Structural evidence of the conversion of nitric oxide (NO) to nitrite ion (NO2-) by lactoperoxidase (LPO): Structure of the complex of LPO with NO2- at 1.89 Å resolution.

Lactoperoxidase (LPO) is a heme containing mammalian enzyme which uses hydrogen peroxide (H2O2) to catalyze the conversion of substrates into oxidized products. LPO is found in body fluids and tissues such as milk, saliva, tears, mucosa and other body secretions. The previous structural studies have shown that LPO converts substrates, thiocyanate (SCN-) and iodide (I-) ions into oxidized products, hypothiocyanite (OSCN-) and hypoiodite (IO-) ions respectively. We report here a new structure of the complex of LPO with an oxidized product, nitrite (NO2-). This product was generated from NO using the two step reaction of LPO by adding hydrogen peroxide (H2O2) in the solution of LPO in 0.1 M phosphate buffer at pH 6.8 as the first step. In the second step, NO gas was added to the above mixture. This was crystallized using 20% (w/v) PEG-3350 and 0.2 M ammonium iodide at pH 6.8. The structure determination showed the presence of NO2- ion in the distal heme cavity of the substrate binding site of LPO. The structure also showed that the propionate group which is linked to pyrrole ring D of the heme moiety was disordered. Similarly, the side chain of Asp108, which is covalently linked to heme moiety, was also split into two components. As a result of these changes, the conformation of the side chain of Arg255 was altered allowing it to form new interactions with the disordered carboxylic group of propionate moiety. These structural changes are indicative of an intermediate state in the catalytic reaction pathway of LPO.

Nanoformulation approach for improved antimicrobial activity of bovine lactoperoxidase.

Lactoperoxidase (LPO) is a glycosylated antimicrobial protein present in milk with a molecular mass of 78 kDa. LPO is included in many biological processes and is well-known to have biocidal actions, acting as an active antibiotic and antiviral agent. The wide spectrum biocidal activity of LPO is mediated via a definite inhibitory system named lactoperoxidase system which plays a potent role in the innate immune response. With the current advancement in nanotechnology, nanoformulations can be developed for stabilizing and potentiating the activity of LPO for several applications. In the research described in this Research Communication, fresh LPO purified from bovine mammary gland secretions was used for nanoparticle synthesis using a simple thermal process at different pH and temperatures. The round-shaped nanoparticles (average size 229 nm) were successfully synthesized at pH 7.0 and a temperature of 75°C. These nanoparticles were tested against four different bacterial species namely S. flexineri, P. aeruginosa, S. aureus, and E. coli. The prepared nanoparticles exhibited strong inhibition of the growth against all four bacterial species as stated by their MIC and ZOI values. These results may help in increasing the efficiency of lactoperoxidase system and will assist in identifying novel avenues to enhance the stability and antimicrobial function of LPO in drug discovery and industrial processes.

Structural evidence of the oxidation of iodide ion into hyper-reactive hypoiodite ion by mammalian heme lactoperoxidase.

Lactoperoxidase (1.11.1.7, LPO) is a mammalian heme peroxidase found in the extracellular fluids of mammals including plasma, saliva, airway epithelial lining fluids, nasal lining fluid, milk, tears, gastric juices, and intestinal mucosa. To perform its innate immune action against invading microbes, LPO utilizes hydrogen peroxide (H2 O2 ) to convert thiocyanate (SCN- ) and iodide (I- ) ions into the oxidizing compounds hypothiocyanite (OSCN- ) and hypoiodite (IO- ). Previously determined structures of the complexes of LPO with SCN- , OSCN- , and I- show that SCN- and I- occupy appropriate positions in the distal heme cavity as substrates while OSCN- binds in the distal heme cavity as a product inhibitor. We report here the structure of the complex of LPO with IO- as the first structural evidence of the conversion of iodide into hypoiodite by LPO. To obtain this complex, a solution of LPO was first incubated with H2 O2 , then mixed with ammonium iodide solution and the complex crystallized by the addition of PEG-3350, 20% (wt/vol). These crystals were used for X-ray intensity data collection and structure analysis. The structure determination revealed the presence of four hypoiodite ions in the substrate binding channel of LPO. In addition to these, six other hypoiodite ions were observed at different exterior sites. We surmise that the presence of hypoiodite ions in the distal heme cavity blocks the substrate binding site and inhibits catalysis. This was confirmed by activity experiments with the colorimetric substrate, ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-sulfonic acid)), in the presence of hypoiodite and iodide ions.

Milk Proteins-Their Biological Activities and Use in Cosmetics and Dermatology.

Milk and colostrum have high biological potential, and due to their natural origin and non-toxicity, they have many uses in cosmetics and dermatology. Research is ongoing on their potential application in other fields of medicine, but there are still few results; most of the published ones are included in this review. These natural products are especially rich in proteins, such as casein, ß-lactoglobulin, α-lactalbumin, lactoferrin, immunoglobulins, lactoperoxidase, lysozyme, and growth factors, and possess various antibacterial, antifungal, antiviral, anticancer, antioxidant, immunomodulatory properties, etc. This review describes the physico-chemical properties of milk and colostrum proteins and the natural functions they perform in the body and compares their composition between animal species (cows, goats, and sheep). The milk- and colostrum-based products can be used in dietary supplementation and for performing immunomodulatory functions; they can enhance the effects of certain drugs and can have a lethal effect on pathogenic microorganisms. Milk products are widely used in the treatment of dermatological diseases for promoting the healing of chronic wounds, hastening tissue regeneration, and the treatment of acne vulgaris or plaque psoriasis. They are also increasingly regarded as active ingredients that can improve the condition of the skin by reducing the number of acne lesions and blackheads, regulating sebum secretion, ameliorating inflammatory changes as well as bestowing a range of moisturizing, protective, toning, smoothing, anti-irritation, whitening, soothing, and antiaging effects.

Structure of a ternary complex of lactoperoxidase with iodide and hydrogen peroxide at 1.77 Å resolution.

Lactoperoxidase (LPO) is a mammalian heme peroxidase which catalyzes the conversion of thiocyanate (SCN¯) and iodide (I-) by hydrogen peroxide (H2O2) into antimicrobial hypothiocyanite (OSCN¯) and hypoiodite (IO-). The prosthetic heme group is covalently attached to LPO through two ester linkages involving conserved glutamate and aspartate residues. On the proximal side, His351 is coordinated to heme iron while His 109 is located in the substrate binding site on the distal heme side. We report here the first structure of the ternary complex of LPO with iodide (I-) and H2O2 at 1.77 Å resolution. LPO was crystallized with ammonium iodide and the crystals were soaked in the reservoir solution containing H2O2. Structure determination showed the presence of an iodide ion and a H2O2 molecule in the substrate binding site. The iodide ion occupied the position which is stabilized by the interactions with heme moiety, His109, Arg255 and Glu258 while H2O2 was held between the heme iron and His109. The presence of I- in the distal heme cavity seems to screen the positive charge of Arg255 thus suppressing the proton transfer from H2O2 to His109. This prevents compound I formation and allows trapping of a stable enzyme-substrate (LPO-I--H2O2) ternary complex. This stable geometrical arrangement of H2O2 in the distal heme cavity of LPO is similar to that of H2O2 in the structure of the transient intermediate of the palm tree heme peroxidase. The biochemical studies showed that the catalytic activity of LPO decreased when the samples of LPO were preincubated with ammonium iodide.

Potassium-induced partial inhibition of lactoperoxidase: structure of the complex of lactoperoxidase with potassium ion at 2.20 Å resolution.

Lactoperoxidase, a heme-containing glycoprotein, catalyzes the oxidation of thiocyanate by hydrogen peroxide into hypothiocyanite which acts as an antibacterial agent. The prosthetic heme moiety is attached to the protein through two ester linkages via Glu258 and Asp108. In lactoperoxidase, the substrate-binding site is formed on the distal heme side. To study the effect of physiologically important potassium ion on the structure and function of lactoperoxidase, the fresh protein samples were isolated from yak (Bos grunniens) colostrum and purified to homogeneity. The biochemical studies with potassium fluoride showed a significant reduction in the catalytic activity. Lactoperoxidase was crystallized using 200 mM ammonium nitrate and 20% PEG-3350 at pH 6.0. The crystals of LPO were soaked in the solution of potassium fluoride and used for the X-ray intensity data collection. Structure determination at 2.20 Å resolution revealed the presence of a potassium ion in the distal heme cavity. Structure determination further revealed that the propionic chain attached to pyrrole ring C of the heme moiety, was disordered into two components each having an occupancy of 0.5. One component occupied a position similar to the normally observed position of propionic chain while the second component was found in the distal heme cavity. The potassium ion in the distal heme cavity formed five coordinate bonds with two oxygen atoms of propionic moiety, Nε2 atom of His109 and two oxygen atoms of water molecules. The presence of potassium ion in the distal heme cavity hampered the catalytic activity of lactoperoxidase.

Structure of Yak Lactoperoxidase at 1.55 Å Resolution.

Lactoperoxidase (LPO) is a heme containing oxido-reductase enzyme. It is secreted from mammary, salivary, lachrymal and mucosal glands. It catalyses the conversion of thiocyanate into hypothiocyanate and halides into hypohalides. LPO belongs to the superfamily of mammalian heme peroxidases which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). The heme prosthetic group is covalently linked in LPO through two ester bonds involving conserved residues Glu258 and Asp108. It was isolated from colostrum of yak (Bos grunniens), purified to homogeneity and crystallized using ammonium iodide as a precipitating agent. The crystals belonged to monoclinic space group P21 with cell dimensions of a = 53.91 Å, b = 78.98 Å, c = 67.82 Å and ß = 92.96°. The structure was determined at 1.55 Å resolution. This is the first structure of LPO from yak. Also, this is the highest resolution structure of LPO determined so far from any source. The structure determination revealed that three segments (Ser1-Cys15), (Thr117-Asn138) and (Cys167-Leu175) were disordered and formed one surface of LPO structure. In the substrate binding site, the iodide ions were observed in three subsites which are formed by (1) heme moiety and residues, Gln105, Asp108, His109, Phe113, Arg255, Glu258, Phe380 and Phe381, (2) residues, Asn230, Lys232, Pro236, Cys248, Phe254, Phe381 and Pro424 and (3) residues, Ser198, Leu199 and Arg202. The structure determination also revealed that the side chain of Phe254 was disordered. It was observed to adopt two conformations in the structures of LPO.

Nanoformulation approach for improved stability and efficiency of lactoperoxidase.

Lactoperoxidase is a glycosylated protein with a molecular mass of 78 kDa, which being excreted in several mammalian secretions. Lactoperoxidase is included in many biological processes and well-known to have biocidal actions, attending as active antibiotics and antiviral agents. This wide-spectrum of biocidal activities mediates via a definite inhibitory system named lactoperoxidase system which acts a potent role in the innate immune response since its activity is not restricted by the antimicrobial effect, but might act a significant role in the hydrolysis of many toxins like aflatoxin. Hence with the current progresses in technology, nanoparticles can offer chances as an active candidate that might be utilized for stabilizing and potentiating the activity of LPO for use in several applications. Due to the variability functions of LPO, this enzyme considers an active target to be encapsulated or coated to NPs for developing novel nanocombinations with controlled surface characteristics. The development of approaches which might enhance conformational stabilization for several weeks of LPO via nanoformulation could improve the biopharmaceutical applicability of this bioactive ingredient. Nanoformulation of LPO enhances novel functions that can be useful in many biotechnological applications like food industry, cosmetic and pharmaceutical applications or to deliver and encapsulate bioactive components.

Immobilization of lactoperoxidase on Fe3O4 magnetic nanoparticles with improved stability.

OBJECTIVE: The study aimed to develop a facile and effectual method to increase the stability of lactoperoxidase (LPO) by using its immobilization on Fe3O4 magnetic nanoparticles (Fe3O4 MNPs). RESULTS: The successful immobilization of LPO on Fe3O4 MNPs was confirmed by using Fourier transform infrared spectroscopy (FT-IR) and field emission scanning electron microscopy (FE-SEM). The Km values of free LPO and LPO immobilized on Fe3O4 were 53.19, 72.46 mM and their Vmax values were 0.629, 0.576 µmol/mL min respectively. The overall results indicated that the stability of the immobilized LPO was significantly improved compared to free LPO. The LPO immobilized on Fe3O4 (LPO- Fe3O4) retained 28% of the initial activity within 30 days at 25 °C whereas the free enzyme lost its activity after 7 days at the same temperature. Moreover, evaluation of the thermal stability of LPO at 75 °C determined the conservation of 19% of the initial activity of LPO in the LPO- Fe3O4 sample after 60 min whereas the free enzyme lost its activity after 5 min. CONCLUSIONS: According to the present results, Fe3O4 magnetic nanoparticles are suitable for the immobilization of LPO.

Preparation and characterization of novel nanocombination of bovine lactoperoxidase with Dye Decolorizing and anti-bacterial activity.

Interaction between nanoparticles (NPs) and protein is particularly important due to the formation of dynamic nanoparticle-protein complex. The current study indicated that silica NPs were able to induce conformational modification in the adsorbed lactoperoxidase (LPO) which in turns degrades the synthetic dyes. The maximum degradation efficiency was recorded for the LPO modified silica NPs in the presence of H2O2 comparing with either free LPO or silica NPs. Degradation efficiency of crystal violet and commassie blue R250 after 6 h was assessed to be 100(%). Also, degradation efficiency of Congo red reached 90.6% and 79.3% in the presence and absence of H2O2, respectively, however methyl red degradation efficiency recorded 85%. The viability assay experiment indicated that the IC50 value of the LPO modified silica NPs on human fibroblast cells reached 2.8 mg/ml after 48 h incubation. In addition to dye removal, the LPO modified silica NPs were able to inhibit the antibiotic resistant bacterial strains (Salmonell typhii, Staphylococcus areus, Pseudomonas aureginosa, E. coli, Proteus sp. and streptococcus sp.) at concentrations up to 2.5 mg/ml with inhibition activity about 95%. These findings emphasized that the ability of LPO for degradation of the synthetic dyes after adsorption on silica NPs besides it could be a promising agent with potent inhibitory effect targeting a wide range of multidrug resistant bacteria.

The Significance of Lactoperoxidase System in Oral Health: Application and Efficacy in Oral Hygiene Products.

Lactoperoxidase (LPO) present in saliva are an important element of the nonspecific immune response involved in maintaining oral health. The main role of this enzyme is to oxidize salivary thiocyanate ions (SCN-) in the presence of hydrogen peroxide (H2O2) to products that exhibit antimicrobial activity. LPO derived from bovine milk has found an application in food, cosmetics, and medical industries due to its structural and functional similarity to the human enzyme. Oral hygiene products enriched with the LPO system constitute an alternative to the classic fluoride caries prophylaxis. This review describes the physiological role of human salivary lactoperoxidase and compares the results of clinical trials and in vitro studies of LPO alone and complex dentifrices enriched with bovine LPO. The role of reactivators and inhibitors of LPO is discussed together with the possibility of using nanoparticles to increase the stabilization and activity of this enzyme.

Catalyzed Oxidation of Carotenoids by Lactoperoxidase in the Presence of Ethanol.

The discovery of the lactoperoxidase system as a biocatalyst in milk was a landmark finding. The activation of this system using hydrogen peroxide (H2O2) raised hopes for oxidation of various organic substrates. The involvement of lactoperoxidase system in the catalyzed-oxidation of carotenoids in the whey proteins, and the effect of various solvents on carotenoids' oxidation reaction rate has been studied. However, there is no evidence for this reaction without the addition of oxidizing agents, such as peroxides. Here, we reveal that carotenoids are oxidized through the addition of just ethanol in the presence of lactoperoxidase. The oxidation of carotenoids through this exquisite strategy is ∼360 times faster than harnessing the lactoperoxidase system in whey proteins via the addition of hydrogen peroxide. Bearing in mind that ethanol is not an oxidizing agent, this observation suggests a potential paradigm shift in our understanding of lactoperoxidase and catalyzed oxidation in biochemical systems.

Structural insights into the camel milk lactoperoxidase: Homology modeling and molecular dynamics simulation studies.

Lactoperoxodase (LPO) is a heme peroxidase enzyme present in mammalian milk. It is an antimicrobial protein with wide range of industrial applications. Although the three dimensional structure of LPO from various mammalian species has been reported, but its structure from camel source is still unknown. So far, the crystallization attempts have not been successful in determining camel LPO (cLPO) structure. Herein, we developed the three dimensional structure of cLPO by homology modeling approach using prime module available in Schrodinger suite. The developed model in complex with ligand hypothiocyanate (OSCN-) was further validated by Ramachandran plot followed by molecular dynamics (MD) simulation studies using Desmond module of Schrodinger. cLPO model exhibited overall structural similarity with template crystal structure, however, it displayed different interaction pattern of amino acid residues with ligand OSCN- in comparison to template crystal structure. Moreover, the ligand binding site environment in cLPO is more polar, less hydrophobic, and harbours more number of charged residues than template crystal structure. The substrate binding pocket environment of cLPO shows a considerable difference from template crystal structure. This subsequently resulted in dissimilar behaviour of ligand during the course of MD simulation studies.

Design, Synthesis, Molecular Modeling, and Biological Evaluation of Novel Thiouracil Derivatives as Potential Antithyroid Agents.

Hyperthyroidism is the result of uncontrolled overproduction of the thyroid hormones. One of the mostly used antithyroid agents is 6-n-propyl-2-thiouracil (PTU). The previously solved X-ray crystal structure of the PTU bound to mammalian lactoperoxidase (LPO) reveals that the LPO-PTU binding site is basically a hydrophobic channel. There are two hydrophobic side chains directed towards the oxygen atom in the C-4 position of the thiouracil ring. In the current study, the structural activity relationship (SAR) was performed on the thiouracil nucleus of PTU to target these hydrophobic side chains and gain more favorable interactions and, in return, more antithyroid activity. Most of the designed compounds show superiority over PTU in reducing the mean serum T4 levels of hyperthyroid rats by 3% to 60%. In addition, the effect of these compounds on the levels of serum T3 was found to be comparable to the effect of PTU treatment. The designed compounds in this study showed a promising activity profile in reducing levels of thyroid hormones and follow up experiments will be needed to confirm the use of the designed compounds as new potential antithyroid agents.

Peroxidase Activity of Human Hemoproteins: Keeping the Fire under Control.

The heme in the active center of peroxidases reacts with hydrogen peroxide to form highly reactive intermediates, which then oxidize simple substances called peroxidase substrates. Human peroxidases can be divided into two groups: (1) True peroxidases are enzymes whose main function is to generate free radicals in the peroxidase cycle and (pseudo)hypohalous acids in the halogenation cycle. The major true peroxidases are myeloperoxidase, eosinophil peroxidase and lactoperoxidase. (2) Pseudo-peroxidases perform various important functions in the body, but under the influence of external conditions they can display peroxidase-like activity. As oxidative intermediates, these peroxidases produce not only active heme compounds, but also protein-based tyrosyl radicals. Hemoglobin, myoglobin, cytochrome c/cardiolipin complexes and cytoglobin are considered as pseudo-peroxidases. Рeroxidases play an important role in innate immunity and in a number of physiologically important processes like apoptosis and cell signaling. Unfavorable excessive peroxidase activity is implicated in oxidative damage of cells and tissues, thereby initiating the variety of human diseases. Hence, regulation of peroxidase activity is of considerable importance. Since peroxidases differ in structure, properties and location, the mechanisms controlling peroxidase activity and the biological effects of peroxidase products are specific for each hemoprotein. This review summarizes the knowledge about the properties, activities, regulations and biological effects of true and pseudo-peroxidases in order to better understand the mechanisms underlying beneficial and adverse effects of this class of enzymes.

Comparative Site-Specific N-Glycosylation Analysis of Lactoperoxidase from Buffalo and Goat Milk Using RP-UHPLC-MS/MS Reveals a Distinct Glycan Pattern.

The N-glycan pattern of lactoperoxidase (LPO) from buffalo and goat milk was analyzed with the corresponding site of attachment. The enzyme was purified from whey on cation exchange chromatography, proteolyzed using chymotrypsin, and the resulting (glyco)peptides were directly analyzed on reverse phase ultrahigh performance liquid chromatography coupled to ESI-Q-TOF MS in tandem mode. N-Glycans such as high mannose, complex, and hybrid types were identified in buffalo and goat LPO. Among sialylated complex and hybrid types, the terminal Neu5Ac linked to either LacNAc/LacdiNAc found exclusively in buffalo, whereas Neu5Gc linked to LacdiNAc was predominant in goat LPO. N-Glycans at Asn6 and Asn349 in buffalo LPO were completely core fucosylated, while these sites in goat LPO showed differential fucosylation. Differential occupancy was observed at Asn112 with or without nonfucosylated complex and hybrid types, whereas mainly high mannose glycans were found in Asn222 in both of the LPOs. The presence of glycan isomers in buffalo and goat LPO was also observed. Despite the presence of distinct complex and hybrid glycans, the common glycosylation features in buffalo and goat LPO were identified and are comparable with those of bovine LPO. This finding could be useful in exploring the beneficial role of these glycans as functional ingredients for food products.

Lactoperoxidase immobilization on silver nanoparticles enhances its antimicrobial activity.

Lactoperoxidase (LPO) is an antimicrobial protein present in milk that plays an important role in natural defence mechanisms during neonatal and adult life. The antimicrobial activity of LPO has been commercially adapted for increasing the shelf life of dairy products. Immobilization of LPO on silver nanoparticles (AgNPs) is a promising way to enhance the antimicrobial activity of LPO. In the current study, LPO was immobilized on AgNPs to form LPO/AgNP conjugate. The immobilized LPO/AgNP conjugate was characterized by various biophysical techniques. The enhanced antibacterial activity of the conjugate was tested against E. coli in culture at 2 h intervals for 10 h. The results showed successful synthesis of spherical AgNPs. LPO was immobilized on AgNPs with agglomerate sizes averaging approximately 50 nm. The immobilized conjugate exhibited stronger antibacterial activity against E. coli in comparison to free LPO. This study may help in increasing the efficiency of lactoperoxidase system and will assist in identifying novel avenues to enhance the stability and antimicrobial function of LPO system in dairy and other industries.

Susceptibility of influenza viruses to hypothiocyanite and hypoiodite produced by lactoperoxidase in a cell-free system.

Lactoperoxidase (LPO) is an enzyme found in several exocrine secretions including the airway surface liquid producing antimicrobial substances from mainly halide and pseudohalide substrates. Although the innate immune function of LPO has been documented against several microbes, a detailed characterization of its mechanism of action against influenza viruses is still missing. Our aim was to study the antiviral effect and substrate specificity of LPO to inactivate influenza viruses using a cell-free experimental system. Inactivation of different influenza virus strains was measured in vitro system containing LPO, its substrates, thiocyanate (SCN-) or iodide (I-), and the hydrogen peroxide (H2O2)-producing system, glucose and glucose oxidase (GO). Physiologically relevant concentrations of the components of the LPO/H2O2/(SCN-/I-) antimicrobial system were exposed to twelve different strains of influenza A and B viruses in vitro and viral inactivation was assessed by determining plaque-forming units of non-inactivated viruses using Madin-Darby canine kidney cells (MDCK) cells. Our data show that LPO is capable of inactivating all influenza virus strains tested: H1N1, H1N2 and H3N2 influenza A viruses (IAV) and influenza B viruses (IBV) of both, Yamagata and Victoria lineages. The extent of viral inactivation, however, varied among the strains and was in part dependent on the LPO substrate. Inactivation of H1N1 and H1N2 viruses by LPO showed no substrate preference, whereas H3N2 influenza strains were inactivated significantly more efficiently when iodide, not thiocyanate, was the LPO substrate. Although LPO-mediated inactivation of the influenza B strains tested was strain-dependent, it showed slight preference towards thiocyanate as the substrate. The results presented here show that the LPO/H2O2/(SCN-/I-) cell-free, in vitro experimental system is a functional tool to study the specificity, efficiency and the molecular mechanism of action of influenza inactivation by LPO. These studies tested the hypothesis that influenza strains are all susceptible to the LPO-based antiviral system but exhibit differences in their substrate specificities. We propose that a LPO-based antiviral system is an important contributor to anti-influenza virus defense of the airways.

Stabilization of bovine lactoperoxidase in the presence of ectoine.

Lactoperoxidase (LPO) is a heme peroxidase with various applications in industry and medicine. In this study, the effects of ectoine, as a compatible solute, on the structure, thermal stability, thermodynamic parameters, activity, and stability of LPO have been investigated. The results showed that the catalytic activity of LPO was improved by increasing ectoine concentration. The UV-visible absorption spectroscopy and FTIR spectra studies indicated that ectoine could bind to the LPO spontaneously. Moreover, ectoine increased the enzyme Tm and Gibbs free energy. The fluorescence measurements showed that LPO fluorescence was quenched in the presence of ectoine. The quenching mechanism was probably a static quenching by forming a ground state complex. The thermodynamic parameters indicated that hydrogen bonding and Vander Waals forces played a key role in the LPO-ectoine interaction process. The findings suggest that ectoine could be used as a lactoperoxidase stabilizing agent for industrial or medical purposes.

Immobilization of lactoperoxidase on graphene oxide nanosheets with improved activity and stability.

OBJECTIVES: The purpose of this study was to develop a facile and efficient method to enhance the stability and activity of lactoperoxidase (LPO) by using its immobilization on graphene oxide nanosheets (GO-NS). METHODS: Following the LPO purification from bovine whey, it was immobilized onto functionalized GO-NS using glutaraldehyde as cross-linker. Kinetic properties and stability of free and immobilized LPO were investigated. RESULTS: LPO was purified 59.13 fold with a specific activity of 5.78 U/mg protein. The successful immobilization of LPO on functionalized GO-NS was confirmed by using dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FT-IR). The overall results showed that the stability of the immobilized LPO was considerably improved compared to free LPO. Apparent Km and Vmax of LPO also indicated that the immobilized enzyme had greater affinity to the substrate than the native enzyme. CONCLUSIONS: Graphene oxide nanosheets are effective means for immobilization of LPO.