacteria and their antibacterial properties.

Bacterial metabolites have been observed to be important in new drug formulation for both plant, animals and human beings. The aim of this study was to identify the different bioactive compounds found in three rhizobacterial isolates (B. amyloliquefaciens, B. thuringiensis and Bacillus sp.) from the rhizosphere of Bambara groundnut and to assay for their antibacterial properties. Gas chromatography mass spectrometry (GC-MS) was used to carry out the analysis using seven extraction solvents. In the GC-MS analysis, 68 compounds were identified based on peak area percentage, retention time and structure. From the bioactive compounds in B. amyloliquefaciens and B. thuringiensis, the peak area percentage shows that dimethylfuvene from ethyl acetate extraction had the highest relative abundance with 89.11% while Formic acid 2-methylpropyl ester from hexane extraction had the lowest with 6.25%. Others are tridecane, acetic acid butyl ester, paraldehyde, s-(+)-1,2 propanediol, tropone, phthalan and p-xylene with relative abundance of 61.72%, 60.41%, 83.79%, 71.53%, 24.06%, 86.72% and 64.33% respectively. These extracts inhibited the growth of the four test organisms, Bacillus cereus, Pseudomonas aeruginosa, Micrococcus cryophilus and Enterococcus feacalis. Butanol extract from B. amyloliquefaciens had 28 mm zone of inhibition against B. cereus compared to 18 mm and 16 mm by Bacillus sp. and B. thuringiensis respectively. Its zone of inhibition was 24 mm zone against M. cryophilus compared to 12 mm and 19 mm by Bacillus sp. and B. thuringiensis respectively. Butanol extract from B. thuringiensis suppressed E. feacalis and P. aeruginosa having 23 mm and 26 mm zones of inhibition respectively. This was higher compared to Bacillus sp. and B. amyloliquefaciens having 18 mm/15 mm and 21 mm/15 mm against E. feacalis and P. aeruginosa respectively. Hexane and ethyl acetate extract from Bacillus sp. suppressed P. aeruginosa with 12 mm and 17 mm inhibition zones respectively compared to no inhibition zones from hexane extract of B. amyloliquefaciens and B. thuringiensis. Zones of inhibition of 2 mm and 6 mm were observed against P. aeruginosa from ethyl acetate extract of B. amyloliquefaciens and B. thuringiensis respectively. These results suggest that the three isolates are quite rich in the production of bioactive compounds that are also very effective antibacterial agents. These volatile organic compounds are promising compounds for more antibacterial bioactivity development.

Ageritin, a Ribotoxin from Poplar Mushroom ( Agrocybe aegerita) with Defensive and Antiproliferative Activities.

Ribotoxins make up a group of extracellular rRNA endoribonucleases produced by ascomycetes that display cytotoxicity toward animal cells, having been proposed as insecticidal agents. Recently, the ribotoxin Ageritin has been isolated from the basidiomycetes Agrocybe aegerita (poplar mushroom), suggesting that ribotoxins are widely distributed among fungi. To gain insights into the protective properties of Ageritin against pathogens and its putative biotechnological applications, we have tested several biological activities of Ageritin, comparing them with those of the well-known ribotoxin α-sarcin, and we found that Ageritin displayed, in addition to the already reported activities, (i) antibacterial activity against Micrococcus lysodeikticus, (ii) activity against the tobacco mosaic virus RNA, (iii) endonuclease activity against a supercoiled plasmid, (iv) nuclease activity against genomic DNA, (v) cytotoxicity to COLO 320, HeLa, and Raji cells by promoting apoptosis, and (vi) antifungal activity against the green mold Penicillium digitatum. Therefore, Ageritin and α-sarcin can induce resistance not only to insects but also to viruses, bacteria, and fungi. The multiple biological activities of Ageritin could be exploited to improve resistance to different pathogens by engineering transgenic plants. Furthermore, the induction of cell death by different mechanisms turns these ribotoxins into useful tools for cancer therapy.

Purification and characterization of lysozyme from Chinese Lueyang black-bone Silky fowl egg white.

Lysozyme, an important antibacterial protein, is an enzyme that cleaves the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine of peptidoglycan in cell walls. The novel lysozyme was purified and characterized from Chinese Lueyang black-bone silky fowl (CBSF) egg white, and its N-terminal amino acid sequence, enzymatic properties, and antibacterial activity were investigated. The CBSF lysozyme was purified using adsorption chromatography, ammonium sulfate precipitation, ion exchange chromatography, and size-exclusion chromatography. The purification fold and yield were 3.28 and 14.69%, respectively. The purified lysozyme was revealed as a single protein band with SDS-PAGE and had a MALDI-TOF/TOF molecular weight of 14305.57 Da and a final specific activity of 3.49 × 105 U/mg protein using Micrococcus lysodeikticus as a substrate. The optimum temperature and pH of the lysozyme were 50 °C and 6.0, respectively. The 20 N-terminal amino acid residues of the purified lysozyme were determined to be KVFGRCELAAAMKRHGLDNY, showing some homology to the N-terminus of the odontophoridae egg white lysozyme. The purified lysozyme exerted a potent antimicrobial activity toward indicator microorganisms, including Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, and Escherichia coli ATCC 25922. However, its inhibition of gram-negative activity was weaker than that of the Gram-positive bacteria.

Absolute Configurations of 14,15-Hydroxylated Prenylxanthones from a Marine-Derived Aspergillus sp. Fungus by Chiroptical Methods.

Determination of the absolute configrations for natural products is one of the most important and challenging tasks, especially when the molecules display high conformational flexibility. In this paper, eight new prenylxanthones, aspergixanthones A-H (1-8), and one known analogue (9), were isolated from the marine-derived fungus Aspergillus sp. ZA-01. The absolute configurations of C-14 and C-15 in 1-8 were difficult to be assigned due to the high conformational flexibility of the chains. To solve this problem, the experimental ECD, ORD, and VCD spectra of 1 were combined for analysis with the corresponding theoretical predictions for its different diastereomers. This study suggested that a concerted application of more than one chiroptical methods could be used as a preferable approach for the stereochemical characterizations of flexible molecules. Compounds 1-9 were evaluated for their cytotoxic and antibacterial activities. Among them, 6 showed cytotoxicity against the A-549 cell line with the IC50 value of 1.1 µM, and 7 exhibited antibacterial activity against Micrococcus lysodeikticus with the MIC value of 0.78 µg/mL.

Onion and Garlic Extracts Potentiate the Efficacy of Conventional Antibiotics against Standard and Clinical Bacterial Isolates.

INTRODUCTION: Onion (Allium cepa L.) and garlic (Allium sativum L.) extracts are traditionally used in many cultures as antimicrobial agents. Nonetheless, there is still a dearth of scientific validation pertaining to the antibacterial and possible antibiotic potentiating activity of these plants. METHODS: Decoction as traditionally used and methanol, ethanol, ethyl acetate, and acetone extracts of onion and garlic were evaluated for their antibacterial activity against 15 bacterial strains (6 ATCC strains and 9 clinical isolates) using the broth microdilution method to establish the minimum inhibitory concentration. The bacteriostatic and bactericidal actions were determined as compared to conventional antibiotics (streptomycin and chloramphenicol). Fractional Inhibitory Concentration (FIC) was determined to establish any synergistic interaction between the extracts and antibiotics using a modified checkerboard assay. RESULTS: The ethyl acetate extract of garlic showed bactericidal effect against 1 ATCC (E. coli) and 2 clinical isolates. Streptomycin produced only indifferent effect (FIC 1< and ≤ 4) when combined with ethyl acetate extract of onion. Chloramphenicol showed synergism with ethyl acetate extract of onion against ATCC S. aureus (FIC 0.27-0.30) and Micrococci species (FIC 0.27-0.32). Streptomycin showed mostly antagonism whereas chloramphenicol showed synergism effects with the ethyl acetate extract of garlic. The observed antibacterial activity might be justified due to the presence of high concentration of phenolic compounds in the extracts. CONCLUSION: This study has provided an opportunity to establish valuable baseline information on the antibiotic potentiating activity of onion and garlic which can be further exploited for the treatment and/or management of infectious diseases.

Antimicrobial Resistance Patterns of the Gram-positive Bacteria Isolated from Children with Bloodstream Infection in an Iranian Referral Hospital: A 6-year Study.

BACKGROUND: Bloodstream infections (BSI) are considered as a serious cause of morbidity and mortality in children. The aim of this study was to report the common Gram-positive bacteria (GPB) responsible for bloodstream infections in children and determine their antimicrobial resistance patterns in Children Medical Center (CMC) Hospital, Tehran, Iran. METHODS: This retrospective study was conducted within a six-year period (March 2011 to September 2016) for pediatric patients with BSI. Standard bacteriological methods were performed for identification of the bacteria. Antimicrobial susceptibility tests were evaluated by using the disk diffusion method according to the CLSI recommendations. RESULTS: Among 68233 blood cultures, 2349 isolates were obtained which 59% of them (N=1393) were GPB and 41% (n=956) were Gram-negative. The most common GPB isolates were Coagulase negative Staphylococcus (CoNS) (N= 609, 44%), followed by Staphylococcus aureus (N=319, 23%), Enterococcus spp. (N=139, 10%), Streptococcus pneumonia (N= 106, 8%), Streptococci viridans (N= 180, 13%) Micrococcus spp. (N=24, 1.7%) and Streptococcus group B (N= 16, 1%). The rate of methicillin resistance in S. aureus and CoNS was 47% (N=116/246) and 91% (N=557/609), respectively. Isolates of S. pneumoniae showed high-level of resistance to trimethoprim/sulfamethoxazole (N=28/33, 85%) and erythromycin (N=59/91, 65%). S. viridans isolates and Micrococcus spp. were highly sensitive to linezolid (100%). All of the tested isolates of Streptococcus group B were sensitive to all the antibiotics used in this study. Among Enterococcus spp., 52% (N=69/133) of the m were resistant to vancomycin. CONCLUSIONS: Our results emphasize the importance of a valuable guide in identifying resistance trends and selecting appropriate antibiotic.

Evaluation of dose dependent antimicrobial activity of self-assembled chitosan, nano silver and chitosan-nano silver composite against several pathogens.

The aim of this investigation to preparation of silver nanoparticles organized chitosan nano polymer, which effective against microbial and pathogens, when apply to liquid medium and edible food products surface, will rescue the growth of microbes. Self-assembly approach used to synthesis of silver nanoparticles and silver nanoparticles organized chitosan nano polymer. Silver nanoparticles and silver nanoparticles organized chitosan nano polymer and film characterized using Ultra-violate visible spectrometer (UV-vis), X-ray diffraction (X-ray), and Scanning electronic microscope (SEM). The crystalline structured protein capped nano silver successfully synthesized at range of 12 nm-29 nm and organized into chitosan nano polymer. Antimicrobial ingredient in liquid medium and food product surface provide to rescue oxidative change and growth of microorganism to provide higher safety. The silver nanoparticles organized chitosan nano polymer caused the death of microorganism. The materials in nano scale synthesized successfully using self-assembly method, which showed good antimicrobial properties.

Insights on the Structure, Molecular Weight and Activity of an Antibacterial Protein-Polymer Hybrid.

Protein-polymer conjugates are attractive biomaterials which combine the functions of both proteins and polymers. The bioactivity of these hybrid materials, however, is often reduced upon conjugation. It is important to determine and monitor the protein structure and active site availability in order to optimize the polymer composition, attachment point, and abundance. The challenges in probing these insights are the large size and high complexity in the conjugates. Herein, we overcome the challenges by combining electron paramagnetic resonance (EPR) spectroscopy and atomic force microscopy (AFM) and characterize the structure of antibacterial hybrids formed by polyethylene glycol (PEG) and an antibacterial protein. We discovered that the primary reasons for activity loss were PEG blocking the substrate access pathway and/or altering protein surface charges. Our data indicated that the polymers tended to stay away from the protein surface and form a coiled conformation. The structural insights are meaningful for and applicable to the rational design of future hybrids.