Nano-hydroxyapatite-assisted enzyme-induced carbonate precipitation enhances Pb-contaminated aqueous solution and loess remediation.

Intensive agricultural activities could cause lead (Pb) bioaccumulation, threatening human health. Although the enzyme-induced carbonate precipitation (EICP) technology has been applied to tackle the aforesaid problem, the urease may denature or even lose its activity when subjected to a significant Pb2+ toxicity effect. To this end, the nano-hydroxyapatite (nHAP)-assisted EICP was proposed to reduce the mobility of Pb2+. Results indicated that a below 30% immobilization efficiency at 60 mM Pb2+ was attained under EICP. nHAP adsorbed the majority of Pb2+, preventing Pb2+ attachment to urease. Further, hydroxylphosphohedyphane or hydroxylpyromorphite was formed at 60 mM Pb2+, followed by the formation of cerussite, allowing hydroxylphosphohedyphane or hydroxylpyromorphite to be wrapped by cerussite. By contrast, carbonate-bearing hydroxylpyromorphite of higher stability (Pb10(PO4)6CO3) was developed at 20 mM Pb2+ as CO3 2- substituted the hydroxyl group in hydroxylpyromorphite. Moreover, nHAP helped EICP to form nucleated minerals. As a result, the EICP-nHAP technology raised the immobilization efficiency at 60 mM Pb2+ up to 70%. The findings highlight the potential of applying the EICP-nHAP technology to Pb-containing water bodies remediation.

Structures, gelatinization properties and enzyme hydrolyses of starches from transparent and floury grains of rices subjected to field natural extreme high temperature.

Three rice varieties underwent the field natural extreme high temperature (EHT) with daily average temperature over 30 °C from 21 to 89 days after sowing, and had transparent, chalky and floury grains. The structures, gelatinization properties and enzyme hydrolyses of starches from transparent and floury grains were investigated. Compared with control transparent grains, floury grains subjected to EHT markedly decreased the contents of amylose molecules, amylopectin A chains and amylopectin B1 chains and increased the contents of amylopectin B2 and B3+ chains and the average branch-chain length of amylopectin. Both transparent and floury grains had A-type starches, but floury grain starches exhibited higher relative crystallinity, gelatinization temperature, retrogradation and pasting viscosities than transparent grain starches. Floury grain starches had lower hydrolysis rates than transparent grain starches. Native starches were more resistant to digestion but gelatinized and retrograded starches were more prone to digestion in floury grains than in transparent grains.

Nucleophilic hydrolysis of dichloroacetonitrile and trichloroacetonitrile disinfection byproducts by peroxymonosulfate: Kinetics and mechanisms.

In this work, it was found that peroxymonosulfate (PMS) could appreciably accelerate the transformation rates of dichloroacetonitrile (DCAN) and trichloracetonitrile (TCAN) in aqueous solutions, especially under alkaline pHs. The impact of reactive oxygen species scavengers (methyl alcohol for sulfate radical, tert-butyl alcohol for hydroxyl radical, and azide for singlet oxygen) and water matrices (chloride (Cl-), bicarbonate (HCO3-), and natural organic matter (NOM)) on DCAN and TCAN transformation by PMS is evaluated, revealing negligible effects. A nucleophilic hydrolysis pathway, as opposed to an oxidation process, was proposed for the transformation of DCAN and TCAN by PMS, supported by the hydrolyzable characteristics of these compounds and validated through density functional theory calculations. Kinetic analysis indicated that the transformation of DCAN and TCAN by PMS adhered to a second-order kinetic law, with higher reaction rates observed at elevated pH levels within the range of 7.0-10.0. Kinetic modeling incorporating the hydrolytic contributions of water, hydroxyl ion, and protonated and deprotonated PMS (i.e., HSO5- and SO52-) effectively fitted the experimental data. Species-specific second-order rate constants reveal that SO52- exhibited significantly higher reactivity towards DCAN ((1.69 ± 0.22) ×104 M-1h-1) and TCAN((6.06 ± 0.18) ×104 M-1h-1) compared to HSO5- ((2.14 ± 0.12) ×102 M-1h-1) for DCAN; and (1.378 ± 0.11) ×103 M-1h-1 for TCAN). Comparative analysis of DCAN and TCAN transformation efficiencies by four different oxidants indicated that PMS rivaled chlorine but falls short of hydrogen peroxide, with peroxydisulfate displaying negligible reactivity. Overall, this study uncovers the nucleophilic hydrolysis characteristics of PMS, supplementing its recognized role as an oxidant precursor or mild oxidant, and underscores its significant implications for environmental remediation.

Enhanced wastewater treatment with an AnF-AAO system for improved internal carbon source utilization.

The main challenge in removing nutrients from municipal wastewater in China is the lack of available carbon sources. While hydrolysis acidification tanks can improve wastewater biodegradability by effectively utilizing internal carbon sources, high sludge concentrations are difficult to control in traditional tank variants. In this study, an innovative anaerobic filter (AnF) hydrolysis acidification reactor composed of a continuously stirred tank reactor (CSTR) and cloth media filter was designed to regulate and maintain high sludge concentrations in the hydrolysis acidifier. The reactor was used as a pretreatment unit for the anaerobic/anoxic/oxic (AAO) units and combined into an AnF-AAO system to explore the effectiveness of internal carbon source utilization in wastewater. The results indicate that as the sludge concentration in the hydrolysis acidifier increased, the hydrolysis and acidification processes became more efficient. The optimal sludge concentration was 40 g/L, which significantly increased the production of soluble chemical oxygen demand and volatile fatty acids. Above this concentration, the efficiency decreased. Compared to traditional AAO processes, the AnF-AAO system achieved superior total nitrogen and phosphorus removal with shorter hydraulic retention times and reduced sludge production by a significant amount of 35%. Due to its capacity for enhancing internal carbon source utilization, the AnF-AAO system constitutes a promising approach for sustainable urban wastewater treatment.

Angiotensin-Converting Enzyme and Renin-Inhibitory Activities of Protein Hydrolysates Produced by Alcalase Hydrolysis of Peanut Protein.

Hypertension is a major controllable risk factor associated with cardiovascular disease (CVD) and overall mortality worldwide. Most people with hypertension must take medications that are effective in blood pressure management but cause many side effects. Thus, it is important to explore safer antihypertensive alternatives to regulate blood pressure. In this study, peanut protein concentrate (PPC) was hydrolyzed with 3-5% Alcalase for 3-10 h. The in vitro angiotensin-converting enzyme (ACE) and renin-inhibitory activities of the resulting peanut protein hydrolysate (PPH) samples and their fractions of different molecular weight ranges were determined as two measures of their antihypertensive potentials. The results show that the crude PPH produced at 4% Alcalase for 6 h of hydrolysis had the highest ACE-inhibitory activity with IC50 being 5.45 mg/mL. The PPH samples produced with 3-5% Alcalase hydrolysis for 6-8 h also displayed substantial renin-inhibitory activities, which is a great advantage over the animal protein-derived bioactive peptides or hydrolysate. Remarkably higher ACE- and renin-inhibitory activities were observed in fractions smaller than 5 kDa with IC50 being 0.85 and 1.78 mg/mL. Hence, the PPH and its small molecular fraction produced under proper Alcalase hydrolysis conditions have great potential to serve as a cost-effective anti-hypertensive ingredient for blood pressure management.

Upcycling soybean meal through enzymatic conversion of insoluble fiber into soluble dietary fiber enhanced by ball milling.

Insoluble dietary fiber (IDF) in soybean meal, due to the insolubility, is one of the major impediments to upcycle the soybean meal for its value-added use. This study converted IDF to soluble dietary fiber (SDF) using ball milling and enzymatic hydrolysis of the IDF. The impact of ball milling and enzymatic hydrolysis on the physicochemical and functional properties of SDF was evaluated. Cellulase, hemicellulase, xylanase, galacturonase, and arabinofuranosidase were employed for hydrolyzing IDF. The results showed that ball milling significantly reduced the particle size of IDF, facilitating enhanced enzymatic hydrolysis and resulting in SDF with lower molecular weight and varied monosaccharide composition. The synergistic effect of ball milling and enzymatic processes with combination of cellulase-xylanase-galacturonase was evident by the improved conversion rates (69.8%) and altered weight-averaged molecular weight (<5900 Da) of the resulting SDF. Rheological and microstructural analyses of the SDF gel indicated that specific enzyme combinations led to SDF gels with distinct viscoelastic properties, pore sizes, and functional capabilities, suitable for varied applications in the food and pharmaceutical sectors. This comprehensive evaluation demonstrates the potential of optimized physical bioprocessing techniques in developing functional ingredients with tailored properties for industrial use.

Enhanced anaerobic digestion of waste-activated sludge by thermal-alkali pretreatment: a pilot-scale study.

The composition of waste-activated sludge (WAS) is complex, containing a large amount of harmful substances, which pose a threat to the environment and human health. The reduction and resource utilization of sludge has become a development demand in sludge treatment and disposal. Based on the technical bottlenecks in the practical application of direct anaerobic digestion technology, this study adopted two different thermal and thermal-alkali hydrolysis technologies to pretreat sludge. A pilot-scale experiment was conducted to investigate the experimental conditions, parameters, and effects of two hydrolysis technologies. This study showed that the optimal hydrolysis temperature was 70 °C, the hydrolysis effect and pH can reach equilibrium with the hydrolysis retention time was 4-8 h, and the optimal alkali concentration range was 0.0125-0.015 kg NaOH/kg dry-sludge. Thermal-alkali combination treatment greatly improved the performance of methane production, the addition of NaOH increased methane yield by 31.2% than that of 70 °C thermal hydrolysis. The average energy consumption is 75 kWh/m3 80% water-content sludge during the experiment. This study provides a better pretreatment strategy for exploring efficient anaerobic digestion treatment technologies suitable for southern characteristic sewage sludge.

Synergistic enzymatic mechanism of lepidolite leaching enhanced by a mixture of Bacillus mucilaginosus and Bacillus circulans.

Numerous studies have demonstrated that the co-leaching of ores by different silicate bacteria significantly improves the performance of bioleaching systems. Nevertheless, the mechanism of different silicate bacteria synergistically or complementarily enhanced the leaching process of lithium-containing silicate remains unclear. This study discussed the leaching impact of the combined presence of two metabolically distinct silicate bacteria on lepidolite, with the aim of comprehending the synergistic effect resulting from the presence of Bacillus mucilaginosus and Bacillus circulans in the leaching process. The results indicated that the polysaccharides and proteins secreted by bacteria-containing functional groups such as -OH and -COOH, which played an important role in the complex decomposition of ores. Organic acids played the role of acid etching and complexation. Bacillus mucilaginosus and Bacillus circulans exhibited low individual leaching efficiency, primarily due to their weak organic acid secretion. Moreover, the prolific polysaccharide production by Bacillus mucilaginosus led to bacterial aggregation, diminishing contact capability with minerals. Bacillus circulans decomposed the excessive polysaccharides produced by Bacillus mucilaginosus through enzymatic hydrolysis in the co-bioleaching process, providing later nutrient supply for both strains. The symbiosis of the two strains enhanced the synthesis and metabolic capabilities of both strains, resulting in increased organic acid secretion. In addition, protein and humic acid production by Bacillus mucilaginosus intensified, collectively enhancing the leaching efficiency. These findings suggested that the primary metabolic products secreted by different bacterial strains in the leaching process differ. The improvement in bioleaching efficiency during co-leaching was attributed to their effective synergistic metabolism. This work contributes to the construction of an efficient engineering microbial community to improve the efficiency of silicate mineral leaching, and reveals the feasibility of microbial co-culture to improve bioleaching.

Recent Advances and Perspectives on Food-Grade Immobilisation Systems for Enzymes.

The use of enzyme immobilisation is becoming increasingly popular in beverage processing, as this method offers significant advantages, such as enhanced enzyme performance and expanded applications, while allowing for easy process termination via simple filtration. This literature review analysed approximately 120 articles, published on the Web of Science between 2000 and 2023, focused on enzyme immobilisation systems for beverage processing applications. The impact of immobilisation on enzymatic activity, including the effects on the chemical and kinetic properties, recyclability, and feasibility in continuous processes, was evaluated. Applications of these systems to beverage production, such as wine, beer, fruit juices, milk, and plant-based beverages, were examined. The immobilisation process effectively enhanced the pH and thermal stability but caused negative impacts on the kinetic properties by reducing the maximum velocity and Michaelis-Menten constant. However, it allowed for multiple reuses and facilitated continuous flow processes. The encapsulation also allowed for easy process control by simplifying the removal of the enzymes from the beverages via simple filtration, negating the need for expensive heat treatments, which could result in product quality losses.

Effect of Hydrochloric Acid Hydrolysis under Sonication and Hydrothermal Process to Produce Cellulose Nanocrystals from Oil Palm Empty Fruit Bunch (OPEFB).

This paper presents an approach for hydrolyzing cellulose nanocrystals from oil palm empty fruit bunch (OPEFB) presented through hydrochloric acid hydrolysis under sonication-hydrothermal conditions. Differences in concentration, reaction time, and acid-to-cellulose ratio affect toward the yield, crystallinity, microstructure, and thermal stability were obtained. The highest yield of cellulose nanocrystals up to 74.82%, crystallinity up to 78.59%, and a maximum degradation temperature (Tmax) of 339.82 °C were achieved through hydrolysis using 3 M HCl at 110 °C during 1 h. X-ray diffraction analysis indicated a higher diffraction peak pattern at 2θ = 22.6° and a low diffraction peak pattern at 2θ = 18°. All cellulose nanocrystals showed a crystalline size of under 1 nm, and it was indicated that the sonication-hydrothermal process could reduce the crystalline size of cellulose. Infrared spectroscopy analysis showed that a deletion of lignin and hemicellulose was demonstrated in the spectrum. Cellulose nanocrystal morphology showed a more compact structure and well-ordered surface arrangement than cellulose. Cellulose nanocrystals also had good thermal stability, as a high maximum degradation temperature was indicated, where CNC-D1 began degrading at temperatures (T0) of 307.09 °C and decomposed (Tmax) at 340.56 °C.

Effect of Glycerol and Sisal Nanofiber Content on the Tensile Properties of Corn Starch/Sisal Nanofiber Films.

Currently, petroleum-derived plastics are widely used despite the disadvantage of their long degradation time. Natural polymers, however, can be used as alternatives to overcome this obstacle, particularly cornstarch. The tensile properties of cornstarch films can be improved by adding plant-derived nanofibers. Sisal (Agave sisalana), a very common low-cost species in Brazil, can be used to obtain plant nanofibers. The goal of this study was to obtain sisal nanofibers using low concentrations of sulfuric acid to produce thermoplastic starch nanocomposite films. The films were produced by a casting technique using commercial corn starch, glycerol, and sisal nanofibers, accomplished by acid hydrolysis. The effects of glycerol and sisal nanofiber content on the tensile mechanical properties of the nanocomposites were investigated. Transmission electron microscopy findings demonstrated that the lowest concentration of sulfuric acid produced fibers with nanometric dimensions related to the concentrations used. X-ray diffraction revealed that the untreated fibers and fibers subjected to acid hydrolysis exhibited a crystallinity index of 61.06 and 84.44%, respectively. When the glycerol and nanofiber contents were 28 and 1%, respectively, the tensile stress and elongation were 8.02 MPa and 3.4%. In general, nanocomposites reinforced with sisal nanofibers showed lower tensile stress and higher elongation than matrices without nanofibers did. These results were attributed to the inefficient dispersion of the nanofibers in the polymer matrix. Our findings demonstrate the potential of corn starch nanocomposite films in the packaging industry.

Impaired ATP hydrolysis in blood plasma contributes to age-related neutrophil dysfunction.

BACKGROUND: The function of polymorphonuclear neutrophils (PMNs) decreases with age, which results in infectious and inflammatory complications in older individuals. The underlying causes are not fully understood. ATP release and autocrine stimulation of purinergic receptors help PMNs combat microbial invaders. Excessive extracellular ATP interferes with these mechanisms and promotes inflammatory PMN responses. Here, we studied whether dysregulated purinergic signaling in PMNs contributes to their dysfunction in older individuals. RESULTS: Bacterial infection of C57BL/6 mice resulted in exaggerated PMN activation that was significantly greater in old mice (64 weeks) than in young animals (10 weeks). In contrast to young animals, old mice were unable to prevent the systemic spread of bacteria, resulting in lethal sepsis and significantly greater mortality in old mice than in their younger counterparts. We found that the ATP levels in the plasma of mice increased with age and that, along with the extracellular accumulation of ATP, the PMNs of old mice became increasingly primed. Stimulation of the formyl peptide receptors of those primed PMNs triggered inflammatory responses that were significantly more pronounced in old mice than in young animals. However, bacterial phagocytosis and killing by PMNs of old mice were significantly lower than that of young mice. These age-dependent PMN dysfunctions correlated with a decrease in the enzymatic activity of plasma ATPases that convert extracellular ATP to adenosine. ATPases depend on divalent metal ions, including Ca2+, Mg2+, and Zn2+, and we found that depletion of these ions blocked the hydrolysis of ATP and the formation of adenosine in human blood, resulting in ATP accumulation and dysregulation of PMN functions equivalent to those observed in response to aging. CONCLUSIONS: Our findings suggest that impaired hydrolysis of plasma ATP dysregulates PMN function in older individuals. We conclude that strategies aimed at restoring plasma ATPase activity may offer novel therapeutic opportunities to reduce immune dysfunction, inflammation, and infectious complications in older patients.

The flavour of wheat gluten hydrolysate after Corynebacterium Glutamicum fermentation: Effect of degrees of hydrolysis and fermentation time.

Corynebacterium glutamicum was used to ferment wheat gluten hydrolysates (WGHs) to prepare flavour base. This study investigated the effect of hydrolysis degrees (DHs) and fermentation time on flavour of WGHs. During fermentation, the contents of amino nitrogen, total acid and small peptides increased, while the protein and pH value decreased. Succinic acid, GMP, and Glu were the prominent umami substances in fermented WGHs. The aromas of WGHs with different DHs could be distinguished by electronic nose and GC-IMS. Based on OAV of GC-MS, hexanal was the main compound in WGHs, while phenylethyl alcohol and acetoin were dominant after fermentation. WGHs with high DHs accumulated more flavour metabolites. Correlation analysis showed that small peptides (<1 kDa) could promote the formation of flavour substances, and Asp was potentially relevant flavour precursor. This study indicated that fermented WGHs with different DHs can potentially be used in different food applications based on flavour profiles.

Valorization of fish processing by-products for protein hydrolysate recovery: Opportunities, challenges and regulatory issues.

Protein-rich fish processing by-products, often called rest raw materials (RRM), account for approximately 60% of the total fish biomass. However, a considerable amount of these RRM is utilized for low-value products such as fish meal and silage. A promising and valuable approach for maximizing the utilization of RRM involves the extraction of bioactive fish protein hydrolysate (FPH). This review assesses and compares different hydrolyzation methods to produce FPH. Furthermore, the review highlights the purification strategy, nutritional compositions, and bioactive properties of FPH. Finally, it concludes by outlining the application of FPH in food products together with various safety and regulatory issues related to the commercialization of FPH as a protein ingredient in food. This review paves the way for future applications by highlighting efficient biotechnological methods for valorizing RRM into FPH and addressing safety concerns, enabling the widespread utilization of FPH as a valuable and sustainable source of protein.

Immunostimulatory Effect of Ovomucin Hydrolysates by Pancreatin in RAW 264.7 Macrophages via Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway.

Ovomucin (OM), which has insoluble fractions is a viscous glycoprotein, found in egg albumin. Enzymatic hydrolysates of OM have water solubility and bioactive properties. This study investigated that the immunostimulatory effects of OM hydrolysates (OMHs) obtained by using various proteolytic enzymes (Alcalase®, bromelain, α-chymotrypsin, Neutrase®, pancreatin, papain, Protamax®, and trypsin) in RAW 264.7 cells. The results showed that OMH prepared with pancreatin (OMPA) produced the highest levels of nitrite oxide in RAW 264.7 cells, through upregulation of inducible nitric oxide synthase mRNA expression. The production of pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-6 were increased with the cytokines mRNA expression. The effect of OMPA on mitogen-activated protein kinase signaling pathway was increased the phosphorylation of p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase in a concentration-dependent manner. Therefore, OMPA could be used as a potential immune-stimulating agent in the functional food industry.

Comprehensive study on pilot nitrification-sludge fermentation coupled denitrification system with extended sludge retention time.

The sludge fermentation-coupled denitrification process, utilized for sludge reduction and nitrogen removal from wastewater, is frequently hindered by its hydrolysis step's efficacy. This study addresses this limitation by extending the sludge retention time (SRT) to 120 days. As a result, the nitrate removal efficiency (NRE) of the nitrification-sludge fermentation coupled denitrification (NSFD) pilot system increased from 67.1 ± 0.2 % to 96.7 ± 0.1 %, and the sludge reduction efficiency (SRE) rose from 40.2 ± 0.5 % to 62.2 ± 0.9 %. Longer SRT enhanced predation and energy dissipation, reducing intact cells from 99.2 % to 78.0 % and decreasing particle size from 135.2 ± 4.6 µm and 19.4 ± 2.1 µm to 64.5 ± 3.5 µm and 15.5 ± 1.6 µm, respectively. It also created different niches by altering the biofilm's adsorption capacity, with interactions between these niches driving improved performance. In conclusion, extending SRT optimized the microbial structure and enhanced the performance of the NSFD system.

Complex Formation and Hydrolytic Processes of Protected Peptides Containing the -SXH- Motif in the Presence of Nickel(II) Ion.

Interactions between metal ions and proteins are considered reversible, such as the coordination of a metal ion to a protein or enzyme, but irreversible processes like the oxidative reactions, aggregation or hydrolytic processes may occur. In the presence of Ni(II)-ions selective hydrolysis of the peptides containing the -SXH- or -TXH- motif was observed. Since the side chain of histidine serves as the metal ion binding site for many native proteins, and very often histidine is present in a -SXH- or -TXH- sequence, to study the complex formation and hydrolytic processes in presence of nickel(II) ion four peptides were synthesised: Ac-SKHM-NH2, A3SSH-NH2, A4SSH-NH2, AAAeKSH-NH2. The Ni(II)-induced hydrolysis of Ac-SKHM-NH2 peptide occurs rapidly in alkaline medium already at room temperature. In two peptides containing -SSH- sequence on the C-termini, the N-terminal part is the major binding site for the nickel(II) ion, but the formation of dinuclear complexes was also observed. In the [Ni2LH-6]2- complex of hexapeptide, the coordination sphere of the metal ions is saturated with deprotonated Ser-O-, which does not result in hydrolysis of the peptide. For A4SSH-NH2, both Ni(II) ions fulfill the conditions for hydrolysis, which was confirmed by HPLC analyses at pH ~ 8.2 and 25 °C.

A composite enzyme derived from papain and chymotrypsin reduces the Allergenicity of Cow's Milk allergen casein by targeting T and B cell epitopes.

Casein, the major allergen in cow's milk, presents a significant challenge in providing nutritional support for children with allergies. To address this issue, we investigated a composite enzyme, comprising papain and chymotrypsin, to reduce the allergenicity of casein. Enzymatic hydrolysis induced substantial structural changes in casein, diminishing its affinity for specific IgE and IgG antibodies. Additionally, in a BALB/c mouse model, casein hydrolysate alleviated allergic symptoms, evidenced by lower serum IgE and IgG levels, reduced plasma histamine, and decreased Th2 cytokine release during cell co-culture. Peptidomic analysis revealed a 52.38% and 60% reduction in peptides containing IgE epitopes in casein hydrolyzed by the composite enzyme compared to papain and chymotrypsin, respectively, along with a notable absence of previously reported T cell epitopes. These results demonstrate the potential of enzyme combinations to enhance the efficiency of epitope destruction in allergenic proteins, providing valuable insights into the development of hypoallergenic dairy products.

Illuminating a biologics development challenge: systematic characterization of CHO cell-derived hydrolases identified in monoclonal antibody formulations.

Monoclonal antibodies (mAb) and other biological drugs are affected by enzymatic polysorbate (PS) degradation that reduces product stability and jeopardizes the supply of innovative medicines. PS represents a critical surfactant stabilizing the active pharmaceutical ingredients, which are produced by recombinant Chinese hamster ovary (CHO) cell lines. While the list of potential PS-degrading CHO host cell proteins (HCPs) has grown over the years, tangible data on industrially relevant HCPs are still scarce. By means of a highly sensitive liquid chromatography-tandem mass spectrometry method, we investigated seven different mAb products, resulting in the identification of 12 potentially PS-degrading hydrolases, including the strongly PS-degrading lipoprotein lipase (LPL). Using an LPL knockout CHO host cell line, we were able to stably overexpress and purify the remaining candidate hydrolases through orthogonal affinity chromatography methods, enabling their detailed functional characterization. Applying a PS degradation assay, we found nine mostly secreted, PS-active hydrolases with varying hydrolytic activity. All active hydrolases showed a serine-histidine-aspartate/glutamate catalytical triad. Further, we subjected the active hydrolases to pH-screenings and revealed a diverse range of activity optima, which can facilitate the identification of residual hydrolases during bioprocess development. Ultimately, we compiled our dataset in a risk matrix identifying PAF-AH, LIPA, PPT1, and LPLA2 as highly critical hydrolases based on their cellular expression, detection in purified antibodies, active secretion, and PS degradation activity. With this work, we pave the way toward a comprehensive functional characterization of PS-degrading hydrolases and provide a basis for a future reduction of PS degradation in biopharmaceutical drug products.

Neokestose suppresses the increase in plasma glucose caused by oral administration of sucrose in a streptozotocin­induced diabetic rat.

Neokestose is considered to have a prebiotic function. However, the physiological activity of neokestose remains unknown. Neokestose has a blastose, a sucrose analog, in its structure. We previously demonstrated that oral administration of blastose to diabetic rats suppressed the increase in plasma glucose (PG) concentration after sucrose administration. Therefore, neokestose might have a similar effect. In this study, we investigated the effects of neokestose on PG concentrations and the mechanism of its action. We first administered neokestose orally to streptozotocin-induced diabetic rats and observed that the expected consequent increase in PG concentration was significantly suppressed. Next, we examined the inhibitory effect of neokestose on glycosidase activity, but observed only a slight inhibitory effect. Therefore, we hypothesized that neokestose might be hydrolyzed by gastric acid to produce blastose. We performed an acid hydrolysis of neokestose using artificial gastric juice. After acid hydrolysis, peaks corresponding to neokestose and its decomposition products including blastose were observed. Therefore, we suggest that neokestose and blastose, a decomposition product, synergistically inhibit glycosidase activity. These findings support the potential use of neokestose as a useful functional oligosaccharide that can help manage plasma glucose concentrations in patients with diabetes mellitus.