Efficient expression of OUC-Sb-lip2 in Yarrowia lipolytica and its comprehensive utilization in the enrichment of DHA and EPA from fish oil.

Lipases are widely used in the modification of functional lipids, particularly in the enrichment of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). In this study, a lipase named OUC-Sb-lip2 was expressed in Yarrowia lipolytica, achieving a promising enzyme activity of 472.6 U/mL by optimizing the culture medium, notably through olive oil supplementation. A significant proportion (58.8%) of the lipase activity was located in the cells, whereas 41.2% was secreted into the supernatant. Both whole-cell and immobilized OUC-Sb-lip2 were used to enrich DHA and EPA from fish oil. The whole-cell approach increased the DHA and EPA contents to 2.59 and 2.55 times that of the original oil, respectively. Similarly, the immobilized OUC-Sb-lip2 resulted in a 2.00-fold increase in DHA and an 1.99-fold increase in EPA after a 6-h hydrolysis period. Whole cell and the immobilized OUC-Sb-lip2 retained 48.7% and 52.7% of their activity after six cycles of reuse, respectively.

Safety evaluation of an extension of use of the food enzyme triacylglycerol lipase from the non-genetically modified Aspergillus luchuensis strain AE-L.

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the non-genetically modified Aspergillus luchuensis strain AE-L by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in one food manufacturing process. Subsequently, the applicant has requested to extend its use to include four additional processes and to revise the previous use level. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of five food manufacturing processes. The dietary exposure to the food enzyme-total organic solids (TOS) was calculated to be up to 0.458 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (1726 mg TOS/kg bw per day, the highest dose tested), the Panel derived a revised margin of exposure of at least 3769. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Residue profiles of peptides with cholesterol esterase and pancreatic lipase inhibitory activities through virtual screening and sequence analysis.

The detailed characterization of the structural features of peptides targeting cholesterol esterase (CEase) or pancreatic lipase (PPL) will benefit the management of hyperlipidemia and obesity. This study employed the Glide SP (standard precision)-peptide method to predict the binding modes of 202 dipeptides and 203 tripeptides to these targets, correlating residue composition and position with binding energy. Strong preferences for Trp, Phe, and Tyr were observed at all positions of potential inhibitory peptides, whereas negatively charged residues Glu and Asp were disfavored. Notably, Arg and aromatic rings significantly influenced the peptide conformation at the active site. Tripeptide IWR demonstrated the high efficacy, with IC50 values of 0.214 mg/mL for CEase and 0.230 mg/mL for PPL. Five novel IWR scaffold-tetrapeptides exhibited promising inhibitory activity. Non-covalent interactions and energy contributions dominated the formation of stable complexes. Our results provide insights for the development of new sequences or peptide-like molecules with enhanced inhibitory activity.

Novel extracellular lipase gene Lip1728 influences nutrient-dependent performance bacterial quorum sensing of Burkholderia pyrrocinia WZ10-3.

Quorum sensing (QS) is a cellular communication mechanism in which bacteria secrete and recognize signaling molecules to regulate group behavior. Lipases provide energy for bacterial cell growth but it is unknown whether they influence nutrient-dependent QS by hydrolyzing substrate. A high-yield lipase-producing strain, Burkholderia pyrrocinia WZ10-3, was previously identified in our laboratory, but the composition of its crude enzymes was not elucidated. Here, we identified a key extracellular lipase, Lip1728, in WZ10-3, which accounts for 99 % of the extracellular lipase activity. Lip1728 prefers to hydrolyze triglycerides at sn-1,3 positions, with pNP-C16 being its optimal substrate. Lip1728 exhibited activity at pH 5.0-10.0 and regardless of the presence of metal ions. It had strong resistance to sodium dodecyl sulfate and short-chain alcohols and was activated by phenylmethanesulfonylfluoride (PMSF). Lip1728 knockout significantly affected lipid metabolism and biofilm formation in the presence of olive oil. Finally, oleic acid, a hydrolysate of Lip1728, influenced the production of the signal molecule N-acyl homoserine lactone (AHL) and biofilm formation by downregulating the AHL synthetase gene pyrI. In conclusion, Lip1728, as a key extracellular lipase in B. pyrrocinia WZ10-3, exhibits superior properties that make it suitable for biodiesel production and plays a crucial role in QS.

Diagnosis and stabilisation of familial chylomicronemia syndrome in two infants presenting with hypertriglyceridemia-induced acute pancreatitis.

Familial chylomicronemia syndrome (FCS) is a rare disorder of triglyceride (TG) metabolism caused by loss of function variants in one of five known canonical genes involved in chylomicron lipolysis and clearance-LPL, APOC2, APOA5, LMF1, and GPIHBP1. Pathogenic variants in LPL, which encodes the hydrolytic enzyme lipoprotein lipase, account for over 80%-90% of cases. FCS may present in infancy with hypertriglyceridemia-induced acute pancreatitis and is challenging to manage both acutely and in the long-term. Here, we report our experience managing two unrelated infants consecutively diagnosed with hypertriglyceridemia-induced acute pancreatitis caused by LPL deficiency. Both had elevated TGs at presentation (205 and 30 mmol/L, respectively) and molecular genetic testing confirmed each infant carried a different homozygous pathogenic variant in the LPL gene, specifically, c.987C>A (p.Tyr329Ter) and c.632C>A (p.Thr211Lys). The more severely affected infant had cutaneous xanthomata, lipemia retinalis and lipemic plasma at presentation, and required management in an intensive care setting. Acute stabilisation was achieved using insulin and heparin infusions together with the iterative implementation of a fat-restricted diet, low in long chain triglycerides (LCT) and supplemented with medium chain triglycerides (MCT). In both cases, provision of adequate caloric intake (~110-120 kcal/kg/day) was also found to be important for a sustained TG reduction during the acute phase of management. In summary, a high index of suspicion is required to diagnose FCS in infants with hypertriglyceridemia-induced acute pancreatitis, management of which can be challenging, highlighting the need for more evidence-based recommendations.

Safety evaluation of an extension of use of the food enzyme triacylglycerol lipase from the non-genetically modified Rhizopus arrhizus strain AE-TL(B).

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the non-genetically modified Rhizopus arrhizus strain AE-TL(B) by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in two food manufacturing processes. Subsequently, the applicant requested to extend its use to include four additional processes and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of six food manufacturing processes. As the food enzyme-total organic solids (TOS) are removed from one food manufacturing process, the dietary exposure to the food enzyme-TOS was estimated only for the remaining five processes. Dietary exposure was calculated to be up to 0.086 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level reported in the previous opinion (1960 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 22,791. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

An sn-2 regioselective lipase with cis-fatty acid preference from Cordyceps militaris: Biochemical characterization and insights into its regioselective mechanism.

Lipase with unique regioselectivity is an attractive biocatalyst for elaborate lipid modification. However, the excavation of novel sn-2 regioselective lipases is difficult due to their scarcity in nature, with Candida antarctica lipase A (CALA) being the pronouncedly reported one. Here, we identified a novel CALA-like lipase from Cordyceps militaris (CACML7) via in silico mining. Through chiral-phase high-performance liquid chromatography, we determined that CACML7 displays sn-2 regioselectivity (>68 %) as does CALA, but exhibits distinctive chain length selectivity and bias against unsaturated fats. Notably, the curvature of the acyl-binding tunnel was expected to contribute to the 2.2-fold higher preference for cis-fatty acid (C18:1, cis-Δ9) over trans-fatty acid (C18:1, trans-Δ9) unlike trans-active CALA. Random pose docking of trioleoylglycerol (TOG) into the active site of a lid-truncated mutant of CACML7 revealed that TOG accepts a tuning fork conformation, of which the precise positioning of the reactive ester group towards the catalytic center was only favorable via sn-2 binding mode. The unique active site morphology, which we refer to as an "acyl-binding tunnel with a narrow entrance," may contribute to the sn-2 regioselectivity of CACML7. Our data provide an attractive model to better understand the mechanism underlying sn-2 regioselectivity.

Increase of serum pancreatic enzymes during hospitalization for acute heart failure.

AIMS: Acute heart failure (AHF) is associated with end-organ dysfunction. The effect of AHF on the pancreas has not been studied. We aim to evaluate serum markers of pancreatic damage during hospitalization for AHF. METHODS AND RESULTS: In data from the Pragmatic Urinary Sodium-based treatment algoritHm in Acute Heart Failure (PUSH-AHF) study, amylase and lipase values were extracted from available serum samples at baseline, and at 24 and 72 h after hospitalization. The differences between pancreatic enzymes between timepoints were evaluated using the Friedman test. Associations with N-terminal pro-B-type natriuretic peptide (NT-proBNP) were tested using linear regression analysis. The study population consisted of 274 patients. Mean age was 73 ± 11 years, and 117 (43%) were women. Mean left ventricular ejection fraction (LVEF) was 38 ± 14%; 53 (19%) patients had HF with a preserved LVEF (≥50%). At baseline, median amylase and lipase were within normal range (47 [33-63] U/L and 30 [21-44] U/L, respectively). Both enzymes significantly increased in the first 72 h (P-value for trend <0.001); mean change was 9 ± 22 U/L for amylase, and 10 ± 22 U/L for lipase. Moreover, NT-proBNP at baseline showed a positive correlation with mean change in pancreatic enzymes in 72 h (P = 0.02 for amylase and P = 0.006 for lipase). CONCLUSION: Patients admitted for AHF exhibited a significant increase in serum values of pancreatic enzymes in the first 72 h, suggesting that an episode of AHF affects the pancreatic tissue. This rise in pancreatic enzymes was associated with HF severity, as reflected by NT-proBNP.

Comparative Study of ImmobilizedBiolipasa-R for Second Generation Biodiesel Production from an Acid Oil.

The primary objective of this work is to develop a sustainable biocatalytic transesterification process for low-grade oils, aligning with EU green technology requirements for the shift to second generation biodiesel. Thus, we investigated the immobilization and subsequent application of the lipase Biolipasa-R on transesterification processes to produce fatty acid methyl esters (FAMEs) from both a sunflower oil and an acid oil which is a bioproduct of the biodiesel industry. The lipase was immobilized on biomaterials, such as diatomaceous earth, with a yield of 60%, and commercial carriers such as methacrylic resins with a yield of 100%. The enzyme demonstrated superior activity when immobilized on diatomaceous earth, particularly in reactions involving the acid oil, outperforming the benchmark enzyme Novozym® 435 (95.1% and 35% conversion respectively). This work highlights the potential of Biolipasa-R as a cost-effective and efficient biocatalyst for biodiesel production and emphasizes the environmental benefits of utilizing industrial byproducts and eco-friendly immobilization techniques. The findings suggest that Biolipasa-R is a promising candidate for industrial applications in biodiesel production, offering a sustainable solution for waste management and energy generation.

Enzyme Activity of Culturable Fungi and Bacteria Isolated from Traditional Agarwood Fermentation Basin Indicate Temporally Significant Lignocellulosic and Lipid Substrate Modulations.

Agarwood oil is one of the costliest essential oils used in perfumery, medicine and aroma. Production of the oil traditionally involves a soaking/fermentation step. Studies have indicated a definite role of the diverse microorganisms growing during the open soaking step, and in the emergent aroma of the essential oil. However, the temporal nature of fermentation and a key functional aspect i.e., the enzymatic properties of the microbes from the fermentation basin have not been studied yet. A total of 20 bacteria and 14 fungi isolated from fermentation basins located in Assam, India, at different soaking periods classified as early (0-20 days), medium (20-40 days) and late (40-60 days) clearly pointed towards an early fungal domination followed by succession of bacteria. The physico-chemical transformations of the wood are controlled by enzymatic properties (cellulase, xylanase, amylase and lipase) of the isolates. The results indicated a strong lignocellulosic substrate modulation potential in the four isolates, viz- Purpureocillium lilacinum (0.354 mg/mL), Mucor circinelloides (0.331 mg/mL), Penicillium citrinum (0.324 mg/mL) and Bacillus megaterium (0.152 mg/mL). The highest culturable abundance (CFU/mL) was found in M. circinelloides (2 × 109) among fungi and B. megaterium (4.5 × 109) among bacteria. The highest cellulase activity was shown by P. lilacinum (0.354 mg/mL) while xylanase and lipase by M. circinelloides (0.873 and 0.128 mg/mL). An interesting revelation was that a substantial proportion of the isolates (70% bacteria and 78% fungi) were positive for lipase activity. This is the first report on the "culturable microbiome" of the agarwood fermentation basin from a temporal and functional bioactivity perspective. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01257-y.

Rapidly screening of pancreatic lipase inhibitors from Clematis tangutica using affinity ultrafiltration-HPLC-QTOFMS technique combined with targeted separation, in vitro validation, and molecular docking.

INTRODUCTION: Screening of novel pancreatic lipase inhibitors from complex natural products is a meaningful task. OBJECTIVES: Through accurately screening and separating pancreatic lipase inhibitors from Clematis tangutica (C. tangutica), to discover new leading compounds for slimming and accelerate the development and utilization of Tibetan medicine resources. METHODS: An integrated strategy that combines affinity ultrafiltration and high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (AU-HPLC-QTOFMS), targeted separation, in vitro validation, and molecular docking was developed to screen pancreatic lipase inhibitors from C. tangutica. The AU-HPLC-QTOFMS technique was performed to fish for the potential active substances. Macroporous resin, preparative liquid chromatography, and high-speed countercurrent chromatography were implemented for the accurate and targeted separation of active compounds. The inhibitory activities of target compounds to pancreatic lipase were detected by the inhibition experiments in vitro. The binding affinities and binding sites were analyzed using molecular docking. RESULTS: A total of eleven kinds of pancreatic lipase inhibitory substances were screened from C. tangutica. Seven triterpenoid saponins were screened for the first time as lipase inhibitors and successfully prepared with purities higher than 97%. Tanguticoside B, clematangoticoside J, hederoside H1, and rutin showed stronger inhibitory effects with IC50 values of 1.539 ± 0.048, 1.661 ± 0.092, 1.793 ± 0.069, and 1.792 ± 0.094 mmol/l. Moreover, they have the lowest ΔG values of -10.84, -9.97, -10.87, and -9.39 kcal/mol to pancreatic lipase. CONCLUSION: The integrated strategy using AU-HPLC-QTOFMS, targeted separation, in vitro validation, and molecular docking was feasible for rapidly screening and directionally isolating pancreatic lipase inhibitors from C. tangutica.

Circumventing Cardiovascular Calamities: The Dawn of ANGPTL3 Blockade in Severe Dyslipidemia Management.

The landscape of severe dyslipidemia treatment is undergoing a remarkable transformation with the advent of angiopoietin-like 3 (ANGPTL3) inhibitors. ANGPTL3, a pivotal regulator of lipoprotein lipase and endothelial lipase, orchestrates the catabolism of triglyceride-rich and high-density lipoproteins, thus playing a critical role in lipid homeostasis. This review article examines the therapeutic potential of ANGPTL3 blockade and its implications for patients with severe dyslipidemias, particularly those unresponsive to traditional lipid-lowering regimens. We delve into the molecular mechanisms by which ANGPTL3 influences lipid metabolism and appraise the clinical utility of emerging therapeutics, such as monoclonal antibodies and antisense oligonucleotides. Moreover, we discuss the impact of ANGPTL3 inhibition on cardiovascular risk factors and project its promising role in reducing cardiovascular morbidity and mortality. The narrative synthesizes data from recent clinical trials, including the efficacy and safety profiles of ANGPTL3 inhibitors, and forecasts the potential of these agents to revolutionize the management of dyslipidemic conditions. The advent of ANGPTL3-targeted therapies signifies a potential breakthrough in the therapeutic armamentarium against complex lipid disorders, heralding a new era of precision medicine in cardiovascular risk mitigation.

Natural shellac-based microcapsules as lipase carriers for recyclable efficient Pickering interfacial biocatalysis.

Enzyme is an important class of catalyst. However, the efficiency of enzyme-catalyzed reactions is constrained by the limited contact between the enzyme and its substrate. In this study, to overcome this challenge, lipase-loaded microcapsules were prepared from natural shellac and nanoparticles using the emulsion template method. These microcapsules can perform dual roles as stabilizers and enzyme carriers to construct a water-in-oil Pickering interfacial biocatalytic system. The results showed that the hydrolytic conversion of the microcapsules could reach 90% within 20 min, which was significantly higher than that of the traditional biphasic system. The catalytic activity was influenced by the oil-to-water volume ratio and the microcapsule content. The microcapsules remained highly catalytic efficiency even after storage for three months or seven cycles of reuse. These microcapsules were prepared without the use of any cross-linkers or harsh solvents. This green and efficient catalytic system has great application prospects in the food industry.

Repurposing promethazine hydrochloride to inhibit biofilm formation against Burkholderia thailandensis.

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei, an intracellular pathogen with a high mortality rate and significant antibiotic resistance. The high mortality rate and resistance to antibiotics have drawn considerable attention from researchers studying melioidosis. This study evaluated the effects of various concentrations (75, 50, and 25 µg/mL) of promethazine hydrochloride (PTZ), a potent antihistamine, on biofilm formation and lipase activity after 24 h of exposure to B. thailandensis E264. A concentration-dependent decrease in both biofilm biomass and lipase activity was observed. RT-PCR analysis revealed that PTZ treatment not only made the biofilm structure loose but also reduced the expression of btaR1, btaR2, btaR3, and scmR. Single gene knockouts of quorum sensing (QS) receptor proteins (∆btaR1, ∆btaR2, and ∆btaR3) were successfully constructed. Deletion of btaR1 affected biofilm formation in B. thailandensis, while deletion of btaR2 and btaR3 led to reduced lipase activity. Molecular docking and biological performance results demonstrated that PTZ inhibits biofilm formation and lipase activity by suppressing the expression of QS-regulated genes. This study found that repositioning PTZ reduced biofilm formation in B. thailandensis E264, suggesting a potential new approach for combating melioidosis.

A carrier free delivery system of a MAGL inhibitor is effective on ovarian cancer.

Monoacylglycerol lipase (MAGL) is a promising target for cancer therapy due to its involvement in lipid metabolism and its impact on cancer hallmarks like cell proliferation, migration, and tumor progression. A potent reversible MAGL inhibitor, MAGL23, has been recently developed by our group, demonstrating promising anticancer activities. To enhance its pharmacological properties, a nanoformulation using nanocrystals coated with albumin was prepared (MAGL23AF). In a previous work, the formulated inhibitor showed to maintain its potency in ovarian and colon cancer cell lines in terms of IC50, and the formulation was tested on mice in order to assess its biocompatibility, organs biodistribution and toxicity. In the present work, we expanded the investigation to assess the potential in vivo application of MAGL23AF. Stability assays in serum and in human derived microsomes showed a good structural stability in physiological conditions of MAGL23AF. Antitumor efficacy tested on mice bearing ovarian cancer tumor highlighted that MAGL23AF has a more potent antitumor efficacy compared to non-formulated drug and leads to a necrosis-driven cancer cell death. In vivo studies revealed that albumin-complexed nanocrystals improved the therapeutic window of MAGL23, exhibiting a favorable biodistribution with slightly increased accumulation in the tumor. In conclusion, the MAGL23AF showed increased in vitro stability in conditions mirroring the bloodstream environment and hepatic metabolism coupled with an optimal antitumor efficacy in vivo. These results not only validates the efficacy of our formulation but also positions it as a promising strategy for addressing challenges related to the solubility of drugs in body fluids.

Crystal structure of lipase from Pseudomonas aeruginosa reveals an unusual catalytic triad conformation.

The Pseudomonas aeruginosa lipase PaL catalyzes the stereoselective hydrolysis of menthyl propionate to produce L-menthol. The lack of a three-dimensional structure of PaL has so far prevented a detailed understanding of its stereoselective reaction mechanism. Here, the crystal structure of PaL was determined at a resolution of 1.80 Å by single-wavelength anomalous diffraction. In the apo-PaL structure, the catalytic His302 is located in a long loop on the surface that is solvent exposed. His302 is distant from the other two catalytic residues, Asp274 and Ser164. This configuration of catalytic residues is unusual for lipases. Using metadynamics simulations, we observed that the enzyme undergoes a significant conformational change upon ligand binding. We also explored the catalytic and stereoselectivity mechanisms of PaL by all-atom molecular dynamics simulations. These findings could guide the engineering of PaL with an improved diastereoselectivity for L-menthol production.

Stabilization of Eversa® Transform 2.0 lipase with sorbitol to enhance the efficiency of ultrasound-assisted biodiesel production.

Ultrasound technology has emerged as a promising tool for enhancing enzymatic biodiesel production, yet the cavitation effect induced can compromise enzyme stability. This study explored the efficiency of polyols in enhancing lipase stability under ultrasound conditions to further improve biodiesel yield. The incorporation of sorbitol resulted in the highest fatty acid methyl ester (FAME) content in the ultrasound-assisted biodiesel production catalyzed by Eversa® Transform 2.0 among the investigated polyols. Furthermore, sorbitol enhanced the stability of the lipase, allowing it to tolerate up to 100 % ultrasound amplitude, compared to 60 % amplitude in its absence. Enzyme activity assays revealed that sorbitol preserved 99 % of the lipase activity, in contrast to 84 % retention observed without sorbitol under an 80 % ultrasound amplitude. Circular dichroism (CD) and fluorescence spectroscopy analyses confirmed that sorbitol enhanced lipase rigidity and preserved its conformational structure under ultrasound exposure. Furthermore, employing a stepwise methanol addition strategy in ultrasound-assisted reactions with sorbitol achieved an 81.2 wt% FAME content in 8 h with only 0.2 wt% enzyme concentration. This promising result highlights the potential of sorbitol as a stabilizing agent in ultrasound-assisted enzymatic biodiesel production, offering a viable approach for enhancing biodiesel yield and enzyme stability in industrial applications.

Investigation on solvent-free esterification of oleic acid by hemp tea waste-immobilized Candida rugosa lipase.

This study aimed at Candida rugosa lipase immobilization on a low-cost and readily available support. Among agro-industrial crops, hemp tea waste was chosen as the carrier because it provides higher immobilization performance than hemp flower and leaf wastes. Support characterization by ATR-FTIR, SEM and elemental analysis and the optimization of the adsorption immobilization process were performed. The lipase adsorption immobilization was obtained by soaking the support with hexane under mild agitation for 2 h and a successively incubating the enzyme for 1 h at room temperature without removing the solvent. The esterification of oleic acid with n-decanol was tested in a solvent-free system by studying some parameters that influence the reaction, such as the substrates molar ratio, the lipase activity/oleic acid ratio, reaction temperature and the presence/absence of molecular sieves. The biocatalyst showed the ability to bring the esterification reaction to equilibrium under 60 min and good reusability (maintaining 60 % of its original activity after three successive esterification reactions) but low conversion (21 %) at the optimized conditions (40 °C, 1:2 substrates molar ratio, 0.56 lipase/oleic acid ratio, without sieves). Comparing the results with those obtained by free lipase form at the same activity (1 U) and experimental conditions, slightly higher conversion (%) appeared for the free lipase. All this highlighted that probably the source of lipase for its carbohydrate-binding pocket and lid structure affected the esterification of oleic acid but certainly, the immobilization didn't induce any lipase conformational change also allowing the reuse of the catalytic material.

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.

Exploring the natural efficacy of spirulina powder for combating obesity, diabetes, and inflammation.

BACKGROUND: An increasing incidence of metabolic disorders emphasizes the need to explore natural treatments. Spirulina, a microalga with a rich nutrient profile, offers a promising solution for obesity, diabetes, and inflammation. This study provides a meticulous analysis of spirulina powder, evaluating its physicochemical attributes and technofunctional properties through the use of advanced analytical techniques. RESULTS: Spirulina powder demonstrated strong flowability, substantial water and oil absorption capacity, and moderate foaming characteristics. The ethanolic extract of spirulina was found to be a repository of phenolic (6.93 mg GAE/g) and flavonoid (7.17 mg QE/g) compounds, manifesting considerable antioxidant activity with a 58.49 g kg-1 inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The extract also exhibited pronounced inhibitory effects on lipase and amylase enzymes, with inhibition percentages of 72.05 g kg-1 and 70.28 g kg-1, respectively, and displayed a glucose retention capacity of 1.28 mg dL-1 (68.52 g kg-1) in a dialysis membrane assay. These results suggest its efficacy in modulating obesity and glycemic control. The powder also showed a potent anti-inflammatory response by mitigating protein denaturation. CONCLUSION: Spirulina powder is a potent natural agent with multiple health benefits, meriting its incorporation into functional foods. It could be suitable for application in the food industry, offering a natural strategy to combat metabolic diseases. This research adds to the scientific literature on spirulina, paving the way for future research into its utilization. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.