Enzymatic Activity Of Lysozyme Research Articles

The Role of Surface Modification of Silica-Coated Fe3O4 Nanoparticles in the Structure and Enzyme Activity of Lysozyme

The Role of Surface Modification of Silica-Coated Fe3O4 Nanoparticles in the Structure and Enzyme Activity of Lysozyme

Evaluation of immunostimulatory attributes of Asparagus racemosus and Withania somnifera supplemented diets in fish, Channa punctatus (Bloch, 1793)

With the progression of aquaculture industry, there has been a spurt in dietary supplementation with economically viable medicinal herbs having enough immunostimulatory potential. This also aids in avoidance of environmentally undesirable therapeutics that are almost inevitable to safeguard fish against an array of diseases in aquaculture practices. The study aims to determine the optimal dose of herbs that can stimulate substantial immune response in fish for reclamation of aquaculture. Immunostimulatory potential of the two medicinal herbs- Asparagus racemosus (Shatavari), Withania somnifera (Ashwagandha), individually, and in combination, with a basal diet was screened up to 60 days in Channa punctatus. 300 laboratory acclimatized healthy fish (14 ± 1 g; 11 ± 1 cm) were divided into ten groups- C, S1, S2, S3, A1, A2, A3, AS1, AS2, and AS3, based on the composition of dietary supplementation, in triplicates, with 10 specimens per group. The hematological index, total protein and lysozyme enzyme activity were performed after 30 and 60 days, while qRT-PCR analysis of lysozyme expression was done after 60 days of the feeding trial. The significant (P < 0.05) increments in hematological indices- (TEC, TLC, DLC, Hb, Hct, MCV, MCH and MCHC), total protein content and serum lysozyme activity, after 30 and 60 days; whereas upregulation of lysozyme transcript levels, both in liver and muscle tissues after 60 days of the feeding trial were recorded in groups- AS1, AS2, and AS3. The maximal increment in lysozyme expression was recorded in AS3, both in liver and muscle tissues, with 3.75 ± 0.13 and 3.21 ± 0.18-folds, respectively. However, increments were non-significant (P > 0.05) for MCV in AS2 and AS3 after 30 days; and for MCHC in AS1 for both the durations; whereas in AS2 and AS3, after 60 days of the feeding trial. A positive correlation (P < 0.05) among lysozyme expression, MCH, lymphocytes, neutrophils, total protein content, and serum lysozyme activity in AS3, after 60 days, conclusively, evinces that a 3% dietary supplementation with both A. racemosus and W. somnifera enhances immunity and health profile of the fish, C. punctatus. The study, thus finds ample scope in augmentation of aquaculture production and also paves the way for more researches for biological screenings of potential immunostimulatory medicinal herbs that can be appropriately incorporated in the fish diet.

Chronic nanoplastic exposure induced oxidative and immune stress in medaka gonad

Nanoplastic (NP) pollution is a global issue because of its widespread occurrence and potential toxicity. Many studies have investigated the impacts of the short-term toxicity of NPs on organisms. Until now, only a few studies have assessed the toxicological effects of prolonged exposure to NPs at low concentrations in fish. In this study, the effects of NPs (nano-polystyrene microspheres, diameter: 100 nm) on immune and oxidative stress response, histopathology, and survival in medaka were evaluated. The effects of different concentrations (0, 10, 104, and 106 particles/L) of nanoplastics were studied in medaka Oryzias latipes after 3 months of exposure. Lysozyme enzyme activity, oxidative stress-related biomarkers (i.e., superoxide dismutase, catalase, and glutathione peroxidase), and malondialdehyde levels were decreased under NP exposure. The gonadal histology showed that high NP exposure (106 particles/L) inhibited the process of spermatogenesis and oogenesis processes, implying delayed maturation of the gonad. Furthermore, the IBR and PCA analysis revealed the potential biotoxicity of NPs and the total survival rate of medaka was significantly reduced due to the long-term exposure to NPs. Overall, prolonged exposure to low concentrations of NPs is harmful to the health of medaka gonads. In the long run, this may threaten the fish reproduction and population, suggesting the need for long-term toxicological studies to predict the aquatic animal health in nature.

Optical Study of Lysozyme Molecules in Aqueous Solutions after Exposure to Laser-Induced Breakdown.

The properties of a lysozyme solution under laser-induced breakdown were studied. An optical breakdown under laser action in protein solutions proceeds with high efficiency: the formation of plasma and acoustic oscillations is observed. The concentration of protein molecules has very little effect on the physicochemical characteristics of optical breakdown. After exposure to optical breakdown, changes were observed in the enzymatic activity of lysozyme, absorption and fluorescence spectra, viscosity, and the sizes of molecules and aggregates of lysozyme measured by dynamic light scattering. However, the refractive index of the solution and the Raman spectrum did not change. The appearance of a new fluorescence peak was observed upon excitation at 350 nm and emission at 434 nm at exposure for 30 min. Previously, a peak in this range was associated with the fluorescence of amyloid fibrils. However, neither the ThT assay nor the circular dichroism dispersion confirmed the formation of amyloid fibrils. Probably, under the influence of optical breakdown, a small part of the protein degraded, and a part changed its native state and aggregated, forming functional dimers or "native aggregates".

Binding of bioactive esculin and esculetin with hen egg white lysozyme: Spectroscopic and computational methods to comprehensively elucidate the binding affinities, interacting forces, and conformational alterations at molecular level

The binding of bioactive esculin and esculetin with lysozyme have been studied using multi-spectroscopic and computational methods. Intrinsic fluorescence of lysozyme was quenched via static quenching mechanism by these ligands and binding was observed to be moderate in nature. Structural alteration of the protein was noted upon ligand binding and enzymatic activity of lysozyme was also influenced in vitro. • Coumarin derivatives, esculin and esculetin quenched intrinsic fluorescence of HEWL through a static quenching mechanism. • Trp residue occupied a more hydrophobic environment upon interactions with esculin and esculetin. • The α-helical content of HEWL decreased in presence of ligands which could have affected the enzymatic activity of HEWL. • Δ G ° values were observed to be negative which indicated complex formation occurred spontaneously. Multi-spectroscopic and computational methods were employed to investigate the binding mechanism of esculin and esculetin with hen egg white lysozyme (HEWL). UV–vis and fluorescence spectroscopy proved static quenching mechanism was involved in the fluorescence quenching process. The binding constant ( K b ) determined for HEWL-esculin/esculetin complex was in the order of 10 4 M −1 . A negative Δ H ° [-(14.19 ± 2.56) kJ mol −1 ] and positive Δ S ° [+(40.98 ± 3.10) J K −1 mol −1 ] for HEWL-esculin complex suggested the presence of hydrophobic forces and hydrogen bonding. Whereas positive values of both Δ H° [+(9.96 ± 4.26) kJ mol −1 ] and Δ S ° [+(120.05 ± 14.14) J K -1 mol −1 ] for HEWL-esculetin complex indicated the presence of hydrophobic forces in the binding process. Blue shifts were observed for HEWL-esculin/esculetin complexes on carrying out synchronous and three-dimensional (3-D) fluorescence. A red edge excitation shift (REES) of 3 nm was observed for HEWL, whereas 6 and 5 nm for HEWL-esculin and HEWL-esculetin complexes, respectively, were also measured. Circular dichroism (CD) studies revealed that the % α-helical content of HEWL (25.56 ± 2.24) decreased in the presence of esculin (17.96 ± 1.54) and esculetin (19.90 ± 2.14). Amide I peak of HEWL, determined from Fourier transform infrared (FTIR) studies, shifted from 1657 cm −1 to 1654 cm −1 and 1652 cm −1 upon complexation with esculin/esculetin, respectively. The binding distance ( r ) for HEWL-esculin (4.40 ± 0.14 nm) and HEWL-esculetin (4.66 ± 0.11 nm) complexes were determined from fluorescence resonance energy transfer (FRET) theory. Thermal analysis resulted in a lower T m value for the protein–ligand complexes (71.8 °C) compared to the native protein (73.3 °C). Molecular docking and molecular dynamic (MD) simulations provided insight into the binding affinity and flexibility of HEWL upon complexation, respectively. In the presence of esculin and esculetin, a change in enzyme activity of HEWL was observed.

CMOS enhanced the mucosal immune function of skin and gill of goldfish (Carassius auratus Linnaeus) to improve the resistance to Ichthyophthirius multifiliis infection

Methodsof supporting mucosal immune barrier integrity and prevention of some pathogenic infections in aquatic species, are key areas of active study, often focusing on feed additives. The objectives of this study were to explore the effects of feeding cMOS (concentrated mannan oligosaccharide) on the gill and skin mucosal barriers of goldfish (Carassius auratus Linnaeus) and evaluate health status during Ichthyophthirius multifiliis infection. After feeding the cMOS-containing diet for 60 days, Hematoxylin and eosin (H&E) staining showed greater length of gill lamella and thicker dermal dense layer, while Alcian Blue and Periodic acid–Schiff (AB-PAS) staining showed higher numbers of mucin cells in cMOS fed fish. Chemical analysis showed that fish fed cMOS had greater enzyme activity of lysozyme (LZM) and alkaline phosphatase (AKP) in gill and skin tissues, while qRT-PCR revealed higher expression of Muc-2 and IL-1β, as well as lower expression of IL-10. After Ichthyophthirius multifiliis challenge, goldfish fed the cMOS diet had lower mortality and infection rates, as well as fewer visible white spots on the body surfaces. Histologically, the gill and skin of these fish presented less tissue damage and fewer parasites, and had a greater number of mucus cells. In addition, the expression of Muc-2 and IL-10 were notably higher while the expression of IL-1β was significantly lower in cMOS fed goldfish than control fed fish. In this study, cMOS fed goldfish had stronger immune barrier function of skin and gill mucous, and better survival following Ichthyophthirius multifiliis infection.

The effect of putrescine on the lysozyme activity and structure: Spectroscopic approaches and molecular dynamic simulation

The present research addressed the influence of polyamine (putrescine) on the compound as well as function of lysozyme; accordingly, UV- Visible, fluorescence spectroscopy and simulation method were applied to fulfill this goal. Lysozyme's structural variability was examined at various putrescine ‌concentrations; also, the putrescine binding to lysozyme was addressed using spectrofluorescence, circular dichroism (CD) and UV-Vis measurements. The obtained results indicated that with raising the putrescine concentration, the intrinsic quenching fluorescence of lysozyme was decreased based on the static mechanism. Analysis of thermodynamic parameters also indicated that van der Waals as well as hydrogen bond forces served a fundamental role in determining the resulting stability; this was in agreement with modeling studies. Measurement of UV absorption spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy also demonstrated that lysozyme's second and tertiary structures were altered in a putrescine concentration-dependent manner. Putrescine inhibited lysozyme's enzymatic activity, displaying its affinity with the lysozyme's active site. Further, molecular simulation conducted revealed that putrescine could have spontaneous binding to lysozyme, changing its structure, thus further emphasizing the experimental results.

A comparative study of the interaction of naringenin with lysozyme by multi-spectroscopic methods, activity comparisons, and molecular modeling procedures

The present study applied steady-state fluorescence, UV–Vis spectrophotometry, molecular docking studies, and circular dichroism (CD) to investigate the interaction of naringenin with lysozyme in an aqueous medium. The UV–Vis measurement indicated the changes in lysozyme secondary and tertiary structure change as a function of the concentration of naringenin. Naringenin could be used to turn the static quenching mechanism into the intrinsic fluorescence of lysozyme. The negative amount of Gibbs free energy (ΔG°) suggested that the binding operation was spontaneous. Fluorescence studies also demonstrated the changes occurring in the Trp microenvironment upon the concatenation into lysozyme. Analysis of thermodynamic parameters also revealed that hydrophobic forces played a fundamental role in determining the complex stability; this was consistent with the previous modeling studies. Circular dichroism also suggested that the alpha-helicity of lysozyme was enhanced as ligand was bound. Naringenin inhibited lysozyme enzymatic activity, displaying its affinity with the lysozyme active site. Further, molecular docking studies demonstrated that naringenin could bind to both residues essential for catalytic activity in the proximity of Trp 62 and Trp 63.

Dietary Effect of Probiotic and Prebiotic on Some Mucus Indices of Silver Carp (Hypophthalmichthys molitrix) Exposed to Silver Nanoparticles.

Prebiotic and probiotic supplements can directly activate fish primary defense mechanisms by acting on responsible receptors. This study aimed to investigate the effect of using Pediococcus acidilactici and Agaricus bisporus separately and in combination with silver carp's mucosal immune system indices and finally the possibility of reducing the toxicity of silver nanoparticles in silver carp. After calculating LC50, the sub-lethal toxicity test was performed to sub-lethal concentration of silver nanoparticles for 14 days. Fish skin mucus was collected in polyethylene bags containing 10 ml of 50 mM sodium chloride for future analysis. Results of skin mucosal indices activity showed significant changes in treatments fed with additives compared to the control treatment after exposure to silver nanoparticles on days 1, 7, and 14 in including lysozyme enzyme activity, total protein, and skin mucosal protein pattern. The addition of additives will improve the relative mucosal safety indices of silver carp. The results of statistical analysis showed that the addition of combination supplements, Pediococous acidilactici, and Agaricus bisporus powder could affect the skin mucosal safety indices. It is effective and ultimately improves the immune system function of silver carp skin in 14-day exposure to silver nanoparticles.

Decreased production of epithelial-derived antimicrobial molecules at mucosal barriers during early life.

Young age is a risk factor for respiratory and gastrointestinal infections. Here, we compared infant and adult mice to identify age-dependent mechanisms that drive susceptibility to mucosal infections during early life. Transcriptional profiling of the upper respiratory tract (URT) epithelium revealed significant dampening of early life innate mucosal defenses. Epithelial-mediated production of the most abundant antimicrobial molecules, lysozyme and lactoferrin, and the polymeric immunoglobulin receptor (pIgR), responsible for IgA transcytosis, was expressed in an age-dependent manner. This was attributed to delayed functional development of serous cells. Absence of epithelial-derived lysozyme and the pIgR was also observed in the small intestine during early life. Infection of infant mice with lysozyme-susceptible strains of Streptococcus pneumoniae or Staphylococcus aureus in the URT or gastrointestinal tract, respectively, demonstrated an age-dependent regulation of lysozyme enzymatic activity. Lysozyme derived from maternal milk partially compensated for the reduction in URT lysozyme activity of infant mice. Similar to our observations in mice, expression of lysozyme and the pIgR in nasopharyngeal samples collected from healthy human infants during the first year of life followed an age-dependent regulation. Thus, a global pattern of reduced antimicrobial and IgA-mediated defenses may contribute to increased susceptibility of young children to mucosal infections.

Characterization of enzymatic activity of lysozyme in lysozyme–ovotransferrin complex before and after treatment with trypsin

AbstractThe native complex of lysozyme and ovotransferrin was isolated after the separation of egg white proteins using non‐denaturing isoelectric focusing, and mobilization toward the cathode by replacement of the cathodic sodium hydroxide solution with a phosphoric acid solution. The treatment of the lysozyme–ovotransferrin complex with trypsin significantly increased the enzymatic activity of lysozyme. Likewise, an increase in the enzymatic activity of lysozyme was also obtained when a mixture of the purified lysozyme and ovotransferrin was treated with trypsin. The increase in lysozyme enzymatic activity after tryptic treatment of the lysozyme–ovotransferrin complex resulted from the liberation of lysozyme from this complex. This was a consequence of the resistance of lysozyme to tryptic digestion and the digestion of ovotransferrin by trypsin into peptide fragments, which do not bind the lysozyme. The developed methodology could be used for the separation and isolation of other protein complexes and for testing their enzymatic and other activities.

Tropine‐Based Ionic Liquid Gel for Adsorption of Protein Ovalbumin: High Capacity, Selectivity and Biocompatibility

AbstractChicken ovalbumin (OVA) and lysozyme (LZ) are important biological products, which come from the egg‐white. However, the separation of OVA and LZ is realized by traditional salting‐out method needing large amount of ammonium sulfate, leading to lysozyme enzyme activity reducing. In this work, we used a tropine‐based ionic liquid (IL) hydrogel to separate OVA and LZ. The adsorption isotherms, thermodynamics and kinetics were investigated. The IL gel showed maximum adsorption capacity of 926 mg g−1 for OVA, nearly 152 % higher than that reported in other literature. Moreover, the secondary structure of desorbed OVA was confirmed by fluorescence spectroscopy (FL), circular dichroism spectra (CD) and differential scanning calorimetry (DSC). The material presented high OVA adsorption capacity, excellent OVA selectivity and good biocompatibility in OVA‐LZ protein system. It could be considered as a promising material for preferential protein adsorption, at least for separation of OVA and LZ.

Dietary peppermint (Mentha piperita) powder affects growth performance, hematological indices, skin mucosal immune parameters, and expression of growth and stress-related genes in Caspian roach (Rutilus caspicus).

Peppermint is a popular herbal medicine due to its several pharmaceutical applications. In this study, peppermint powder was used as a feed additive to evaluate growth performance, hematological parameters, protein profile of skin mucus, and immune parameters, as well as growth hormone (GH), insulin-like growth factor (IGF), and 70kDa heat shock protein (HSP70) gene expression in Caspian roach (Rutilus caspicus). The fingerlings (average weight of 2.40 ± 0.12g) were fed with diet containing 0 (control), 2, 3, and 4g/kg peppermint for 8weeks. The addition of peppermint significantly enhanced the growth parameters and decreased the food conversion ratio. Hematological indices of fish fed with peppermint-supplemented diets were significantly different from the control group (P < 0.05). Soluble protein, alkaline phosphatase, and lysozyme enzyme activity in mucus samples showed an incremental trend by increasing the peppermint levels in the diet. Evaluation of mucosal immunity indicated a remarkable difference between the protein profile in treatments fed with peppermint-supplemented diets and the control group. A new protein band (approximately 27kDa) was also found in the skin mucus of fish fed with the diet containing 4g/kg peppermint, and the highest band density was observed in this treatment. The highest IGF and GH gene expression were observed in 4g/kg peppermint treatment. There was a significant difference in HSP70 expression between the fish fed with peppermint powder and the control group (P < 0.05). Overall, the results showed that dietary oral administration of peppermint at 4g/kg of feed can act as a growth promoter and immunostimulant.

Efficient Expression of Human Lysozyme Through the Increased Gene Dosage and Co-expression of Transcription Factor Hac1p in Pichia pastoris.

In this work, the high-level expression of the human lysozyme (HLY) was achieved by both optimization of the gene copy number and co-expression of the transcription factor Hac1p for the unfolded protein response (UPR) in the host strain Pichia pastoris KM71H. A series of recombinant constructs with various numbers of HLY expression cassettes was generated for the production of recombinant strains integrated with different copies of the HLY gene. The copy number of the HLY gene was determined by real-time quantitative polymerase chain reaction, and the recombinant strains of P. pastoris carrying one, two, three, four, or six copies of the HLY gene were obtained. Maximum extracellular protein and lysozyme enzyme activity reached 436.99 ± 26.08μg/mL and 61,900 ± 2036.47 U/mL, respectively, in the recombinant strain HLYH4-3 with the four copies of the HLY gene after shaking flask fermentation. Moreover, the co-expression of the transcription factor Hac1p in the recombinant strains further enhanced the HLY yields. Extracellular protein and lysozyme enzyme activity, respectively, reached 517.82 ± 4.19μg/mL and 78,600 ± 1134.95 U/mL by using the Hac1p co-expression strain HLYH4-3/Hac1p. These values are the highest recorded level of human lysozyme expressed by P. pastoris in shaking flask fermentation so far.

Dietary Glycyrrhiza uralensis extracts supplementation elevated growth performance, immune responses and disease resistance against Flavobacterium columnare in yellow catfish (Pelteobagrus fulvidraco)

The present study was conducted to evaluate the influence of Glycyrrhiza uralensis (G. uralensis) extracts on the growth performance, histological structure, immune response and disease resistance against Flavobacterium columnare (F. columnare) of yellow catfish. Fish were fed with two different diets, i.e., basal diet as control group (CG) and diet containing G. uralensis extracts as experimental group (GG). After 60 days feeding, growth performance of GG fish was significantly improved, with increased WG and SGR but decreased FCR compared to CG fish. Fish were then challenged with F. columnare for two times, as fish showed rare mortality after the first infection. GG fish showed significantly lower cumulative mortality during F. cloumnare infection than CG fish after 21 days infection (dpi). Epithelial cell exfoliation and obvious cellular vacuolization in the skin and congestion of gill lamellae were detected in CG fish, while GG fish showed increased width of epidermis and mucous cells number in skin, and increased length of secondary lamina in gill. GG fish also exhibited higher enzyme activity of lysozyme in serum and mRNA expression of lysozyme in head kidney than CG fish at most time points post infection. G. uralensis extracts supplementation also induced earlier serum anti-oxidative responses, with increased superoxide dismutase activity and total antioxidant capacity in GG fish at 1 dpi. Compared to CG fish, GG fish showed increased expression level of genes involved in TLRs-NFκB signaling (TLR2, TLR3, TLR5, TLR9, Myd88, and p65NFκB), resulting in higher expression levels of pro-inflammatory cytokines (IL-1β and IL-8) in the head kidney post infection. However, these genes showed deviation in the gill of GG fish, which increased at some time points but decreased at other time points. Moreover, G. uralensis extracts supplementation also significantly unregulated the expression levels of IgM and IgD in head kidney, and the expression levels of IgM in the gill of yellow catfish, suggesting the elevated humoral immune response during F. columnare infection. All these results contributed to the elevated disease resistance ability against F. cloumnare infection of yellow catfish after dietary G. uralensis extracts supplementation.

Identification and expression pattern of a new digestive invertebrate-type lysozyme from the earthworm.

The invertebrate type (i-type) lysozyme not showing a clear homology with the known types of lysozyme was first demonstrated from a marine bivalve, conch and earthworm by N-terminal sequence. An i-type lysozyme isolated from the earthworm found to be up-regulated upon bacterial challenge, suggesting this lysozyme to function as an inducible immune factor. However, information on the i-type lysozyme related with digestive function is very limited in the earthworm. The objective of this study is to investigate the molecular characteristics and function of the new i-type lysozyme from the earthworm. To identify a new i-type lysozyme, multiple amino acid sequence alignment and phylogenetic analyses were employed. Its mRNA expression pattern was observed by fluorescent in situ hybridization (FISH). A new i-type lysozyme (Ea-iLys) from an earthworm, Eisenia andrei with the open reading frame of 678bp (226 amino acid residues) appeared to comprise conserved 14 cysteine residues for disulfide bridges and amino acid residues for the enzyme activities of lysozyme and isopeptidase, of which mRNA expression is mainly localized in the lining of midgut epithelium. No significant expression signal was detected in immune competent sites such as chloragogue tissue, typhlosole region, body coelom and muscle layers. Our results suggest that this enzyme primarily acts as a digestive enzyme rather than an innate immune factor.

Influence of humic acid on transport, deposition and activity of lysozyme in quartz sand

Interaction with natural organic matter (NOM) is hypothesized to impact the fate and bioavailability of enzymes and some hazardous proteins in terrestrial and aquatic environments. By using saturated column transport experiments the transport and deposition of the model enzyme lysozyme (LSZ), in the absence and presence of purified Aldrich humic acid (PAHA), was investigated at a series of mass ratios PAHA/LSZ at pH 5 and 8 and two ionic strength values (0.5 mM and 50 mM KCl solution). PAHA decreased LSZ transport under all conditions. The shapes of breakthrough curves (BTCs) and retention profiles (RPs) during cotransport of both colloids evolved from symmetrical to blocking with time and from flat to hyper-exponential with depth, respectively, in response to increases in mass ratio PAHA/LSZ. The results indicated that the “size-selective retention” and concurrent homo- and hetero-aggregation induced straining, which resulted in preferential retention of relatively large PAHA-LSZ aggregates in the column and elution of relatively small ones. Due to differences in aggregate size, in general, the enzyme activity of LSZ in the effluent was larger and that of the retained LSZ was smaller than that of the influent. Therefore, protein transport process could partially increase the enzyme activity and bring potential environmental hazards.

Eco-friendly synthesis of CuInS2 and CuInS2@ZnS quantum dots and their effect on enzyme activity of lysozyme.

We report on the green and facile aqueous microwave synthesis of glutathione (GSH) stabilized luminescent CuInS2 (CIS, size = 2.9 nm) and CuInS2@ZnS core–shell (CIS@ZnS, size = 3.5 nm) quantum dots (QDs). The core–shell nanostructures exhibited excellent photo- and water/buffer stability, a long photoluminescence (PL) lifetime (463 ns) and high PL quantum yield (PLQY = 26%). We have evaluated the comparative enzyme kinetics of these hydrophilic QDs by interacting them with the model enzyme lysozyme, which was probed by static and synchronous fluorescence spectroscopy. The quantification of the QD–lysozyme binding isotherm, exchange rate, and critical flocculation concentration was carried out. The core–shell QDs exhibited higher binding with lysozyme yielding a binding constant of K = 5.04 × 109 L mol−1 compared to the core-only structures (K = 6.16 × 107 L mol−1), and the main cause of binding was identified as being due to hydrophobic forces. In addition to the enzyme activity being dose dependent, it was also found that core–shell structures caused an enhancement in activity. Since binary QDs like CdSe also show a change in the lysozyme enzyme activity, therefore, a clear differential between binary and ternary QDs was required to be established which clearly revealed the relevance of surface chemistry on the QD–lysozyme interaction.

Effects of freeze-drying and spray-drying on donkey milk volatile compounds and whey proteins stability

In this work, the profile of the volatile compounds has been determined for the first time in fresh donkey milk and in donkey milk subjected to accelerated shelf-life test, freeze-drying and spray-drying processes. The effects of freeze-drying and spray-drying processes in donkey milk have been analyzed also on three representative whey proteins, β-lactoglobulin, α-lactalbumin and lysozyme, by determining the total amount, the enzymatic activity and the thermal stability. Considering the volatile compounds in fresh donkey milk, 2-heptanone, 1,3-bis (1,1-dimethylethyl)-benzene, nonanal, d-limonene, octanoic acid, and 1-octanol were detected. The accelerated shelf-life test revealed a constant increase of octanoic acid, 2-heptanone, and nonanal compared to fresh donkey milk (P < 0.001) and the appearance of ethyl octanoate, ethyl dodecanoate, 2-nonanone and 2-undecanone, indicating that these compounds may be highly eligible markers of donkey milk shelf-life. Freeze-drying and spray-drying affected β-lactoglobulin and lysozyme content in donkey milk. In particular, spray-drying process decreased significantly lysozyme enzymatic activity (58% of residual activity) and β-lactoglobulin content (6.43 mg/mL in fresh milk vs 5.51 mg/mL in spry-dried milk) due to the high temperature to which donkey milk is subjected.

Oxidative Folding of Conopeptides Modified by Conus Protein Disulfide Isomerase.

Protein disulfide isomerase is a type of enzyme that catalyses the oxidation, isomerization and reduction of disulfide bonds. Conotoxins that containing disulfide bonds are likely substrates of protein disulfide isomerise. Here, we cloned 12 protein disulfide isomerise genes from 12 different cone snail species that inhabited the sea near Sanya in China. The full-length amino acid sequences of these protein disulfide isomerase genes share a high degree of homology, including the same -CGHC- active site sequence and -RDEL- endoplasmic reticulum retention signal. To obtain enough conus protein disulfide isomerase for functional studies, we constructed the expression vector pET28a-sPDI. Conus protein disulfide isomerase was successfully expressed using Escherichia coli expression system and purified using chromatography method of affinity chromatography. The recombinant conus protein disulfide isomerase showed the ability to catalyse disulfide bond formation and rearrangement in the lysozyme enzyme activity assay. The role of conus protein disulfide isomerase in the in vitro oxidative folding of conotoxins was investigated using synthetic linear conotoxin lt14a, a peptide composed of 13 amino acids. It was confirmed by high performance liquid chromatography and mass spectrometry analysis that conus protein disulfide isomerase can catalyse the disulfide bond formation of linear lt14a. Then, conus protein disulfide isomerase was acted as a fusion partner during the production of engineered peptidyl-prolyl cis-trans isomerase and lt14a derived from cone snails. It was shown that peptidyl-prolyl cis-trans isomerase and conotoxin lt14a are successfully expressed in a highly soluble form by fusion with conus protein disulfide isomerase. Thus, conus protein disulfide isomerase functions not only as an enzyme that catalyses oxidative process but also a fusion partner in recombinant conotoxin expression.