Vmax Values Research Articles

Immobilization of Xylanase on ZnO nanoparticles obtained by green synthesis from Eupatorium cannabinum L. and its application in enrichment of fruit juices

Juice production is one of the main components of the nutritional food industry. Therefore, improving fruit juice quality and preventing the formation of negative by-products is important for human health. The role of environmentally friendly, safe and economically more affordable green nanocarriers in improving the quality of fruit juice is increasing day by day. For this purpose, zinc oxide nanoparticles (ZnONPs) were prepared from the aqueous extract of the Eupatorium cannabinum L. using the green synthesis method. After immobilization, structural and morphological characterizations of the prepared ZnONPs and xylanase (Xyl)@ZnONPs were carried out, which confirmed that both the synthesis of ZnONPs and the immobilization of Xyl enzyme on ZnONPs were successfully carried out. The biochemical parameters of Xyl@ZnONPs such as optimum pH and temperature, kinetic parameters, thermal stability, reusability, and storage stability were investigated and compared with free Xyl. After immobilization, the optimum pH of free Xyl shifted from 5.0 to an alkaline pH of 8.0, while no change in the optimum temperature (70 °C) was observed for both enzymes. While the activation energy of free Xyl was 5.56 kJ/mol, it was found to be 7.27 kJ/mol for Xyl@ZnONPs. Xyl@ZnONPs outperformed free Xyl due to its long-term storage stability and reusability. Moreover, Xyl@ZnONPs retained 50 % of its activity after 5 reuses and 87 % of its activity after 4 weeks of storage at room temperature. In addition, a decrease in Km value and an increase in Vmax value were observed after immobilization, which means the increased catalytic activity of the Xyl@ZnONPs. As a result of re-using Xyl@ZnONPs in orange juice for 6 cycles, clarification yield of 66.43 % was obtained under optimized conditions. Overall, this study prepared a suitable, environmentally friendly, and robust carrier support for Xyl immobilization and developed a biocatalyst that will provide high performance in the industrial field.

Kinetic studies on optimized extracellular laccase from Trichoderma harzianum PP389612 and its capabilities for azo dye removal

BackgroundAzo dyes represent a common textile dye preferred for its high stability on fabrics in various harsh conditions. Although these dyes pose high-risk levels for all biological forms, fungal laccase is known as a green catalyst for its ability to oxidize numerous dyes.MethodsTrichoderma isolates were identified and tested for laccase production. Laccase production was optimized using Plackett–Burman Design. Laccase molecular weight and the kinetic properties of the enzyme, including Km and Vmax, pH, temperature, and ionic strength, were detected. Azo dye removal efficiency by laccase enzyme was detected for Congo red, methylene blue, and methyl orange.ResultsEight out of nine Trichoderma isolates were laccase producers. Laccase production efficiency was optimized by the superior strain T. harzianum PP389612, increasing production from 1.6 to 2.89 U/ml. In SDS-PAGE, purified laccases appear as a single protein band with a molecular weight of 41.00 kDa. Km and Vmax values were 146.12 μmol guaiacol and 3.82 μmol guaiacol/min. Its activity was stable in the pH range of 5–7, with an optimum temperature range of 40 to 50 °C, optimum ionic strength of 50 mM NaCl, and thermostability properties up to 90 °C. The decolorization efficiency of laccase was increased by increasing the time and reached its maximum after 72 h. The highest efficiency was achieved in Congo red decolorization, which reached 99% after 72 h, followed by methylene blue at 72%, while methyl orange decolorization efficiency was 68.5%.ConclusionTrichoderma laccase can be used as an effective natural bio-agent for dye removal because it is stable and removes colors very well.

#521 Increasing levels of aortic peak wave velocity along with prolonged higher CaxP product is associated with mortality in peritoneal dialysis

Abstract Background and Aims Valvular calcification is concordant with vascular calcification and develops faster in end-stage renal disease patients than in the average population. There are contradictory reports about the pathophysiology and the predictive value of valvular calcification regarding mortality in this patient population. Aortic peak wave velocity (Vmax) measurement is a non-invasive and reliable marker for the diagnosis of valve calcification. Our study analyzes cardiovascular risk prediction effect of increasing levels of Vmax at different time points in peritoneal dialysis (PD) patients. Method We conducted a retrospective, single-center study of incident PD patients under follow-up between January 1992 and January 2023 in our clinic. Demographics, laboratory, and echocardiographic findings were obtained from medical records. Patients without a baseline echocardiography were excluded. ΔVmax is defined as the last Vmax minus baseline Vmax levels. The patients were divided into two subgroups: Vmax-stable and Vmax-increased. Results The baseline demographics, laboratory, and clinical characteristics, including daily elemental calcium and calcitriol usage, were similar between Vmax-stable and Vmax-increased groups. Baseline calcium (Ca), phosphate (P), and annual CaXP levels were significantly higher in Vmax-increased group [p = 0.01, p = 0.02, and (F(1,27) = 10.415, p = 0.003), respectively]. Cardiovascular mortality was 9.8% in Vmax-stable group and 26.8% in Vmax-increased group (p = 0.02). Conclusion During a follow-up of 20 years, increasing Vmax values were associated with higher CaxP levels in PD patients. Increased Vmax levels, along with higher CaxP levels, are associated with cardiovascular mortality. Even without clinical manifestations, Vmax follow-up is a non-invasive, easily accessible method and might identify the high-risk group regarding cardiovascular mortality in the PD population.

Assessing the nitrate and phosphate uptake kinetics potential and growth performance of Gracilaria corticata var. cylindrica in shrimp farm water (SFW)

Gracilaria corticata var. cylindrica (GCC) was studied as a biofilter in shrimp farm water (SFW) and its growth and biochemical attributes in the medium were evaluated. GCC showed significant nutrient absorption from SFW, with maximum nitrate and phosphate uptake observed after the first 48 h. Nitrate depletion happened faster than phosphate, thus showing a higher affinity for nitrate uptake (78.4% removal) by GCC. Nutrient uptake kinetics followed the Michaelis–Menten curve, with Vmax values of 43.16 μM g dw−1 h−1 for nitrate and 67.50 μM g dw−1 h−1 for phosphate. Also, GCC’s low Km values (12.53 µM for nitrate, 1.91 µM for phosphate) indicated efficient nutrient utilization. GCC showed rapid growth in SFW, with a daily growth rate of 2.93 ± 0.19% d−1, comparable to the commercial MP1 media (3.54 ± 0.19% d−1) and greater to seawater (2.23 ± 0.13% d−1). Proximate analysis revealed consistent biochemical compositions across all three media, with carbohydrate and protein contents of 2.24 ± 0.41% and 0.76 ± 0.12%, respectively, in SFW. Pigment analysis revealed that pigment concentrations varied, with SFW having the greatest R-PE concentration (182.25 ± 32.77 µg g−1), followed by MP1 media and saltwater. R-PC concentrations followed a similar pattern, with SFW having the greatest concentration (85.73 ± 19.33 µg g−1). These findings highlight GCC’s potential as an efficient biofilter for reducing nitrate and phosphate levels in SFW, therefore offering a sustainable solution for aquaculture effluent management and the potential integration of GCC into shrimp farming practices.

Fabrication of immobilized lipases from Candida rugosa on hierarchical mesoporous silica for enzymatic enrichment of ω-3 polyunsaturated fatty acids by selective hydrolysis

In this study, lipase from Candida rugosa was immobilized on hydrophobic hierarchical porous hollow silica microsphere (HPHSM-C3) via adsorption. The prepared biocatalyst HPHSM-C3@CRL exhibited higher activity, thermal and pH stability. HPHSM-C3@CRL remained 70.2% of initial activity after 30 days of storage at 24 °C and 50.4% of initial activity after 10 cycles. Moreover, HPHSM-C3@CRL was utilized in enzymatic enrichment of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in glycerides, achieving ω-3 PUFAs content of 53.42% with the hydrolysis rate of 48.78% under optimal condition. The Km and Vmax value of HPHSM-C3@CRL was 42.2% lower and 63.5% higher than those of CRL, respectively. The 3D structure analysis of CRL, substrates and pore structure of HPHSM-C3 suggested that the hierarchical pore improved activity and selectivity of immobilized lipase. This result demonstrated that HPHSM-C3@CRL may be an effective biocatalyst for the enzymatic enrichment of ω-3 PUFAs in food industries.

Ionic liquid-assisted pretreatment of lignocellulosic biomass using purified Streptomyces MS2A cellulase for bioethanol production

In recent years, the process of producing bioethanol from lignocellulosic biomass through biorefining has become increasingly important. However, to obtain a high yield of ethanol, the complex structures in the feedstock must be broken down into simple sugars. A cost-effective and innovative method for achieving this is ionic liquid pre-treatment, which is widely used to efficiently hydrolyze the lignocellulosic material. The study aims to produce a significant profusion of bioethanol via catalytic hydrolysis of ionic liquid-treated lignocellulose biomass. The current study reports the purification of Streptomyces sp. MS2A cellulase via ultrafiltration and gel permeation chromatography. The kinetic parameters and the biochemical nature of the purified cellulase were analyzed for the effective breakdown of the EMIM[OAC] treated lignocellulose chain. The two-step cellulase purification resulted in 6.28 and 12.44 purification folds. The purified cellulase shows a Km value of 0.82 ± 0.21 mM, and a Vmax value of 85.59 ± 8.87 μmol min−1 mg−1 with the catalytic efficiency of 1.027 S−1. The thermodynamic parameters like ΔH, ΔS, and ΔG of the system were studied along with the thermal deactivation kinetics of cellulase. The optimal temperature and pH of the purified cellulase enzyme for hydrolysis was found to be 40 °C and 7. The rice husk and wheat husk used in this study were pretreated with the EMIM [OAC] ionic liquid and the change in the structure of lignocellulosic biomass was observed via HRSEM. The ionic liquid treated biomass showed the highest catalytic hydrolysis yield of 106.66 ± 0.19 mol/ml on the third day. The obtained glucose was fermented with Saccharomyces cerevisiae to yield 23.43 g of ethanol/l of glucose from the rice husk (RH) and 24.28 g of ethanol/l of glucose from the wheat husk (WH).

Purification and characterization of glutamate dehydrogenase from rainbow trout (Oncorhynchus mykiss) liver and molecular docking studies.

Glutamate dehydrogenase (GDH) participates in the energy metabolism of proteins and the synthesis of metabolites important for the organism. In this study, GDH enzyme was purified from the liver of rainbow trout (Oncorhynchus mykiss) by 2',5'-ADP Sepharose 4B affinity chromatography in one step. As a result of this purification process, GDH enzyme was purified 171-fold with 5.83U/mg protein-specific activity. The characterization experiments presented that the storage stability of the purified GDH enzyme was determined as -80°C; optimum temperature 40°C; it was determined that the optimum ionic strength was 100mM phosphate buffer and the optimum pH was 8.00. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and PAGE studies showed that the natural molar mass of the purified GDH enzyme was 346.74kDa, and the molar mass of its subunits was 53.71kDa. Km and Vmax values for substrates and coenzymes of GDH enzyme purified from rainbow trout liver were calculated, and the lowest Km value was found in NAD+ (1.86mM) and the highest Vmax value in NH4 + (1.79U/mL). The effects of some metal ions, vitamins, and solvents on the activity of the purified GDH enzyme were investigated and also IC50 values and inhibition types. The metal ion with the lowest IC50 value is Ag+ (8.65 ± 1.68μM), and the vitamin is B6 (0.77 ± 0.04mM). The binding affinities of inhibitors were investigated with molecular docking, based on the conformational state of GDH.

Purification and Characterization of a Small Thermostable Protease from Streptomyces sp. CNXK100.

Proteases derived from Streptomyces demonstrate numerous commendable properties, rendering it extensively applicable in biotechnology and various industrial sectors. This study focused on the purification and characterization of the thermostable protease obtained from Streptomyces sp. CNXK100. The purified protease exhibited an estimated molecular weight of 27 kDa, with optimal activity at 75°C and pH 8.0. Notably, the enzyme remained active even without any metal ions and fully active in the presence of Na+, K+, Mg2+, and Cu2+metal ions. The kinetic parameters were determined with a KM value of 3.13 mg/ml and a Vmax value of 3.28 × 106 U/mg. Furthermore, the protease has demonstrated notable stability when subjected to a treatment temperature of up to 65°C for 60 minutes, and across a broad pH range extending from 5.0 to 10.0. This protease also demonstrated resilience against a spectrum of harsh conditions, including exposure to organic solvents, surfactants, bleaching agents, and proteolytic enzymes. Additionally, the enzyme maintained its activity following treatment with commercial detergents, accomplishing complete thrombus lysis at a concentration of 2.50 mg/ml within 4 hours. Remarkably, the protease exhibited stability in terms of activity and protein concentration for 70 days at 4°C. These findings underscore the potential industrial applications of the thermostable protease from Streptomyces sp. CNXK100.

Chemical kinetics and thermodynamics of PPO activity, colour changes and microbial degradation during blanching of the sugarcane billets.

Sugarcane juice, which has a short shelf life, is a popular thirst-quenching and rejuvenating beverage worldwide. The limited shelf life is a result of changes in polyphenol oxidase (PPO) activity, total plate count (TPC) and color attributes (L*, a* and b*-values). We hypothesized that chemical kinetics and thermodynamics of blanched sugarcane cane juice causing alterations in PPO, TPC, and L, a* and b*-values will address the challenges of sugarcane juice preservation. Sugarcane billets were blanched at variable time-temperature combinations in the range 0-20 min and 70-90 °C. Reaction rates increased with increasing temperature; PPO activity, TPC and colour followed first-order kinetics. PPO activity had an activation energy (Ea) of 81 kJ mol-1. The half life (t½) dropped from 16.5 to 3.47 min and decimal reduction time (D-values) dropped from 54.83 to 11.52 min. Thus reactions were temperature-sensitive. Thermodynamic studies indicated an endothermic (positive enthalpy values, ΔH > 0; 78.10 kJ mol-1) and reversible process (negative entropy values ΔS < 0; -0.044 kJmol-1 K-1). Michaeli-Menten constant (Km) and maximum velocity (Vmax) of PPO activity were determined by adding variable lemon juice concentrations in sugarcane juice. As the Km values increased (from 5.53 to 15.81 mm) and Vmax values decreased (from 666.67 to 384.61 UmL-1), a Lineweaver-Burk plot suggested decreased PPO affinity of sugarcane juice. The results of the present study indicate that studies on chemical kinetics and thermodynamics (PPO, TPC and L, a* and b*-values) of blanched sugarcane cane juice shall mitigate challenges of sugarcane juice preservation. © 2024 Society of Chemical Industry.

Structural and functional insights of starch processing α-amylase from hyperthermophilic archaeon Pyrococcusabyssi

The genomic screening of hyper-thermophilic Pyrococcus abyssi showed uncharacterized novel α-amylase sequences. Homology modelling analysis revealed that the α-amylase from P. abyssi consists of an N-terminal GH57 catalytic domain, α-amylase central, and C-terminal domain. Current studies emphasize in-silico structural and functional analysis, recombinant expression, characterization, structural studies through CD spectroscopy, and ligand binding studies of the novel α-amylase from P. abyssi. The soluble expression of PaAFG was observed in the E. coli Rosetta™ (DE3) pLysS strain upon incubation overnight at 18 °C in an orbital shaker. The optimum temperature and pH of the PaAFG were observed at 90 °C in 50 mM phosphate buffer pH 6. The Km value for PaAFG against wheat starch was determined as 0.20 ± 0.053 mg while the corresponding Vmax value was 25.00 ± 0.67 μmol min−1 mg−1 in the presence of 2 mM CaCl2 and 12.5 % glycerol. The temperature ramping experiments through CD spectroscopy reveal no significant change in the secondary structures and positive and negative ellipticities of the CD spectra showing the proper folding and optimal temperature of PaAFG protein. The RMSD and RMSF of the PaAFG enzyme determined through molecular dynamic simulation show the significant protein's stability and mobility. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various industrial applications.

Cloning, heterologous expression and purification of the novel thermo-alkalistable cellulase from Geobacillus sp. TP-3 and its molecular characterisation

BackgroundThermophilic cellulases are essential for effectively degrading cellulose, which is a significant part of lignocellulosic waste. In this study, we focused on a cellulase gene (~ 1.2 kb) obtained from Geobacillus sp. TP-3, a thermo-alkalophilic bacterium isolated from the hot springs of Tapovan (Uttarakhand, India). Cellulase gene (~ 1.2 kb) was amplified via PCR, cloned into pET-28a (+) vector, transferred to Escherichia coli DH5α cells and expressed in Escherichia coli BL21 (DE3). The recombinant cellulase (rCel_TP) was purified using Ni2+-NTA affinity chromatography.ResultsThe purified rCel_TP enzyme exhibited optimal activity at 50 ºC and pH 8, displaying stability even after 3 h of incubation at 50 ºC. The molecular weight of the purified 6 × His-tagged rCel_TP was determined to be ~ 40.2 kDa. Under conditions of 50 ºC and pH 8, the kinetic parameters of the purified enzyme were determined, with Km and Vmax values of 116.78 mg/mL and 44.05 µmolmg−1 min−1, respectively. The activity of the rCel_TP cellulase was significantly improved by Hg2+, Cu2+ and Co2+. However, it was suppressed by dithiothreitol and β-mercaptoethanol. Ethylenediaminetetraacetic acid and solvents also had a slight inhibitory effect.ConclusionThese results suggest the potential applications of the recombinant cellulase in biomass conversion processes for the production of fuels and other industrial operations. The study contributes valuable insights into the properties and applicability of cellulases derived from extremophilic microorganisms.Graphical abstract

Mechanistic evaluation of contrasting modulation in conformation and catalytic performance of urease by surface interaction of graphene oxide and amine- functionalized graphene oxide nanosheets

The increasing use of graphene-based nanomaterials raises concerns about their potential environmental impact as ecotoxicants. This article examines the interaction and effects of graphene oxide (GO) and its amine-derived (GONH2) 2-D nanosheets on urease dynamics, a crucial enzyme in soil nutrient cycling, through spectroscopic, microscopic, and computational analyses. The findings reveal the distinct interaction mechanisms: GO formed ground - and excited-state complexes with urease; thus, fluorescence quenching was static and dynamic via hydrophobic interaction. In contrast, GONH2 nanosheets formed a ground-state complex by static quenching involving Van der Waals and hydrogen bonding. These interactions were energetically favored and altered tryptophan (Trp) residues’ microenvironment more than tyrosine (Tyr). The average exciton lifetime of urease in the presence of GO have decreased from 4.34 ns to 3.647 ns at the mere concentration of 7.11 µM, while for GONH2, no discernable change (4.16 ns) was observed even at the concentration of 16.75 µM. Primarily, the strong interaction of GO leads to the rearrangement of polypeptide structure, reduced α-helices to 12 %, and increased β-sheets by six times. In contrast, with GONH2, α-helices were reduced to 79 %, and β-sheets were increased by 1.8 times at the similar concentration. Molecular docking revealed that neither GO nor GONH2 interacted with the active site of urease, and binding was primarily via hydrophobic, Van der Waals, and hydrogen bonding, concurrent with in-vitro incubation results. Positive total energy (3.85 kJ mol−1) during molecular docking analysis of urease-GO interaction and inhibition in enzyme activity to 62 %, altered Vmax value (0.04606 ± 0.001 mM/s), without affecting the Km (4.78045 ± 0.12 mM) suggested that inhibition was noncompetitive and steric clashes due to disruptive binding might drove the inhibition. Meanwhile, negative total internal energy (-3.97 kJ mol−1) during urease + GONH2 binding and enhanced enzymatic activity by 32 % in-vitro without altering Vmax (0.063091 ± 0.003 mM/s) and Km (4.953943 ± 0.2 mM) values suggested that urease's binding site was well-complemented by GONH2 nanosheets, serving as a redox-mediator and radical-quencher.

Switchable CO2-Responsive Janus Nanoparticle for Lipase Catalysis in Pickering Emulsion.

The use of convertible immobilized enzyme carriers is crucial for biphasic catalytic reactions conducted in Pickering emulsions. However, the intense mechanical forces during the conversion process lead to enzyme leakage, affecting the stability of the immobilized enzymes. In this study, a CO2-responsive switchable Janus (CrSJ) nanoparticle (NP) was developed using silica NP, with one side featuring aldehyde groups and the other side adsorbing N,N-dimethyldodecylamine. A switchable Pickering emulsion catalytic system for biphasic interface reactions was prepared by covalently immobilizing lipase onto the CrSJ NPs. The CO2-responsive nature of the CrSJ NPs allowed for rapid conversion of the Pickering emulsion, and covalent immobilization substantially reduced lipase leakage while enhancing the stability of the immobilization during the conversion process. Impressively, after repeated transformations, the Pickering emulsion still maintains its original structure. Following 10 consecutive cycles of esterification and hydrolysis reactions, the immobilized enzyme's activity remains at 77.7 and 79.5% of its initial activity, respectively. The Km of the CrSJ catalytic system showed no significant change compared to the free enzyme, while its Vmax values were 1.2 and 1.6 times that of the free enzyme in esterification and hydrolysis reactions, respectively.

MOF-based Ag NPs/Co3O4 nanozyme for colorimetric detection of thiophanate-methyl based on analyte-enhanced sensing mechanism.

Silver nanoparticles supported on metal-organic framework (ZIF-67)-derived Co3O4 nanostructures (Ag NPs/Co3O4) were synthesized via a facile in situ reduction strategy. The resulting materials exhibited pH-switchable peroxidase/catalase-like catalytic activity. Ag NP doping greatly enhanced the catalytic activity of Ag NPs/Co3O4 towards 3,3',5,5'-tetramethylbenzidine (TMB) oxidation and H2O2 decomposition which were 59 times (A652 of oxTMB) and 3 times (A240 of H2O2) higher than that of ZIF-67, respectively. Excitingly, thiophanate-methyl (TM) further enhanced the peroxidase-like activity of Ag NPs/Co3O4 nanozyme due to the formation of Ag(I) species in TM-Ag NPs/Co3O4 and generation of more radicals resulting from strong interaction between Ag NPs and TM. The TM-Ag NPs/Co3O4 nanozyme exhibited lower Km and higher Vmax values towards H2O2 when compared with Ag NPs/Co3O4 nanozyme. A simple, bioelement-free colorimetric TM detection method based on Ag NPs/Co3O4 nanozyme via analyte-enhanced sensing strategy was successfully established with high sensitivity and selectivity. Our study demonstrated that hybrid noble metal NPs/MOF-based nanozyme can be a class of promising artificial nanozyme in environmental and food safety applications.

Degradation of textile polyazodyes by Polyporus ciliatus laccase produced in peanut shell solid medium: purification and characterization

This study describes decolorization of reactive sulfonated azoic dye Direct Blue 71, one of the recalcitrant and highly used industrial dyes, by Polyporus ciliatus derived laccase. Agro-industrial wastes are increasingly used as substrates for laccase production from white rot fungi through solid-state fermentation (SSF). Peanut shell (PS) is an attractive substrate for laccase production, and SSF is an adequate strategy for that purpose. It has been employed as a novel support-substrate for laccase production by Polyporus ciliatus under solid-state fermentation. The highest laccase yield of 1260 U/ml was attained using PS after 13 days of incubation at 30°C. The purified laccase (55 kDa) showed maximum activity at pH 3 and 60°C. The extracellular laccase enzyme was purified 67.6-fold to a specific activity of 1466 U/mg by a Mono-Q anion-exchange column. The purified enzyme Km value was found to be 24 µM, while the Vmax value was observed to be 0.96 µM s-1 with ABTS as a substrate. It had high thermotolerance and pH stability and was resistant to several metal ions including copper, cadmium, and iron. P. ciliatus pure laccase showed high potential for the decolorization and detoxification of the reactive sulfonated azoic dye Direct Blue 71, which suggested that this enzyme could be used for textile effluent treatment.

Facile Synthesis of Copper Telluride Nanoparticles as a Robust Nanoenzymatic Catalyst for Intrinsic Peroxidase‐Like Activity

AbstractCopper telluride nanoparticles are classified as the family of metal chalcogenides and were synthesized using a simple solvothermal method using a new source of telluride, ditelluride diphenyl. The characterized techniques revealed that the atomic composition of copper tellurides is Cu2Te and particles are spherical. This study is greatly examined by different designed experiments for the proof of the intrinsic peroxidase‐like activity of copper telluride nanoparticles and its effectiveness in a variety of similar structures. Cu2Te nanoparticles can convert peroxidase substrates (TMB and OPD) to colored products in the presence of H2O2. In addition, the as‐prepared nanoparticles have the potential to oxidize DPPH and ABTS substrates. Michaelis–Menten constant KM value for TMB and H2O2 was found to be 12 mM and 0.60 mM for H2O2 and the Vmax value of the Cu2Te was found to be TMB and H2O2 was 2.50×10−7 and 0.82×10−8 MS−1. These data are found to be almost comparable with natural peroxidase HRP and other artificial enzymes reported in the literature. The natural peroxidase‐like activities of Cu2Te nanoparticles originated from the catalytic decomposition of H2O2 into ⋅OH radical, which was confirmed from the ESR spin‐trapping technique and using a fluorescence probe, terephthalic acid.

Purification, functional characterization and enhanced production of serratiopeptidase from Serratia marcescens MES-4: An endophyte isolated from Morus rubra

Serratiopeptidase, a proteolytic enzyme serves as an important anti-inflammatory and analgesic medication. Present study reports the production and purification of extracellular serratiopeptidase from an endophyte, Serratia marcescens MES-4, isolated from Morus rubra. Purification of the enzyme by Ion exchange chromatography led to the specific activity of 13,030 U/mg protein of serratiopeptidase, showcasing about 3.1 fold enhanced activity. The catalytic domain of the purified serratiopeptidase, composed of Zn coordinated with three histidine residues (His 209, His 213, and His 219), along with glutamate (Glu 210) and tyrosine (Tyr 249). The molecular mass, as determined by SDS-PAGE was ∼51 kDa. The purified serratiopeptidase displayed optimal activity at pH 9.0, temperature 50°C. Kinetic studies revealed Vmax and Km values of 33,333 U/mL and 1.66 mg/mL, respectively. Further, optimized conditions for the production of serratiopeptidase by Taguchi design led to the productivity of 87 U/mL/h with 87.9 fold enhanced production as compared to the previous conditions.

Isolation and biochemical characterization of novel acid phosphatase and zinc-dependent acid phosphatase from the chicken's brain

The AcPase exhibits a specific activity of 31.32 U/mg of protein with a 728-fold purification, and the yield of the enzyme is raised to 3.15 %. The Zn2+-dependent AcPase showed a purification factor of 1.34 specific activity of 14 U/mg of proteins and a total recovery of 5.14. The SDS-PAGE showed a single band corresponding to a molecular weight of 18 kDa of AcPase and 29 kDa of Zn2+-dependent AcPase. The AcPase enzyme has shown a wide range of substrate specificity for p-NPP, phenyl phosphate and FMN, while in the case of ZnAcPase α and β-Naphthyl phosphate and p-NPP were proved to be superior substrates. The divalent metal ions like Mg2+, Mn2+, and Ca2+ increased the activity, while other substrates decreased the enzyme activity. The Km (0.14 mM) and Vmax (21 μmol/min/mg) values of AcPase were higher than those of Zn2+-AcPase (Km = 0.5 mM; Vmax = 9.7 μmol/min/mg). The Zn2+ ions activate the Zn2+-AcPase while Fe3+, Al3+, Pb2+, and Hg2+ showed inhibition on enzyme activity. Molybdate, vanadate and phosphate were found to be competitive inhibitors of AcPase with Ki values 316 μM, 185 μM, and 1.6 mM, while in Zn2+-AcPase tartrate and phosphate also showed competitive inhibition with Ki values 3 mM and 0.5 mM respectively.

Exploration of antimicrobial and anticancer activities of L-amino acid oxidase from Egyptian Naja haje venom

Hepatocellular carcinoma and bacterial resistance are major health burdens nowadays. Thus, providing new therapies that overcome that resistance is of great interest, particularly those derived from nature rather than chemotherapeutics to avoid cytotoxicity on normal cells. Venomous animals are among the natural sources that assisted in the discovery of novel therapeutic regimens. L-amino acid oxidase Nh-LAAO (140 kDa), purified from Egyptian Naja haje venom by a successive two-step chromatography protocol, has an optimal pH and temperature of 8 and 37 °C. Under standard assay conditions, Nh-LAAO exhibited the highest specificity toward L-Arg, L-Met and L-Leu, with Km and Vmax values of 3.5 mM and 10.4 μmol/min/ml, respectively. Among the metal ions, Ca+2, Na+, and K+ ions are activators, whereas Fe+2 inhibited LAAO activity. PMSF and EDTA slightly inhibited the Nh-LAAO activity. In addition, Nh-LAAO showed antibacterial and antifungal activities, particularly against Gentamicin-resistant P. aeruginosa and E. coli strains with MIC of 18 ± 2 μg/ml, as well as F. proliferatum and A. parasiticus among the selected human pathogenic strains. Furthermore, Nh-LAAO exhibited anti-proliferative activity against cancer HepG2 and Huh7 cells with IC50 of 79.37 and 60.11 μg/ml, respectively, with no detectable effect on normal WI-38 cells. Consequently, the apoptosis % of the HepG2 and Huh7 cells were 12 ± 1 and 34.5 ± 2.5 %, respectively, upon Nh-LAAO treatment. Further, the Nh-LAAO arrested the HepG2 and Huh7 cell cycles in the G0/G1 phase. Thus, the powerful selective cytotoxicity of L-amino acid oxidase opens up the possibility as a good candidate for clinical cancer therapy.

Role of organic anion transporter NPT4 in renal handling of uremic toxin 3-indoxyl sulfate

Sodium-phosphate transporter NPT4 (SLC17A3) is a membrane transporter for organic anionic compounds localized on the apical membranes of kidney proximal tubular epithelial cells and plays a role in the urinary excretion of organic anionic compounds. However, its physiological role has not been sufficiently elucidated because its substrate specificity is yet to be determined. The present study aimed to comprehensively explore the physiological substrates of NPT4 in newly developed Slc17a3−/− mice using a metabolomic approach. Metabolomic analysis showed that the plasma concentrations of 11 biological substances, including 3-indoxyl sulfate, were more than two-fold higher in Slc17a3−/− mice than in wild-type mice. Moreover, urinary excretion of 3-indoxyl sulfate was reduced in Slc17a3−/− mice compared to that in wild-type mice. The uptake of 3-indoxyl sulfate by NPT4-expressing Xenopus oocytes was significantly higher than that by water-injected oocytes. The calculated Km and Vmax values for NPT4-mediated 3-indoxyl sulfate uptake were 4.52 ± 1.18 mM and 1.45 ± 0.14 nmol/oocyte/90 min, respectively. In conclusion, the present study revealed that 3-indoxyl sulfate is a novel substrate of NPT4 based on the metabolomic analysis of Slc17a3−/− mice, suggesting that NPT4 regulates systemic exposure to 3-indoxyl sulfate by regulating its urinary excretion.