Soluble Enzyme Research Articles

Protein tyrosine phosphatase 1B (PTP1B) function, structure, and inhibition strategies to develop antidiabetic drugs.

Tyrosine protein phosphatase non-receptor type 1 (PTP1B; also known as protein tyrosine phosphatase 1B) is a member of the protein tyrosine phosphatase (PTP) family and is a soluble enzyme that plays an essential role in different physiological processes, including the regulation of metabolism, specifically in insulin and leptin sensitivity. PTP1B is crucial in the pathogenesis of type 2 diabetes mellitus and obesity. These biological functions have made PTP1B validated as an antidiabetic and anti-obesity, and potentially anticancer, molecular target. Four main approaches aim to inhibit PTP1B: orthosteric, allosteric, bidentate inhibition, and PTPN1 gene silencing. Developing a potent and selective PTP1B inhibitor is still challenging due to the enzyme's ubiquitous expression, subcellular location, and structural properties. This article reviews the main advances in the study of PTP1B since it was first isolated in 1988, as well as recent contextual information related to the PTP family to which this protein belongs. Furthermore, we offer an overview of the role of PTP1B in diabetes and obesity, and the challenges to developing selective, effective, potent, bioavailable, and cell-permeable compounds that can inhibit the enzyme.

Role of neuraminidase in promoting the shedding of endothelial mechanosensing structures in type 2 diabetes

Endothelial dysfunction represents a causal factor in the pathogenesis of cardiovascular disease associated with type 2 diabetes (T2D). Previous work shows that endothelial mechanosensing structures are degraded in T2D, resulting in impaired shear stress mechanotransduction and decreased endothelial-derived nitic oxide (NO) bioavailability. Recent evidence suggests that glypican-1, a well-known mechanosensor, is a substrate of the proinflammatory enzyme a disintegrin and metalloproteinase-17 (ADAM17). A key step in ADAM17 activation is externalization of phosphatidylserine (PS) to the outer leaflet of the plasma membrane, which can be mediated by the phospholipid scramblase anoctamin-6 (ANO6). However, whether ANO6-mediated activation of ADAM17 leads to glypican-1 shedding in endothelial cells remains unknown. Also unknown are the mechanisms by which this pathway becomes overactive in T2D. We recently reported that T2D is associated with increased circulating levels of neuraminidase, a soluble enzyme that cleaves sialic acid residues. We also showed that intraluminal exposure of isolated arteries to neuraminidase impairs shear stress mechanotransduction. Herein, we tested the hypothesis that neuraminidase-induced impaired shear stress mechanotransduction is mediated by ANO6-induced ADAM17 activation and consequent shedding of glypican-1. All reported differences are significant at P<0.05. In support of our hypothesis, we show that neuraminidase and ADAM17 activities are increased, while nitrite (NO surrogate) is decreased, in plasma of men and women with T2D who also display evidence of impaired shear stress mechanotransduction ( i.e., diminished flow-mediated dilation). In cultured endothelial cells, we show that neuraminidase exposure decreases sialic acid content and increases intracellular Ca2+ mobilization, adhesion of lactadherin (a peptide that binds externalized PS), ADAM17 activity, and shedding of glypican-1. Also in cultured endothelial cells, overexpression of ADAM17 increases shedding of glypican-1 and impairs shear stress-induced activation of endothelial NO synthase. Congruently, intraluminal exposure of human omental arteries to active ADAM17 recombinant protein impairs flow-mediated dilation. Furthermore, silencing of ANO6 reduces ADAM17 activity, leading to increased presence of glypican-1 on the endothelial cell surface. Taken together, we provide evidence that neuraminidase, an enzyme increased in the circulation of individuals with T2D, promotes ANO6-dependent externalization of PS in endothelial cells, which in turn leads to ADAM17 activation and consequent shedding of glypican-1. Thus, this work supports the idea that the neuraminidase-ANO6-ADAM17 axis should be considered as a potential target for restoring endothelial mechanosensation and improving NO bioavailability in T2D. R01HL151384 (to LAML and JP), R01HL153264 (to LAML and JP), R01HL137769 (to JP), and R21DK116081 (to CM-A). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Methylomonas defluvii sp. nov., a type I methane-oxidizing bacterium from a secondary sedimentation tank of a wastewater treatment plant.

An aerobic methanotroph was isolated from a secondary sedimentation tank of a wastewater treatment plant and designated strain OY6T. Cells of OY6T were Gram-stain-negative, pink-pigmented, motile rods and contained an intracytoplasmic membrane structure typical of type I methanotrophs. OY6T could grow at a pH range of 4.5-7.5 (optimum pH 6.5) and at temperatures ranging from 20 °C to 37 °C (optimum 30 °C). The major cellular fatty acids were C14 : 0, C16 : 1ω7c/C16 : 1ω6c and C16 : 1ω5c; the predominant respiratory quinone was MQ-8. The genome size was 5.41 Mbp with a DNA G+C content of 51.7 mol%. OY6T represents a member of the family Methylococcaceae of the class Gammaproteobacteria and displayed 95.74-99.64 % 16S rRNA gene sequence similarity to the type strains of species of the genus Methylomonas. Whole-genome comparisons based on average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) confirmed that OY6T should be classified as representing a novel species. The most closely related type strain was Methylomonas fluvii EbBT, with 16S rRNA gene sequence similarity, ANI by blast (ANIb), ANI by MUMmer (ANIm) and dDDH values of 99.64, 90.46, 91.92 and 44.5 %, respectively. OY6T possessed genes encoding both the particulate methane monooxygenase enzyme and the soluble methane monooxygenase enzyme. It grew only on methane or methanol as carbon sources. On the basis of phenotypic, genetic and phylogenetic data, strain OY6T represents a novel species within the genus Methylomonas for which the name Methylomonas defluvii sp. nov. is proposed, with strain OY6T (=GDMCC 1.4114T=KCTC 8159T=LMG 33371T) as the type strain.

Fulvic acid foliar application: a novel approach enhancing antioxidant capacity and nutritional quality of pistachio (Pistacia vera L.)

BackgroundThe global growth of pistachio production has prompted exploration into sustainable agricultural practices, on the application of humic substances such as fulvic acid in enhancing the quality of horticultural crops. The present study was carried out in Qom province, Iran, on 20 years old pistachio (Pistacia vera L. cv. Kaleh-Ghoochi) trees and investigated the impact of foliar spraying of fulvic acid at varying concentrations (1.5, 3, and 4.5 g L− 1) on the antioxidant and quality properties of pistachio. The different concentrations of fulvic acid were applied at two key stages: at the initiation of pistachio kernel formation (late June) and the development stage of pistachio kernel (late August), as well as at both time points. Following harvest at the horticulturally mature phase, various parameters, including total phenols, flavonoids, soluble proteins, soluble carbohydrate content, antioxidant capacity, and antioxidant enzyme activity, were assessed.ResultsResults indicated that foliar application of fulvic acid, particularly at 1.5 g L− 1 during both late June and August, effectively increased phenolic compounds (31.8%) and flavonoid content (24.53%). Additionally, this treatment also augmented antioxidant capacity and heightened the activity of catalase (CAT) (37.56%), ascorbate peroxidase (APX) (63.86%), and superoxide dismutase (SOD) (76.45%). Conversely, peroxidase (POX) (41.54%) activity was reduced in fulvic acid-treated nuts compared with controls. Moreover, the content of chlorophyll (45%) and carotenoids (46.7%) was enhanced using this organic fertilizer. In terms of mineral elements, the increment was observed in zinc (Zn) (58.23%) and potassium (K) (28.12%) amounts in treated nuts. Additionally, foliar application of fulvic acid led to elevated levels of soluble carbohydrates and proteins in treated nuts.ConclusionsIn the present study, application of fulvic acid resulted in enhancement of antioxidant activity and quality traits of pistachio nut through an increase in total phenol, flavonoids, chlorophyll, carotenoids, K, Zn, and also activity of antioxidant enzymes. Therefore, use of fulvic acid emerges as a promising strategy to enhance the quality and nutritional attributes of pistachios, contributing to sustainable agricultural practices and improved crop outcomes.

Broad-spectrum pH functional chitosan-phosphatase beads for the generation of plant-available phosphorus: utilizing the insoluble P pool.

Utilization of organic phosphates and insoluble phosphates for the gradual generation of plant-available phosphorus (P) is the only sustainable solution for P fertilization. Enzymatic conversions are one of the best sustainable routes for releasing P to soil. Phosphatase enzyme aids in solubilizing organic and insoluble phosphates to plant-available P. We herein report the preparation of highly functional chitosan beads co-immobilized with acid phosphatase and alkaline phosphatase enzymes via a glutaraldehyde linkage. The dual enzyme co-immobilized chitosan beads were characterized using Fourier-transform infrared (FTIR), thermogravimetric (TGA), and scanning electron microscopy-energy dispersive x-ray (SEM-EDX) analyses to confirm the immobilization. The co-immobilized system was found to be active for a broader pH range of ∼4-10 than the individually bound enzymes and mixed soluble enzymes. The bound matrix exhibited pH optima at 6 and 9, respectively, for acid and alkaline phosphatase and a temperature optimum at 50°C. The phosphate-solubilizing abilities of the chitosan-enzyme derivatives were examined using insoluble tri-calcium phosphate (TCP) for wide pH conditions of 5.5, 7, and 8.5 up to 25 days. The liberation of phosphate was highest (27.20mg/mL) at pH 5.5 after the defined period. The residual soil phosphatase activity was also monitored after 7days of incubation with CBE for three different soils of pH ∼5.5, 7, and 8.5. The residual phosphatase activity increased for all the soils after applying the CBE. The germination index of the Oryza sativa (rice) plant was studied using different pH buffer media upon the application of the CBE in the presence of tri-calcium phosphate as a phosphate source. Overall, the dual-enzyme co-immobilized chitosan beads were highly effective over a wide pH range for generating plant-available phosphates from insoluble phosphates. The chitosan-enzyme derivative holds the potential to be used for sustainable phosphorus fertilization with different insoluble and organic phosphorus sources.

The trade-off between root growth redundancy and premature senescence under different straw returning modes affects boll formation and seedcotton yield

The indeterminate growth habit of cotton (Gossypium hirsutum L.) makes a coordinated rootshoot relationship critical for increasing yield, which requires comprehensive consideration of root growth redundancy and premature senescence. Although recent evidence suggests that straw management affects root growth and thus seedcotton yield, there is limited documentation on the dynamics of root physiological activity and the rootshoot relationship of cotton under different straw returning modes. The present study compared four treatments: straw removal (CK), straw mulching (SM), straw incorporation (SI), and strawderived biochar incorporation (SBI) in an 8year field experiment. The growth dynamics of roots, shoots and reproductive organs, root physiological activity and seedcotton yield were investigated. The results showed that longterm straw application significantly enhanced seedcotton yield, but the effect varied significantly with straw returning modes. The seedcotton yield increased by 30.5%, 17.8% and 1.8% under SBI, SI and SM respectively in the 8th year of straw management, compared to CK. SBI and SI significantly enhanced the biomass of cotton roots and shoots while had higher growth rate of shoots relative to roots from peak squaring stage to peak bollsetting stage. This reduced root growth redundancy and increased the boll loading and boll capacity of root system. Besides, SBI increased the root activity, soluble protein content and antioxidant enzyme activities, and reduced the root malondialdehyde content from peak bollsetting stage to boll opening stage. These resulted in the greatest increase in boll number (53.862.1%) and boll biomass (39.480.4%) at the boll opening stage under SBI compared to CK. Overall, strawderived biochar improved the boll retention capacity of cotton roots by balancing the growth redundancy and premature senescence of the root system, and was a more suitable straw returning strategy to promote boll formation and increase cotton yield.

Various Strategies for the Immobilization of a Phospholipase C from Bacillus cereus for the Modulation of Its Biochemical Properties.

In this study, the effect of various immobilization methods on the biochemical properties of phospholipase C (PLC) from Bacillus cereus obtained from the oily soil located in Sfax, Tunisia, was described. Different supports were checked: octyl sepharose, glyoxyl agarose in the presence of N-acetyl cysteine, and Q-sepharose. In the immobilization by hydrophobic adsorption, a hyperactivation of the PLCBc was obtained with a fold of around 2 times. The recovery activity after immobilization on Q-sepharose and glyoxyl agarose in the presence of N-acetyl cysteine was 80% and 58%, respectively. Furthermore, the biochemical characterization showed an important improvement in the three immobilized enzymes. The performance of the various immobilized PLCBc was compared with the soluble enzyme. The derivatives acquired using Q-sepharose, octyl sepharose, and glyoxyl agarose were stable at 50 °C, 60 °C, and 70 °C. Nevertheless, the three derivatives were more stable in a large range of pH than the soluble enzyme. The three derivatives and the free enzyme were stable in 50% (v/v) ethanol, hexane, methanol, and acetone. The glyoxyl agarose derivative showed high long-term storage at 4 °C, with an activity of 60% after 19 days. These results suggest the sustainable biotechnological application of the developed immobilized enzyme.

Plasma Soluble Angiotensin-Converting Enzyme 2 and Risk of Recurrent Stroke: A Nested Case-Control Analysis

Introduction: The angiotensin-converting enzyme 2 (ACE-2) and its shedding product (soluble ACE-2 [sACE-2]) are implicated in adverse cardiovascular outcomes. However, the relationship between sACE-2 and stroke recurrence is unknown. Herein, we examined the relationship of sACE-2 with stroke recurrence in patients with ischemic stroke or transient ischemic attack. Methods: Data were obtained from the Third China National Stroke Registry (CNSR-III). Eligible cases consisted of 494 patients who developed recurrent stroke within 1-year follow-up, and 494 controls were selected using age- and sex-matched with a 1:1 case-control ratio. Conditional logistic regressions were used to evaluate the association between sACE-2 and recurrent stroke. The main outcomes were recurrent stroke within 1 year. Results: Among 988 patients included in this study, the median (interquartile range) of sACE-2 was 25.17 (12.29–45.56) ng/mL. After adjustment for conventional confounding factors, the odds ratio (OR) with 95% confidence interval (CI) in the highest quartile versus the lowest quartile was 1.68 (1.12–2.53) for recurrent stroke within 1-year follow-up. Subgroup analysis showed that the association between elevated plasma level of sACE-2 and stroke recurrence was significant in patients with higher systemic inflammation, as indicated by high-sensitivity C-reactive protein ≥ 2 mg/L (adjusted OR: 2.33 [95% CI, 1.15–4.72]) and neutrophil counts ≥ median (adjusted OR: 2.66 [95% CI, 1.35–5.23]) but not significant in patients with lower systemic inflammation. Discussion/Conclusion: Elevated plasma sACE-2 concentration was associated with increased risk of recurrent stroke.

Improving the stability of an unstable lipase by applying different immobilization strategies for the selective hydrolysis of fish oil

AbstractRhizopus oryzae lipase (ROL) is known to present high selectivity in chemical reactions. However, the poor stability of ROL effectively limits its industrial applications. In this study, several immobilization protocols, such as hydrophobic adsorption, covalent immobilization, multi‐point covalent attachment, ionic adsorption/cross‐linking, and ionic interaction, were applied to improve the stability of ROL. Heterogeneous modification of aspartic and glutamic acid residues on the surface of ROL was carried out by 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC) to introduce new amine groups with lower pKb. The highest immobilization yield of 89% was achieved for octyl‐agarose, producing specific activity of 45 U/mg, which is 15 folds higher than the specific activity of the soluble enzyme. Improved stability of ROL was observed, in particular for those derivatives obtained by multi‐point covalent attachment of ROL on glyoxyl‐agarose (Gx‐ROL) and aminated ROL on glyoxyl‐agarose (Gx‐NH2‐ROL) by retaining 28%–36% of their initial activities after 24 h incubation at 60°C. Immobilization also altered the co‐solvent stability profile of the immobilized derivatives producing biocatalysts with varied co‐solvent stabilities. Furthermore, utilization of the immobilized preparations in fish oil hydrolysis revealed the selective release of cis‐5,8,11,14,17‐eicosapentaenoic acid (EPA) and cis‐4,7,10,13,16,19‐docosahexaenoic acid (DHA) in favor of EPA. The highest EPA/DHA selectivity of 33 was observed for the hydrophobically immobilized ROL on octyl‐sepharose.

The Relationship between Soluble Angiotensin-Converting Enzyme Level and Coronavirus Disease 2019 in Children: A Prospective Cohort Study

Abstract Objective Our objective was to investigate soluble angiotensin-converting enzyme (sACE) levels in pediatric patients with coronavirus disease 2019 (COVID-19) and to identify factors associated with the occurrence and severity of pediatric COVID-19. Methods This was a prospective cohort study conducted between April 2020 and July 2020. The study population consisted of 143 children (between 1 month and 18 years old), 103 of whom had COVID-19 and 40 of whom were negative for COVID-19 (randomly selected). The sACE levels and other laboratory data of all participants were measured at admission (day 0, baseline). Repeat measurements were performed in patients on the 5th day. Disease severity was documented at baseline and on the 5th day, and the change in severity between these time points was recorded. Results Age and sex distribution were similar in the two groups. At baseline, 31 (30.1%) of the patients were asymptomatic, 58 (56.3%) had mild disease, and 14 (13.6%) had moderate disease. Baseline sACE levels were similar in the groups (p = 0.120). Higher weight was independently associated with low sACE levels in children (p = 0.037). The sACE level of patients on the 5th day was significantly lower compared with baseline (p = 0.007). Patients who experienced a decrease in disease severity were compared with those who did not demonstrate a decrease. Baseline sACE levels were significantly lower in those who experienced decreased severity (p = 0.039). Multiple linear regression revealed that COVID-19 severity at baseline was independently associated with the low sACE level at baseline (p = 0.023). Conclusion Lower sACE at diagnosis was associated with COVID-19 severity in children. However, no strong evidence was found that could suggest the sACE level as an important predictor for the occurrence or severity of COVID-19 in children.

UCST-Type Soluble Immobilized Cellulase: A New Strategy for the Efficient Degradation and Improved Recycling Performance of Wastepaper Cellulose.

This paper reports an innovative study that aims to address key issues in the efficient recycling of wastepaper cellulose. The research team utilized the temperature-responsive upper critical solution temperature (UCST) polymer P(NAGA-b-DMA) in combination with the LytA label's affinity for choline analogs. This innovative approach enabled them to successfully develop a novel soluble immobilized enzyme, P(NAGA-b-DMA)-cellulase. This new enzyme has proven highly effective, significantly enhancing the degradation of wastepaper cellulose while demonstrating exceptional stability. Compared with the traditional insoluble immobilized cellulase, the enzyme showed a significant improvement in the pH, temperature stability, recycling ability, and storage stability. A kinetic parameter calculation showed that the enzymatic effectiveness of the soluble immobilized enzyme was much better than that of the traditional insoluble immobilized cellulase. After the immobilization reaction, the Michaelis constant of the immobilized enzyme was only increased by 11.5%. In the actual wastepaper degradation experiment, the immobilized enzyme was effectively used, and it was found that the degradation efficiency of wastepaper cellulose reached 80% of that observed in laboratory conditions. This novel, thermosensitive soluble immobilized cellulase can efficiently catalyze the conversion of wastepaper cellulose into glucose under suitable conditions, so as to further ferment into environmentally friendly biofuel ethanol, which provides a solution to solve the shortage of raw materials and environmental protection problems in the paper products industry.

Resilient Enzymes through Immobilisation: Stable NDP Polyphosphate Phosphotransferase from Ruegeria pomeroyi for Nucleotide Regeneration

Immobilisation plays an important role in the industrial application of enzymes. The stabilisation and reusability of immobilised enzymes reduce the cost of the catalyst and facilitate their use in continuously operated reactors. For this purpose, an applicable type of immobilisation needs to be identified. In this study, we investigate the conversion of CDP and PolyP to CTP by NDP polyphosphate phosphotransferase 3 from Ruegeria pomeroyi (RpPPK2-3) and describe the covalent immobilisation of RpPPK2-3. In order to select a suitable carrier for the immobilisation of RpPPK2-3, a screening with different amino methacrylate (glutaraldehyde-pre-activated) and epoxy methacrylate carriers was carried out. The epoxy methacrylate carrier ECR8209M (Purolite®) was found to be the most suitable. With a half-life of 462 d when stored at 6 °C and a 50-fold reusability with a 93% residual activity, the immobilised enzyme showed a higher stability compared to the soluble enzyme with a half-life of 0.04 d. Although the half-life of the soluble enzyme could be increased to 32 d by adding PPi, it could not reach the stability of the immobilisate. Due to the resilience of the immobilisate, it is suitable for application in continuous reactor set-ups, e.g., packed-bed reactors.

Engineering a "muco-trapping" ACE2-immunoglobulin hybrid with picomolar affinity as an inhaled, pan-variant immunotherapy for COVID-19.

Soluble angiotensin-converting enzyme 2 (ACE2) can act as a decoy molecule that neutralizes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by blocking spike (S) proteins on virions from binding ACE2 on host cells. Based on structural insights of ACE2 and S proteins, we designed a "muco-trapping" ACE2-Fc conjugate, termed ACE2-(G4S)6-Fc, comprised of the extracellular segment of ACE2 (lacking the C-terminal collectrin domain) that is linked to mucin-binding IgG1-Fc via an extended glycine-serine flexible linker. ACE2-(G4S)6-Fc exhibits substantially greater binding affinity and neutralization potency than conventional full length ACE2-Fc decoys or similar truncated ACE2-Fc decoys without flexible linkers, possessing picomolar binding affinity and strong neutralization potency against pseudovirus and live virus. ACE2-(G4S)6-Fc effectively trapped fluorescent SARS-CoV-2 virus like particles in fresh human airway mucus and was stably nebulized using a commercial vibrating mesh nebulizer. Intranasal dosing of ACE2-(G4S)6-Fc in hamsters as late as 2 days postinfection provided a 10-fold reduction in viral load in the nasal turbinate tissues by Day 4. These results strongly support further development of ACE2-(G4S)6-Fc as an inhaled immunotherapy for COVID-19, as well as other emerging viruses that bind ACE2 for cellular entry.

(133) Investigating Actinidin as a Potential Collagenase on Human Peyronie’s Disease Cells

Abstract Introduction Peyronie's Disease (PD) is characterized by a connective tissue disorder in the penile tunica albuginea and the formation of collagen-rich plaques, leading to penile curvature, painful erections, and erectile dysfunction. Intralesional Collagenase Clostridium histolyticum injections are no longer available in Canada, making treatment options for PD limited. Actinidin is a soluble enzyme that has the ability to hydrolyze various types of collagen and fibrinogen. We have previously reported the efficacy of actinidin on reducing collagen in in vitro human PD models. Objective To determine the dose-response efficacy and cytotoxicity of actinidin in reducing the intercellular bound collagen in human PD plaque models. Methods Fibroblast cells are isolated from human PD tissue (N ≥ 3) and 2D models are cultured. These cells were subjected to various treatment groups including media-only control and 5 concentrations of actinidin treatment ranging from 0.5 mg/ml to 30 mg/ml. The collagenase treatment was prepared from Hayward kiwi by isolating actinidin by filtration and pH adjustment. A dose-response curve was generated at different time points at various actinidin concentrations using an MTT assay. After 24 hours of treatment, collagen extraction was performed and quantified using a Soluble Collagen Quantification Assay Kit and a fluorescent microplate reader. The effects of the treatments on the extracellular matrix were further investigated by staining the cells with DAPI for the nucleus and Phalloidin-conjugated FITC for actin filaments. A twelve-hour timelapse was conducted using a fluorescent confocal microscope to capture FITC and DAPI fluorescein images. The total fluorescent area of each well was measured, and actin signals were normalized by the DAPI signals. Results The dose-response curve revealed that the lethal concentration affecting 50% of the cells after a 48-hour period is approximately 15-20 mg/ml. Treatment of 1 mg/mL actinidin significantly reduced cellular collagen of human PD fibroblasts compared to the control group (P < 0.001). The normalized stained β-actin signal was measured as the ratio between intracellular actin staining and nuclei count in each microscopic field. The high concentration actinidin treatment (30 mg/mL) exhibited a significantly higher normalized actin signal compared to other treatment groups (P < 0.0001). These findings suggest that actinidin may possess mechanisms that affect the plasma membrane integrity. Conclusions Our continuation of preliminary results demonstrates the ability of actinidin to break down PD cells by compromising extracellular collagen and the cellular membrane. High doses of actinidin cause cell destruction and we suggest that treatment should not exceed 10 mg/ml. Further studies will investigate the molecular mechanism of actinin in hydrolyzing collagen and to evaluate the effects on an animal model. Given the absence of durable FDA-approved treatments for PD in Canada, this novel option holds future treatment potential. Disclosure Yes, this is sponsored by industry/sponsor: KiwiEnzyme.com Ltd. Clarification: No industry support in study design or execution.

State of the Art Technologies for High Yield Heterologous Expression and Production of Oxidoreductase Enzymes: Glucose Oxidase, Cellobiose Dehydrogenase, Horseradish Peroxidase, and Laccases in Yeasts P. pastoris and S. cerevisiae

Oxidoreductase (OXR) enzymes are in high demand for biocatalytic applications in the food industry and cosmetics (glucose oxidase (GOx) and cellobiose dehydrogenase (CDH)), bioremediations (horseradish peroxidase (HRP) and laccase (LAC)), and medicine for biosensors and miniature biofuel cells (GOx, CDH, LAC, and HRP). They can be used in a soluble form and/or within the yeast cell walls expressed as chimeras on the surface of yeast cells (YSD), such as P. pastoris and S. cerevisiae. However, most of the current studies suffer from either low yield for soluble enzyme expression or low enzyme activity when expressed as chimeric proteins using YSD. This is always the case in studies dealing with the heterologous expression of oxidoreductase enzymes, since there is a requirement not only for multiple OXR gene integrations into the yeast genome (super transformations), and codon optimization, but also very careful design of fermentation media composition and fermentation conditions during expression due to the need for transition metals (copper and iron) and metabolic precursors of FAD and heme. Therefore, scientists are still trying to find the optimal formula using the above-mentioned approaches; most recently, researcher started using protein engineering and directed evolution to increase in the yield of recombinant enzyme production. In this review article, we will cover all the current state-of-the-art technologies and most recent advances in the field that yielded a high expression level for some of these enzymes in specially designed expression/fermentation systems. We will also tackle and discuss new possibilities for further increases in fermentation yield using cutting-edge technologies such as directed evolution, protein and strain engineering, high-throughput screening methods based on in vitro compartmentalization, flow cytometry, and microfluidics.

Targeting Efficient Features of Urate Oxidase to Increase Its Solubility.

With the demand for mass production of protein drugs, solubility has become a serious issue. Extrinsic and intrinsic factors both affect this property. A homotetrameric cofactor-free urate oxidase (UOX) is not sufficiently soluble. To engineer UOX for optimum solubility, it is important to identify the most effective factor that influences solubility. The most effective feature to target for protein engineering was determined by measuring various solubility-related factors of UOX. A large library of homologous sequences was obtained from the databases. The data was reduced to six enzymes from different organisms. On the basis of various sequence- and structure-derived elements, the most and the least soluble enzymes were defined. To determine the best protein engineering target for modification, features of the most and least soluble enzymes were compared. Metabacillus fastidiosus UOX was the most soluble enzyme, while Agrobacterium globiformis UOX was the least soluble. According to the comparison-constant method, positive surface patches caused by arginine residue distribution are appropriate targets for modification. Two Arg to Ala mutations were introduced to the least soluble enzyme to test this hypothesis. These mutations significantly enhanced the mutant's solubility. While different algorithms produced conflicting results, it was difficult to determine which proteins were most and least soluble. Solubility prediction requires multiple algorithms based on these controversies. Protein surfaces should be investigated regionally rather than globally, and both sequence and structural data should be considered. Several other biotechnological products could be engineered using the data reduction and comparison-constant methods used in this study.

Campylobacter jejuni Surface-Bound Protease HtrA, but Not the Secreted Protease nor Protease in Shed Membrane Vesicles, Disrupts Epithelial Cell-to-Cell Junctions.

Fundamental functions of the intestinal epithelium include the digestion of food, absorption of nutrients, and its ability to act as the first barrier against intruding microbes. Campylobacter jejuni is a major zoonotic pathogen accounting for a substantial portion of bacterial foodborne illnesses. The germ colonizes the intestines of birds and is mainly transmitted to humans through the consumption of contaminated poultry meat. In the human gastrointestinal tract, the bacterium triggers campylobacteriosis that can progress to serious secondary disorders, including reactive arthritis, inflammatory bowel disease and Guillain-Barré syndrome. We recently discovered that C. jejuni serine protease HtrA disrupts intestinal epithelial barrier functions via cleavage of the tight and adherens junction components occludin, claudin-8 and E-cadherin. However, it is unknown whether epithelial damage is mediated by the secreted soluble enzyme, by HtrA contained in shed outer-membrane vesicles (OMVs) or by another mechanism that has yet to be identified. In the present study, we investigated whether soluble recombinant HtrA and/or purified OMVs induce junctional damage to polarized intestinal epithelial cells compared to live C. jejuni bacteria. By using electron and confocal immunofluorescence microscopy, we show that HtrA-expressing C. jejuni bacteria trigger efficient junctional cell damage, but not soluble purified HtrA or HtrA-containing OMVs, not even at high concentrations far exceeding physiological levels. Instead, we found that only bacteria with active protein biosynthesis effectively cleave junctional proteins, which is followed by paracellular transmigration of C. jejuni through the epithelial cell layer. These findings shed new light on the pathogenic activities of HtrA and virulence strategies of C. jejuni.

Immobilization of α-amylase onto functionalized molybdenum diselenide nanoflowers (MoSe2-NFs) as scaffolds: Characterization, kinetics, and potential applications in starch-based industries

The current study presents the application of molybdenum diselenide nanoflowers (MoSe2-NFs) as an innovative substrate for immobilizing α-amylase by glutaraldehyde activation. This approach results in the development of a nanobiocatalyst that exhibits remarkable advantages compared to a standalone enzyme. Several physical methods, such as fluorescence microscopy, FT-IR, SEM, TEM, XRD, AFM, and Raman spectroscopy, were used to confirm that α-amylase was successfully attached to MoSe2-NFs. By employing the Box-Behnken design of the RSM, the parameters were optimized, resulting in an immobilization efficiency of roughly 87.33%. The immobilized variant of α-amylase demonstrated superior thermostability, pH stability, reusability, and storage stability in comparison to the soluble enzyme. The catalytic activity of α-amylase was highest when immobilized on MoSe2-NFs at the same pH and temperature as the soluble enzyme. However, there was an expansion in the range of parameters in which this activity was observed. Furthermore, the immobilized enzyme exhibited a retention of nearly 80% residual activity following 12 successive reuses. The immobilized enzyme exhibited around 82% residual activity after being stored for 120 days. It is possible that the immobilization process changed the Michaelis-Menten constant, which means that the substrate could no longer reach certain active sites on the enzyme because it had become longer. The study's findings suggest that the α-amylase-MoSe2-NFs system could be useful in industry because it can work in a wider range of temperature and pH conditions. Furthermore, the intrinsic non-toxic characteristics of the matrix, along with its ability to be kept for prolonged periods and recycled, render nano biocatalysts very well-suited for the effective synthesis of maltose in the food and pharmaceutical industries.

Enhancing salinity tolerance in cucumber through Selenium biofortification and grafting

BackgroundSalinity stress is a major limiting factor for plant growth, particularly in arid and semi-arid environments. To mitigate the detrimental effects of salinity stress on vegetable production, selenium (Se) biofortification and grafting onto tolerant rootstocks have emerged as effective and sustainable cultivation practices. This study aimed to investigate the combined effects of Se biofortification and grafting onto tolerant rootstock on the yield of cucumber grown under salinity stress greenhouse conditions. The experiment followed a completely randomized factorial design with three factors: salinity level (0, 50, and 100 mM of NaCl), foliar Se application (0, 5, and 10 mg L-1 of sodium selenate) and grafting (grafted and non-grafted plants) using pumpkin (Cucurbita maxima) as the rootstock. Each treatment was triplicated.ResultsThe results of this study showed that Se biofortification and grafting significantly enhanced salinity tolerance in grafted cucumbers, leading to increased yield and growth. Moreover, under salinity stress conditions, Se-Biofortified plants exhibited increased leaf relative water content (RWC), proline, total soluble sugars, protein, phenol, flavonoids, and antioxidant enzymes. These findings indicate that Se contributes to the stabilization of cucumber cell membrane and the reduction of ion leakage by promoting the synthesis of protective compounds and enhancing antioxidant enzyme activity. Moreover, grafting onto pumpkin resulted in increased salinity tolerance of cucumber through reduced Na uptake and translocation to the scion.ConclusionIn conclusion, the results highlight the effectiveness of Se biofortification and grafting onto pumpkin in improving cucumber salinity tolerance. A sodium selenate concentration of 10 mg L-1 is suggested to enhance the salinity tolerance of grafted cucumbers. These findings provide valuable insights for the development of sustainable cultivation practices to mitigate the adverse impact of salinity stress on cucumber production in challenging environments.

Characterisation and beneficial effects of a Lupinus angustifolius protein hydrolysate obtained by immobilisation of the enzyme alcalase®.

Bioactive peptides have been considered potential components for the future functional foods and nutraceuticals generation. The enzymatic method of hydrolysis has several advantages compared to those of chemical hydrolysis and fermentation. Despite this fact, the high cost of natural and commercial proteases limits the commercialization of hydrolysates in the food and pharmacological industries. For this reason, more efficient and economically interesting techniques, such as the immobilisation of the enzyme, are gaining attention. In the present study, a new protein hydrolysate from Lupinus angustifolius was generated by enzymatic hydrolysis through the immobilisation of the enzyme alcalase® (imLPH). After the chemical and nutritional characterization of the imLPH, an in vivo study was carried out in order to evaluate the effect of 12 weeks treatment with imLPH on the plasmatic lipid profile and antioxidant status in western-diet-fed apolipoprotein E knockout mice. The immobilisation of alcalase® generated an imLPH with a degree of hydrolysis of 29.71 ± 2.11%. The imLPH was mainly composed of protein (82.50 ± 0.88%) with a high content of glycine/glutamine, arginine, and aspartic acid/asparagine. The imLPH-treatment reduced the amount of abdominal white adipose tissue, total plasma cholesterol, LDL-C, and triglycerides, as well as the cardiovascular risk indexes (CRI) -I, CRI-II, and atherogenic index of plasma. The imLPH-treated mice also showed an increase in the plasma antioxidant capacity. For the first time, this study demonstrates the beneficial in vivo effect of a lupin protein hydrolysate obtained with the alcalase® immobilised and points out this approach as a possible cost-effective solution at the expensive generation of the hydrolysate through the traditional batch conditions with soluble enzymes.