High Residual Activity Research Articles

Simultaneous carbon dioxide sequestration and utilization for cadaverine production using dual promoters in engineered Escherichia coli strains

Human carbonic anhydrase II (hCAII) is a rapid-acting zinc-metalloenzyme that catalyzes CO2 hydration reversibly, with encouraging applications in carbon capture, sequestration, and utilization (CCSU). However, biocatalyst durability is a major challenge. Herein, hCAII is emphasized in 4 different Escherichia coli strains and designated under dual promoters from sigma factor 70 (σ70) and heat shock protein (HSP70A) to suppress the usage of inducer and stimulate activity in heat environments. As a result, hCAII under high-efficient dual promoters regulation retained high residual activity in CO2 biomineralization of 68.8 % after 4 cycles at 40 °C. Moreover, co-expression of CAC9 with lysine decarboxylase (CadA) simultaneously sequestered CO2 release up to 95.7 % and increased cadaverine titer from 18.0 to 36.7 g/L by using E. coli MG1655. The remnant biomass from cadaverine synthesis sustained converting CO2 to 57.9 mg-CaCO3. Thus, the dual promoters design demonstrated the promising potential for CCSU through simultaneous CO2 utilization and cadaverine synthesis.

Activity of AC electrokinetically immobilized horseradish peroxidase.

Dielectrophoresis (DEP) is an AC electrokinetic effect mainly used to manipulate cells. Smaller particles, like virions, antibodies, enzymes, and even dye molecules can be immobilized by DEP as well. In principle, it was shown that enzymes are active after immobilization by DEP, but no quantification of the retained activity was reported so far. In this study, the activity of the enzyme horseradish peroxidase (HRP) is quantified after immobilization by DEP. For this, HRP is immobilized on regular arrays of titanium nitride ring electrodes of 500nm diameter and 20nm widths. The activity of HRP on the electrode chip is measured with a limit of detection of 60fg HRP by observing the enzymatic turnover of Amplex Red and H2 O2 to fluorescent resorufin by fluorescence microscopy. The initial activity of the permanently immobilized HRP equals up to 45% of the activity that can be expected for an ideal monolayer of HRP molecules on all electrodes of the array. Localization of the immobilizate on the electrodes is accomplished by staining with the fluorescent product of the enzyme reaction. The high residual activity of enzymes after AC field induced immobilization shows the method's suitability for biosensing and research applications.

Glucose Tolerance, Antiprotease Activity and Total Oxidant/Antioxidant Capacity Studies of β-Glucosidase Hybrid Nanoflower for Industrial Applications.

β-Glycosidases, which catalyse the hydrolysis of glycoside bonds, have a wide spectrum of industrial applications. However, the reaction product glucose inhibits the activities of many β-glucosidases. Consequently, the reduced catalytic activities of the enzyme limit the industrial applications of the enzymes. For that reason, the studies dealing with maintaining the activities of the relevant enzymes at high glucose concentrations are a great interest among the researchers. In this context, herein, protein-inorganic hybrid nanoflowers were synthesized using β-glucosidase and copper ion by fast sonication method for 10 min. After characterization of synthesized nanoflowers, pH/temperature studies, glucose tolerance, anti-protease activity, recyclability and total antioxidant and total oxidative capacity levels were estimated. Accordingly, the optimum pHs of free β-glucosidase and hybrid nanoflower (β-GNF) were found to be 6 and 5, respectively, and the optimum temperature values for both hybrid nanoflowers and free enzyme were 40 °C. β-GNF exhibited better activity than free enzyme in low acidic and alkaline environment and at high temperature. The nanoflower retained nearly all (99 %) of its initial activity at all glucose concentrations (0.01, 0.05 and 0.1 mg/mL), especially at pH 5 and 6. Also, β-GNF maintained more than 90 % of initial activity at 0.01 and 0.05 mg/mL glucose at pH 4 and 7. It also displayed about 96 % high residual activity after proteinase K treatment for 3 h at 37 °C, while that of the free β-glucosidase was about 87 %. The reusability studies showed that β-GNF only lost ∼28 % of its initial activities at the end of five cycles. The hybrid nanoflowers at 5 mg/mL concentration exhibited the high total antioxidant capacity. In addition, low total oxidant capacity and oxidative stress index levels were recorded at the same concentration of the hybrid nanoflower. The findings of the present study revealed that β-GNFs may be evaluated as a candidate for various industrial applications due to its high glucose tolerance, anti-protease activity, reusability and resistance to low acidic/alkaline environment and high temperature.

Synthesis and characterizations of sugar-glass nanoparticles mediated protein delivery system for tissue engineering application

The present work focuses on the synthesis and characterization of a sugar-glass nanoparticle (SGnP) based reservoir type protein delivery system pertinent to tissue engineering applications. The SGnP nanocarriers were prepared via inverse micelle of sodium bis(2-ethylhexyl) sulfosuccinate based on an anionic surfactant and subsequent flash-freezing technique. Initially, a total of five different grades of protein-free SGnPs have been prepared to examine the effects of systematic changes in starting concentrations of the aqueous phase, organic solvent, the molar ratio of water, and surfactant in controlling the size, shape, and uniformity of micelles. Evidently, the Fourier transform infrared (FTIR) and scanning electron microscope (SEM) results confirmed that the SGnP can be successfully prepared. Subsequently, SGnP based protein depot has been validated using bovine serum albumin (BSA), horseradish peroxidase (HRP) and growth and differentiation factor-5 (GDF-5). The particle size, morphology, protein encapsulation efficiency and in vitro release kinetics were assessed using SEM, FTIR, UV–visible spectroscopy and Bradford protein assays. Excellent encapsulation efficiency (93%–94%) and sustained release behaviour of BSA (∼22% protein release after 14 d) and GDF-5 proteins (∼29% protein release after 30 d) were exhibited by the optimal grades of SGnP constructs with an average particle size of 266 nm and 93 nm, respectively. Furthermore, FTIR, differential scanning calorimeter (DSC), polyacrylamide gel electrophoresis (PAGE) and NATIVE-PAGE studies results confirm successful encapsulation, stability and preserving the structural integrity of proteins placed into the core of the SGnP constructs. Evidently, a very high (93%) residual HRP enzyme activity signifies the capability of our SGnP system to protect the encapsulated proteins from process-related stresses. In vitro cytotoxicity and fluorescence cell morphology analyses using human adipose-derived mesenchymal stem cells affirmed good cytocompatibility of protein encapsulated SGnP. Overall, the study findings indicate SGnP nanocarrier-mediated protein delivery systems as a promising approach complementary to conventional techniques in tissue engineering and therapeutic applications.

The prevalence of Fabry disease among 1009 unrelated patients with hypertrophic cardiomyopathy: a Russian nationwide screening program using NGS technology

BackgroundThere is a vast number of screening studies described in the literature from the beginning of the twenty-first century to the present day. Many of these studies are related to the estimation of Fabry disease (FD) morbidity among patients from high-risk groups, including adult patients with hypertrophic cardiomyopathy (HCM) and left ventricular hypertrophy (LVH). These studies show diverse detection frequencies (0–12%) depending on the methodology. Our study is the only example of large-scale selective FD screening based on the implementation of next-generation sequencing technology (NGS) as a first-level test to estimate FD morbidity in the Russian population over 18 years of age burdened with HCM.MethodsThe study included 1009 patients (578 males and 431 females), with a median age of 50 years, who were diagnosed with HCM according to current clinical guidelines. In the first stage of screening, all patients underwent molecular genetic testing (NGS method) of target regions. These regions included the coding sequences of 17 genes and mutations that can lead to the development of HCM. Lysosomal globotriaosylsphingosine (lyso-Gb3) concentrations and α-galactosidase A (α-gal A) enzyme activity were measured in the second stage of screening to reveal pathogenic or likely pathogenic variants in the GLA gene.ResultsWe revealed 8 (0.8%) patients (3 (37.5%) males and 5 (62.5%) females) with an average age of 59 ± 13.3 years who had pathogenic, likely pathogenic variants and variants of uncertain significance (VUS) in the GLA gene (NM_000169.2) as a result of selective screening of 1009 Russian patients with HCM. FD was confirmed via biochemical tests in a male with the pathogenic variant c.902G > A, p.R301Q as well as in two females with likely pathogenic variants c.897C > A, p.D299E and c.1287_1288dup, p.*430Fext*?. These tests showed reduced enzymatic activity and increased substrate concentration. However, a female with the pathogenic variant c.416A > G, p.N139S and with normal enzymatic activity only had increased substrate concentrations. The revealed nucleotide variants and high values of biochemical indicators (lyso-Gb3) in these 4 patients allowed us to estimate the FD diagnosis among 1009 Russian patients with HCM. Mild extracardiac manifestations were observed in these four patients; however, both biochemical values within the reference range in females with the c.971T > G, p.L324W (VUS) variant. α-gal A activity and lyso-Gb3 concentrations were also within the normal range in two males with hemizygous variants, c.546T > C, p.D182D and c.640-794_640-791del (we regarded them as VUS), and in one female with the c.427G > A, p.A143T variant (with conflicting interpretations of pathogenicity).ConclusionThe prevalence rate of FD among 1,009 adult Russian patients with HCM was 0.4%. We recommend FD screening among adult patients of both sexes with HCM and an undefined genetic cause via NGS method with subsequent analysis of α-gal A activity and lyso-Gb3 concentration in patients with pathogenic, likely pathogenic variants, and VUS. This strategy identifies patients with an atypical form of FD that is characterized by high residual activity of α-gal A, low concentrations of lyso-Gb3, and minor extracardiac manifestations.

Effect of the morphology and composition of trimetallic PtCuAu/C catalysts on the activity and stability of the methanol oxidation reaction

A study on the influence of the method for obtaining trimetallic PtCuAu/C catalysts on their activity in the oxidation of methanol has been carried out. The structural characteristics of the obtained trimetallic catalysts have been studied by X-ray diffraction and transmission electron microscopy. The nanoparticles of the material obtained by the galvanic synthesis method had a size twice as large (~ 6 nm) than the nanoparticles of the material obtained by the co-deposition of metal precursors. According to the results from the accelerated stress testing of catalysts, it was found that the material obtained by the galvanic method of substitution of copper atoms with gold had a higher residual activity in the oxidation of methanol than the commercial Pt/C analogue. This study shows the potential of obtaining and using multicomponent platinum-containing nanoparticles deposited on a carbon carrier as effective catalysts for use in methanol fuel cells.

Characterization of Cell-Envelope Proteinases from Two Lacticaseibacillus casei Strains Isolated from Parmigiano Reggiano Cheese.

Simple SummaryLactic acid bacteria are nutritionally fastidious microorganisms typically used in the production of fermented dairy foods. The lactic acid bacteria’s proteolytic system is crucial for their growth in milk, and plays a paramount role in developing the organoleptic and healthy properties of fermented dairy foods. Cell-envelope proteinases are the first component of this system, responsible for the degradation of caseins into short peptides. In the present work, the cell-envelope proteinases of two highly proteolytic Lacticaseibacillus casei strains, previously isolated from ripened Parmigiano Reggiano cheese, were characterized from a genetic and biochemical point of view. Two different prt genes existed in the genomes of both strains, but only one, named prtR1, was expressed. PrtR1 proteins extracted from both strains displayed the highest activity at 40 °C and pH 7. Interestingly, PrtR1 extracted from Lacticaseibacillus casei PRA205 retained some residual activity at 5 °C and at pH 4. These important biotechnological features can be exploited in the production of fermented dairy products. Peptidomic analysis revealed that both proteinases were able to release β- and αS1-casein-derived bioactive peptides, suggesting that Lacticaseibacillus casei can be a source of new proteinases that can be exploited for the formulation of dairy beverages or hydrolysates enriched in bioactive peptides.In the present work, two cell-envelope proteinases (CEPs) from Lacticaseibacillus casei strains PRA205 and 2006 were characterized at both the biochemical and genetic levels. The genomes of both L. casei strains included two putative CEPs genes prtP2 and prtR1, but only prtR1 was transcribed. The extracted PrtR1 proteinases were serine proteinases with optimal activity at 40 °C and pH 7.5, and were activated by Ca2+ ions. Interestingly, PrtR1 from L. casei PRA205 exhibited high residual activity at pH 4 and at 5 °C, suggesting its possible exploitation for fermented food production. The caseinolytic activity against αS1- and β-casein indicated that both PrtR1s belonged to the PI/PIII type. These PrtR1s cleaved β-casein peptide bonds preferentially when amino acid M or N was present at the P1 subsite and amino acids A and D were at the P1′ subsite. Several bioactive peptides were found to be released from PrtR1 after αs1- and β-casein hydrolysis.

Biochemical characterization of LysVpKK5 endolysin from a marine vibriophage

Endolysins have been proposed as a potential antibacterial alternative for aquaculture, especially against Vibrio; the bacterial-agents that most frequently cause disease. Although multiple marine vibriophages have been characterized to date, research on vibriophage endolysins is recent. In this study, biochemical characterization of LysVpKK5 endolysin encoded by Vibrio parahaemolyticus-infecting VpKK5 phage was performed. In silico analysis revealed that LysVpKK5 possesses a conserved amidase_2 domain with a zinc-binding motif of high structural similarity to T7 lysozyme (RMSD = 0.107 Å). Contrary to expectations, the activity was inhibited with Zn2+ and was improved with other divalent cations, especially Ca2+. It showed optimal muralytic activity at pH 10, and curiously, no lytic activity at pH ≤ 7 was recorded. As for the thermal stability test, the optimal activity was recorded at 30 °C; the higher residual activity was recorded at 4 °C, and was lost at ≥ 50 °C. On the other hand, increasing NaCl concentrations reduced the activity gradually; the optimal activity was recorded at 50 mM NaCl. On the other hand, the enzymatic activity at 0.5 M NaCl was approx 30% and of approx 50% in seawater. LysVpKK5 endolysin exhibited a higher activity on V. parahaemolyticus ATCC-17802 strain, in comparison with AHPND + strains.

Precision medicine in Fabry disease.

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A (GLA) gene, leading to a deficiency in α-galactosidase A. The lysosomal accumulation of glycosphingolipids, primarily globotriaosylceramide (Gb3) and its deacylated form, globotriaosylsphingosine (lyso-Gb3), results in progressive renal failure, cardiomyopathy associated with cardiac arrhythmia and recurrent cerebrovascular events, significantly limiting life expectancy in affected patients. In male patients, a definitive diagnosis of FD involves demonstrating a GLA deficiency in leucocytes. In females, because of the potential high residual enzymatic activity, the diagnostic gold standard requires molecular genetic analyses. The current treatment options for FD include recombinant enzyme replacement therapies (ERTs) with intravenous agalsidase-α (0.2 mg/kg body weight) or agalsidase-β (1 mg/kg body weight) every 2 weeks as well as an oral pharmacological chaperone (migalastat 123 mg every other day) that selectively and reversibly binds to the active sites of amenable mutant forms of the GLA enzyme. These therapies facilitate cellular Gb3 clearance and an overall improvement of disease burden. However, ERT can lead to infusion-associated reactions, as well as the formation of neutralizing anti-drug antibodies in ∼40% of all ERT-treated males, leading to an attenuation of therapy efficacy. This article reviews the clinical presentation, diagnosis and interdisciplinary clinical management of FD and discusses the therapeutic options, with a special focus on precision medicine, accounting for individual variability in genetic mutations, Gb3 and lyso-Gb3 levels, allowing physicians to predict more accurately which prevention and treatment strategy is best for which patient.

A comprehensive review on molecular mechanism of defective dry-cured ham with excessive pastiness, adhesiveness, and bitterness by proteomics insights.

Excessive bitterness, pastiness, and adhesiveness are the main organoleptic and textural defects of dry-cured ham, which often cause a lot of financial losses to manufacturers and seriously damage the quality of the product. These sensory and textural defects are related to the protein degradation of dry-cured ham. Proteomics shows great potential to improve our understanding of the molecular mechanism of sensory and textural defects and identify biomarkers for monitoring their quality traits. This review presents some of the major achievements and considerations in organoleptic and textural defects of dry-cured ham by proteomics analysis in the recent decades and gives an overview about how to correct sensory and textural defects of dry-cured ham. Proteomics reveals that muscle proteins derived from myofibril and cytoskeleton and involved in metabolic enzymes and oxygen transport have been identified as potential biomarkers in defective dry-cured ham. Relatively high residual activities of cathepsin B and L are responsible for the excessive degradation of these protein biomarkers in defective dry-cured ham. Ultrasound-assisted mild thermal or high-pressure treatment shows a good correction for the organoleptic and textural defects of dry-cured ham by changing microstructure and conformation of muscle proteins by accelerating degradation of proteins and polypeptides into free amino acids.

Health Characteristics of Patients with Cystic Fibrosis whose Genotype Includes a Variant of the Nucleotide Sequence c.3140-16T>A and Functional Analysis of this Variant.

Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease, associated with pathogenic variants in the CFTR gene. The splicing variant c.3140-16T>A (3272-16T>A) has been described previously and, according to the Russian CF Patients Registry, occurs with a frequency of 0.34%. The phenotypic features of CF patients with the c.3140-16T>A variant were compared with those of patients with the genotype F508del/F508del. Patients with the allele c.3140-16T>A had higher average age and age at diagnosis, and the allele was present in a greater proportion of adults. Patients carrying the c.3140-16T>A allele were characterised by better physical development indicators, both in adults and in children, had preserved pancreatic function, as well as the absence of a number of complications, and required pancreatic enzyme replacement therapy less often than patients with the F508del/F508del genotype. Sweat test values also were lower in patients with the c.3140-16T>A genotype. According to the results of clinical and laboratory studies, the phenotype of patients with the genetic variant c.3140-16T>A can be considered “mild”. Functional CFTR protein activity in the presence of c.3140-16T>A was evaluated using intestinal current measurements (ICM) and the forskolin-induced swelling assay on organoids obtained from patients’ rectal biopsies. c.3140-16T>A had high residual CFTR channel activity and was amenable to effective pharmacological correction with thea VX-770 potentiator. To evaluate the effect of the variant on CFTR pre-mRNA splicing we performed a minigene assay, as well as RT-PCR analysis of RNA isolated from the nasal epithelium and rectal biopsy of patients. We showed that the c.3140-16T>A variant creates a novel acceptor AG dinucleotide within CFTR intron 19, resulting in a 14-nucleotide extension of exon 20. This frameshift produces a premature termination codon and triggers mRNA degradation by the nonsense-mediated decay (NMD) mechanism. Moreover, we observed that the c.3140-16T>A allele could produce a residual amount of normally spliced transcript, thus explaining the patient’s mild phenotype.

Functional tests for assessment of residual CFTR channel activity and personalized selection of efficacious CFTR-modulators for cystic fibrosis patients with ‘mild’ and ‘severe’ genetic variants

Intestinal current measurement (ICM) and forskolin-induced swelling (FIS) assay in human intestinal organoids from rectal biopsies of cystic fibrosis (CF) patients are the new functional tests for assessment of CFTR channel activity that are widely used in the leading laboratories worldwide for scientific and clinical studies.The aim of the study was to assess the use of the new functional tests in adult CF patients with identified N1303K and R334W CFTR gene variants.Methods. Rectal suction biopsies were obtained from the two CF patients aged 36 and 27 years with N1303K/3821delT and R334W/F508del CFTR mutations, respectively. Results of the ICM and FIS assay in intestinal organoids were compared to the clinical data.Results. ICM has demonstrated that R334W is a ‘mild’ genetic variant with high residual CFTR channel activity. At the same time, N1303K is a ‘severe’ genetic variant and leads to a severe loss of CFTR channel function. These findings correlate with the clinical data. CFTR modulators compensate for the reduced activity of the R334W CFTR variant, as shown by the FIS assay. But there was a limited response of the forskolin-stimulated organoids to VX-770 potentiator and VX-809 corrector in the cells with N1303K genetic variant.Conclusion. ICM and FIS assay in human intestinal organoids are reliable methods for quantification of CFTR channel activity. They can also predict the efficacy of the targeted therapy in CF patients in vivo.

Fabry Disease: The Current Treatment Landscape.

Fabry disease (FD) is a rare X-linked lysosomal storage disease based on a deficiency of α-galactosidase A (AGAL) caused by mutations in the α-galactosidase A gene (GLA). The lysosomal accumulation of glycosphingolipids, especially globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3, deacylated form), leads to a multisystemic disease with progressive renal failure, cardiomyopathy with potentially malignant cardiac arrhythmias, and strokes, which considerably limits the life expectancy of affected patients. Diagnostic confirmation in male patients is based on the detection of AGAL deficiency in blood leukocytes, whereas in women, due to the potentially high residual enzymatic activity, molecular genetic detection of a causal mutation is required. Current treatment options for FD include recombinant enzyme replacement therapy (ERT) with intravenous agalsidase-alfa (0.2 mg/kg body weight) or agalsidase-beta (1 mg/kg body weight) every 2 weeks and oral chaperone therapy with migalastat (123 mg every other day), which selectively and reversibly binds to the active site of AGAL, thereby correcting the misfolding of the enzyme and allowing it to traffic to the lysosome. These therapies enable cellular Gb3 clearance and improve the burden of disease. However, in about 40% of all ERT-treated men, ERT can lead to infusion-associated reactions and the formation of neutralizing antidrug antibodies, which reduces the efficacy of therapy. In chaperone therapy, there are carriers of amenable mutations that show limited clinical success. This article provides a brief overview of the clinical picture in FD patients, diagnostic confirmation, and interdisciplinary clinical management of FD. The focus is on current and future therapeutic options.

Effects of glass transition and hydration on the biological stability of dry yeast.

The purpose of this study was to determine the effects of glass transition and hydration on the storage stability of baker's dry yeast (Saccharomyces cerevisiae). The glass transition temperature (Tg ) of the yeast decreased with increase in water activity (aw ), and aw at which glass transition occurs at 25 °C was determined as the critical aw (awc ). From mechanical relaxation measurements at 25 °C, the yeast exhibited a large mechanical relaxation above the awc , and the degree of mechanical relaxation increased gradually with increasing aw . This behavior corresponded to a gradual increase in molecular mobility with increasing aw in the rubbery liquid state. Freezable water was observed from aw ≥0.810, and the proportion of freezable water increased with increasing aw . Examination of the effect of aw on the residual biological activity of yeast samples stored at 25 °C for 30 days revealed maximum residual biological activity at aw =0.225 to 0.432. In the lower aw range, the residual biological activity decreased because of oxidation of lipids. In the higher aw range, the residual biological activity decreased gradually with increasing aw . The yeast samples maintained a relatively high residual biological activity, because they could maintain relatively low molecular mobility even in the rubbery liquid state, as suggested by their mechanical relaxation behavior. At aw ≥0.809, residual activity decreased to a negligible value. This could be explained by the appearance of secondary hydrate water (freezable water). Hydrate water protects yeast cells from lipid oxidation but reduces the Tg . As a result, the yeast cells are stabilized maximally only at the awc . PRACTICAL APPLICATION: Although the growth rate of yeast cells becomes negligible below a certain aw , the biological activity of dry yeast decreases gradually during storage. The fact that dry yeast can be maximally stabilized at the awc is practically useful as a criterion for controlling storage stability. In addition, it was found that a remarkable reduction in the molecular mobility, which is otherwise ordinarily increased due to the glass-to-rubber transition, is prevented in yeast. It is possible that the crystallization of amorphous sugar can be prevented by yeast extract. The suggested effect is expected to result in enhanced quality of carbohydrate-based foods.

Radioembolization With Holmium-166 Polylactic Acid Microspheres: Distribution of Residual Activity in the Delivery Set and Outflow Dynamics During Planning and Treatment Procedures

Purpose: To evaluate the microsphere outflow dynamics and residual Ho-166 activity during and after transarterial radioembolization planning and treatment procedures, and to assess the distribution and predilection sites of residual activity in the proprietary delivery set and the microcatheter. Materials and Methods: Fifteen planning and 12 therapeutic radioembolization procedures were performed with poly-l-lactic acid microspheres loaded with Ho-166. The amount and distribution of residual activity was assessed by dose calibrator measurements and SPECT imaging. The activity flow profile from the microcatheter was assessed dynamically. For planning procedures, different injection methods were evaluated in order to attempt to decrease the residual activity. Results: The median residual activities for planning and treatment procedures using standard injection methods were 31.2% (range 17.3%–44.1%) and 4.3% (range 3.5%–6.9%), respectively. Planning residual activities could be decreased significantly with 2 injection methods similar to treatment procedures, to 17.5% and 10.9%, respectively (P = 0.002). Main predilection sites of residual microspheres were the 3-way stopcock and the outflow needle connector. During treatment procedures, more than 80% of the injected activity is transferred during the first 3 injection cycles. Conclusion: After treatment procedures with holmium-loaded microspheres, mean residual activity in the delivery set is reproducibly low and between reported values for glass and resin microspheres. The majority of microspheres is transferred to the patient during the second and third injection cycle. An estimated residual waste of 3% to 4% may be included in the treatment activity calculation. For planning procedures, a modified injection technique should be used to avoid high residual activities.

Immobilized enzyme-driven value enhancement of lignocellulosic-based agricultural byproducts: Application in aroma synthesis

We have combined the advantages of immobilization and spouted bed drying to enhance lipase activity and stability for further use of this enzyme in esterification. Through covalent binding, Cercospora kikuchii lipase was immobilized onto agricultural byproducts activated with glutaraldehyde, sodium periodate, or epichlorohydrin. The immobilized derivatives showed high lipase activity retention (from 83.8 to 116.9%) and low residual moisture (average of 3.96%) and water activity (average of 0.19). The kinetic parameters KM and Vmax of the free lipase were 0.212 mM and 160.3 μmol/min/mg, respectively; lipase immobilized onto the support activated with 1.5% glutaraldehyde had lower KM (0.121 mM) and higher Vmax (275.4 μmol/min/mg). Lipase immobilized onto rice husk retained 60.3% of its initial enzyme activity after five reuse cycles, and its residual activity was high 88.7% after storage at 5 °C for six months. This immobilized derivative system was used to catalyze butyl butyrate synthesis. Under the best conditions for butyl butyrate production (temperature of 50 °C and enzyme concentration of 0.5 g), the esterification yields reached 95.0% and afforded a butyl butyrate concentration of 15.2 g/L. The enzyme stability, high activity retention after reuse cycles, and ability to catalyze butyl butyrate synthesis from butyric acid strongly indicate that the innovative one-step immobilization protocol reported herein is feasible for lipase immobilization onto the surface of eco-friendly supports.

Carryover de herbicidas utilizados no controle de soqueiras do algodoeiro sobre o milho cultivado em sucessão

São limitadas as informações sobre a atividade residual de herbicidas aplicados no controle das soqueiras do algodoeiro e o seu potencial para afetar o desenvolvimento e a produtividade do milho semeado na safra seguinte. O objetivo desse trabalho foi identificar o efeito carryover promovido pela aplicação única e sequencial de herbicidas utilizados no controle das soqueiras do algodoeiro e estimar o intervalo de segurança entre o manejo químico da soqueira do algodoeiro e a semeadura do milho. Foram conduzidos simultaneamente dois experimentos em casa de vegetação, um para aplicação única e outro para aplicação sequencial dos tratamentos herbicidas. Os experimentos foram instalados em esquema fatorial 15 x 5, delineados em blocos casualizados com quatro repetições. O primeiro fator foi composto por tratamentos herbicidas e o segundo fator por cinco épocas de semeadura do milho após a aplicação (0, 30, 60, 90 e 120 dias). As parcelas experimentais foram compostas de vasos plásticos com capacidade para 3 dm3 preenchidos com solo de textura argilosa (470 g kg-1 de argila), seco e peneirado. Os herbicidas utilizados foram 2,4-D, glyphosate, saflufenacil, [imazapic+imazapyr], dicamba, fluroxypyr e sulfentrazone. Conclui-se que a aplicação sequencial da maioria dos tratamentos proporcionou um aumento na atividade residual dos herbicidas avaliados. Os tratamentos 2,4-D (1340 g ha-1), 2,4-D + glyphosate (1340 + 720 g ha-1) e 2,4-D + glyphosate + saflufenacil (1340 + 720 + 105 g ha-1) apresentaram os menores intervalos de segurança e foram aqueles que apresentaram o menor risco de afetar o milho semeado em sucessão, mesmo em aplicações sequenciais. Contudo, os tratamentos constituídos pela associação com herbicidas de maior atividade residual no solo como o dicamba, fluroxypyr, sulfentrazone e [imazapic+imazapyr], apresentaram maior potencial de carryover para a cultura do milho, sendo observado intervalo de segurança superior a 94 dias após a aplicação sequencial de 2,4-D + glyphosate + sulfentrazone.

Non-thermal Processing of Pineapple (Ananas comosus [L.] Merr.) Juice Using Continuous Pressure Change Technology (PCT): Effects on Physical Traits, Microbial Loads, Enzyme Activities, and Phytochemical Composition

A comprehensive study using continuous pressure change technology (PCT) for the non-thermal processing of fresh pineapple juice on pilot scale was conducted (1 L/min, 50 MPa, argon, 3 min, <35 °C). The immediate effects of a single and a twofold PCT treatment on the most important quality parameters were examined and compared with those of fresh and thermally pasteurised (90 °C) juices. In comparison to the fresh juice, both PCT-treated samples exhibited slightly brighter and less yellowish colour (CIE L*a*b*). A significant reduction in the mean particle size resulted in diminished centrifugable pulp contents and enhanced cloud stability. Moreover, a slightly improved microbial quality (−0.9 to −1.2 log10 CFU/mL) in terms of total aerobic and yeast and mould counts was attained. Noteworthy, PCT retained a high bromelain activity (−3 to −15% reduction) and efficiently inactivated polyphenol oxidase. Water-soluble vitamins, phenolic compounds, and all further constituents assessed were mostly preserved. However, the high residual peroxidase activity (−10 to −23%) and microbial loads are likely to affect juice quality during storage. In contrast, thermal pasteurisation ensured a complete reduction in both microbial counts (−4.4 to −4.5 log10 CFU/mL) and effective inactivation of peroxidase. However, bromelain activity was strongly affected (−83%) by heat treatment, and colour was darkened and even less yellowish. Overall, this study highlighted the potential of PCT for the production of fresh-like pineapple juices; however, its current limitations were revealed as well.

Development of l-asparaginase@hybrid Nanoflowers (ASNase@HNFs) Reactor System with Enhanced Enzymatic Reusability and Stability

Hybrid nanoflowers materials have recently received great attention in enzyme immobilization applications because of the advantages such as their large surface area, excellent stability, simple, eco-friendly, and cost-effective synthesis. In this study, l-asparaginase which is an important commercial enzyme in the medicine and food industry was selected as a model enzyme. To the best of our knowledge, this study is the first report of designing l-asparaginase@hybrid nanoflowers to enhance its enzymatic performance. l-asparaginase@hybrid nanoflowers were synthesized using ASNase as an organic component and Cu(II) ion as inorganic component. They were characterized by their morphology and chemical point of view by using different techniques. The synthesized l-asparaginase@hybrid nanoflowers exhibited high residual activity at broad pH and high temperature ranges in comparison to free form. Moreover, l-asparaginase@hybrid nanoflowers possessed good reusability and excellent long-time storage stability. Especially, l-asparaginase@hybrid nanoflowers-3 maintained nearly 51 and 75% of its original activity, respectively, after nine consecutive catalytic cycles and storage at 30 °C for 4 weeks. The results indicated that these hybrid nanoflowers will be promising carrier matrix for the immobilization of ASNase in biotechnological applications with improved catalytic properties.

Screening of macromolecular cross-linkers and food-grade additives for enhancement of catalytic performance of MNP-CLEA-lipase of hevea brasiliensis

Skim latex from Hevea brasiliensis (rubber tree) consist of many useful proteins and enzymes that can be utilized to produce value added products for industrial purposes. Lipase recovered from skim latex serum was immobilized via cross-linked enzyme aggregates (CLEA) technology, while supported by magnetic nanoparticles for properties enhancement, termed ‘Magnetic Nanoparticles CLEA-lipase’ (MNP-CLEA-lipase). MNP-CLEA-lipase was prepared by chemical cross-linking of enzyme aggregates with amino functionalized magnetic nanoparticles. Instead of using glutaraldehyde as cross-linking agent, green, non-toxic and renewable macromolecular cross-linkers (dextran, chitosan, gum Arabic and pectin) were screened and the best alternative based on highest residual activity was chosen for further analysis. Same goes for additives; the traditional use of expensive BSA was replaced with low cost, food grade additives (egg protein, soy protein and milk protein). The optimum condition parameters such as concentration of cross-linker, concentration of additive and concentration of precipitant were determined using one factor at a time (OFAT) method and structural analysis was done by FTIR. The screening results showed that pectin was the best cross-linker (118 % residual activity) and soy protein was the best additive (129 % residual activity) for MNP-CLEA-lipase with optimum conditions of 180 mg/ml pectin, 0.6 % w/v soy protein and 80 % saturation ammonium sulphate. FTIR of MNP-CLEA-lipase showed an Amide I and Amide V bands which originated from silanized magnetic nanoparticles (MNP) and a new band, Amide II was formed prior to the successful CLEA immobilization of lipase enzyme with MNPs.