Macromolecular Interactions of Lipoprotein Lipase (LPL).

Lipoprotein lipase (LPL) is a critical enzyme in humans that provides fuel to peripheral tissues. LPL hydrolyzes triglycerides from the cores of lipoproteins that are circulating in plasma and interacts with receptors to mediate lipoprotein uptake, thus directing lipid distribution via catalytic and non-catalytic functions. Functional losses in LPL or any of its myriad of regulators alter lipid homeostasis and potentially affect the risk of developing cardiovascular disease-either increasing or decreasing the risk depending on the mutated protein. The extensive LPL regulatory network tunes LPL activity to allocate fatty acids according to the energetic needs of the organism and thus is nutritionally responsive and tissue dependent. Multiple pharmaceuticals in development manipulate or mimic these regulators, demonstrating their translational importance. Another facet of LPL biology is that the oligomeric state of the enzyme is also central to its regulation. Recent structural studies have solidified the idea that LPL is regulated not only by interactions with other binding partners but also by self-associations. Here, we review the complexities of the protein-protein and protein-lipid interactions that govern LPL structure and function.

Exploring selection signatures in the divergence and evolution of lipid droplet (LD) associated genes in major oilseed crops.

BACKGROUND: Oil bodies or lipid droplets (LDs) in the cytosol are the subcellular storage compartments of seeds and the sites of lipid metabolism providing energy to the germinating seeds. Major LD-associated proteins are lipoxygenases, phospholipaseD, oleosins, TAG-lipases, steroleosins, caleosins and SEIPINs; involved in facilitating germination and enhancing peroxidation resulting in off-flavours. However, how natural selection is balancing contradictory processes in lipid-rich seeds remains evasive. The present study was aimed at the prediction of selection signatures among orthologous clades in major oilseeds and the correlation of selection effect with gene expression. RESULTS: The LD-associated genes from the major oil-bearing crops were analyzed to predict natural selection signatures in phylogenetically close-knit ortholog clusters to understand adaptive evolution. Positive selection was the major force driving the evolution and diversification of orthologs in a lineage-specific manner. Significant positive selection effects were found in 94 genes particularly in oleosin and TAG-lipases, purifying with excess of non-synonymous substitution in 44 genes while 35 genes were neutral to selection effects. No significant selection impact was noticed in Brassicaceae as against LOX genes of oil palm. A heavy load of deleterious mutations affecting selection signatures was detected in T-lineage oleosins and LOX genes of Arachis hypogaea. The T-lineage oleosin genes were involved in mainly anther, tapetum and anther wall morphogenesis. In Ricinus communis and Sesamum indicum > 85% of PLD genes were under selection whereas selection pressures were low in Brassica juncea and Helianthus annuus. Steroleosin, caleosin and SEIPINs with large roles in lipid droplet organization expressed mostly in seeds and were under considerable positive selection pressures. Expression divergence was evident among paralogs and homeologs with one gene attaining functional superiority compared to the other. The LOX gene Glyma.13g347500 associated with off-flavor was not expressed during germination, rather its paralog Glyma.13g347600 showed expression in Glycine max. PLD-α genes were expressed on all the tissues except the seed,δ genes in seed and meristem while ß and γ genes expressed in the leaf. CONCLUSIONS: The genes involved in seed germination and lipid metabolism were under strong positive selection, although species differences were discernable. The present study identifies suitable candidate genes enhancing seed oil content and germination wherein directional selection can become more fruitful.

Reducing the lipase LIPE in mutant α-synuclein mice improves Parkinson-like deficits and reveals sex differences in fatty acid metabolism.

Impaired lipid metabolism is a risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) and can shift the physiological α-synuclein (αS) tetramer-monomer (T:M) ratio toward aggregation prone monomers. A resultant increase in phospho-serine 129+ αS monomers associating with excess mono- and polyunsaturated fatty acids contributes to the αS aggregation. We previously reported that decreasing the release of monounsaturated fatty acids (MUFAs) by reducing or inhibiting the hormone sensitive lipase (LIPE) reversed pathologic αS phosphorylation and improved soluble αS homeostasis in cultured αS triplication PD neurons and reduced DAergic neurodegeneration in a C.elegans αS model. However, assessing LIPE as a potential therapeutic target for progressive PD motor phenotypes has not been investigated. 3K αS mice, representing a biochemical and neuropathological amplification of the E46K fPD-causing mutation, have decreased αS T:M ratios, lipidic aggregates, and a L-DOPA responsive PD-like motor syndrome. Here, we reduced LIPE by crossings of 3K mice with LIPE null mice, which attenuated motor deficits in male LIPE+/- knockdown (LKD)-3K mice. Heterozygous LIPE reduction was associated with an improved αS T:M ratio, and dopaminergic neurotransmitter levels and fiber densities. In female 3K-LKD mice, an increase in pS129+ and larger lipid droplets (LDs) likely decreased the benefits seen in males. Reducing LIPE decreased MUFA release from neutral lipid storage, thereby reducing MUFA in phospholipid membranes with which αS interacts. Our study highlights fatty acid turnover as a therapeutic target for Lewy body diseases and support LIPE as a promising target in males. LIPE regulation represents a novel approach to mitigate PD and DLB risk and treat disease.

Amylase and lipase levels in the metabolic syndrome and type 2 diabetes: A longitudinal study in rhesus monkeys.

Latent associations between low serum amylase and reduced plasma insulin levels and increased adiposity have been described previously in a small study of asymptomatic middle-aged humans. In the present study, we sought to determine the nature of such changes during the longitudinal progression from metabolically normal to overt type 2 diabetes mellitus (T2DM) in nonhuman primates (NHPs), a disease that appears to be the same in both pathophysiology and underlying mechanisms as that which most commonly develops in middle-aged adult humans. Amylase and lipase levels were characterized in 157 unrelated adult rhesus monkeys (Macaca mulatta); 38% developed T2DM while under study. In all monkeys, multivariable linear regression analysis revealed that amylase could be negatively predicted by % body fat (ß -0.29; p = 0.002), age (ß -0.27; p = 0.005), and HbA1c (ß -0.18; p = 0.037). Amylase levels were positively predicted by lipase levels (ß = 0.19; p = -0.024) in all NHPs included in the study. Amylase was significantly lower in NHPs with metabolic syndrome (p < 0.001), prediabetes (PreDM) (p < 0.001), and T2DM (p < 0.001) compared to metabolically normal adult NHPs. Lipase increased in NHPs with PreDM (p = 0.005) and T2DM (p = 0.04) compared to normal NHPs. This is the first longitudinal study of any species, including humans, to show the dynamics of amylase and lipase during the metabolic progression from normal to metabolic syndrome, to PreDM and then to overt T2DM. The extraordinary similarity between humans and monkeys in T2DM, in pancreatic pathophysiology and in metabolic functions give these findings high translational value.

An efficient and high-water-content enzymatic esterification method for the synthesis of ß-sitosterol conjugated linoleate via a sodium citrate-based three-liquid-phase system.

Three-liquid-phase systems (TLPSs) are novel interfacial enzymatic reaction systems that have been successfully applied in many valuable reactions. However, these systems are suitable only for hydrolysis reactions and not for more widely used esterification reactions. Surprisingly, our recent research revealed that two water-insoluble substrates (ß-sitosterol and conjugated linoleic acid) could be rapidly esterified in this system. The initial rate of the esterification reaction in the TLPS based on sodium citrate was enhanced by approximately 10-fold relative to that in a traditional water/n-hexane system. The special emulsion structure (S/W1/W2 emulsion) formed may be vital because it not only provides a larger reaction interface but also spontaneously generates a middle phase that might regulate water activity to facilitate esterification. Furthermore, the lipase-enriched phase could be reused at least 8 times without significant loss of catalytic efficiency. Therefore, this TLPS is an ideal enzymatic esterification platform for ester synthesis because it is efficient, convenient to use, and cost-effective.

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 the food enzyme triacylglycerol lipase from the non-genetically modified Penicillium caseifulvum strain AE-LRF.

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the non-genetically modified Penicillium caseifulvum strain AE-LRF by Amano Enzyme Inc. The food enzyme was free from viable cells of the production organism. It is intended to be used in four food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.013 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 69 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 5308. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. However, the Panel noted that traces of ■■■■■, used in the manufacture of the triacylglycerol lipase, may be found in the food enzyme. The Panel considered that the risk of allergic reactions upon dietary exposure could not be excluded, particularly in individuals sensitised to fish. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

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.

Leptin-mediated suppression of lipoprotein lipase cleavage enhances lipid uptake and facilitates lymph node metastasis in gastric cancer.

BACKGROUND: Lymph node metastasis (LNM) is the primary mode of metastasis in gastric cancer (GC). However, the precise mechanisms underlying this process remain elusive. Tumor cells necessitate lipid metabolic reprogramming to facilitate metastasis, yet the role of lipoprotein lipase (LPL), a pivotal enzyme involved in exogenous lipid uptake, remains uncertain in tumor metastasis. Therefore, the aim of this study was to investigate the presence of lipid metabolic reprogramming during LNM of GC as well as the role of LPL in this process. METHODS: Intracellular lipid levels were quantified using oil red O staining, BODIPY 493/503 staining, and flow cytometry. Lipidomics analysis was employed to identify alterations in intracellular lipid composition following LPL knockdown. Protein expression levels were assessed through immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assays. The mouse popliteal LNM model was utilized to investigate differences in LNM. Immunoprecipitation and mass spectrometry were employed to examine protein associations. In vitro phosphorylation assays and Phos-tag sodium dodecyl-sulfate polyacrylamide gel electrophoresis assays were conducted to detect angiopoietin-like protein 4 (ANGPTL4) phosphorylation. RESULTS: We identified that an elevated intracellular lipid level represents a crucial characteristic of node-positive (N+) GC and further demonstrated that a high-fat diet can expedite LNM. LPL was found to be significantly overexpressed in N+ GC tissues and shown to facilitate LNM by mediating dietary lipid uptake within GC cells. Leptin, an obesity-related hormone, intercepted the effect exerted by ANGPTL4/Furin on LPL cleavage. Circulating leptin binding to the leptin receptor could induce the activation of inositol-requiring enzyme-1 (IRE1) kinase, leading to the phosphorylation of ANGPTL4 at the serine 30 residue and subsequently reducing its binding affinity with LPL. Moreover, our research revealed that LPL disrupted lipid homeostasis by elevating intracellular levels of arachidonic acid, which then triggered the cyclooxygenase-2/prostaglandin E2 (PGE2) pathway, thereby promoting tumor lymphangiogenesis. CONCLUSIONS: Leptin-induced phosphorylation of ANGPTL4 facilitates LPL-mediated lipid uptake and consequently stimulates the production of PGE2, ultimately facilitating LNM in GC.

Clinical characterization and mutation spectrum of patients with hypertriglyceridemia in a German outpatient clinic.

BACKGROUND: Severe hypertriglyceridemia (HTG) has predominantly multifactorial causes (MCS). Yet a small subset of patients have the monogenetic form (FCS). It remains a challenge to distinguish patients clinically, since decompensated MCS might mimic FCS´s severity. Aim of the current study was to determine clinical criteria that could sufficiently distinguish both forms as well as to apply the FCS score proposed by Moulin and colleagues. METHODS: We retrospectively studied 72 patients who presented with severe HTG in our clinic during a time span of seven years and received genetic testing. We classified genetic variants (ACMG-criteria), followed by genetic categorization into MCS or FCS. Clinical data were gathered from the medical records and the FCS score was calculated for each patient. RESULTS: Molecular genetic screening revealed eight FCS patients and 64 MCS patients. Altogether, we found 13 pathogenic variants of which four have not been described before. The FCS patients showed a significantly higher median triglyceride level compared to the MCS. The FCS score yielded a sensitivity of 75% and a specificity of 93.7% in our cohort, and significantly differentiated between the FCS and MCS group (p<0.001). CONCLUSIONS: In our cohort we identified several variables that significantly differentiated FCS from MCS. The FCS score performed similar to the original study by Moulin, thereby further validating the discriminatory power of the FCS score in an independent cohort.

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 2h and a successively incubating the enzyme for 1h 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 60min 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 (1U) 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.

Insights on the role of blocking agent on the properties of the lipase from Thermomyces lanuginosus immobilized on heterofunctional support for hydroesterification reactions.

Here, we report a study of the effect of the blocking agent on the properties of the lipase from Thermomyces lanuginosus (TLL) immobilized on a heterofunctional support (Purolite C18-ethylnediamina (EDA)- vinyl sulfone (VS)-TLL-blocking agent) in different reactions. The performance of the biocatalysts was compared to those immobilized on standard hydrophobic support (Purolite C18-TLL) and the commercial one (TLL-IM). The nature of the blocking agent (Cys, Gly and Asp) altered the enzyme features. TLL-IM always gave a comparatively worse performance, with its specificity for the oil being very different to the Purolite biocatalysts. Under optimized conditions, Purolite C18-TLL yielded 97 % of hydrolysis conversion after 4 h using a water/waste cooking soybean oil (WCSO) mass ratio of 4.3, biocatalyst load of 6.5 wt% and a temperature of 44.2 °C (without buffer or emulsification agent). In esterification reactions of the purified free fatty acids (FFAs) obtained from WCSO, the best TLL biocatalysts depended on the utilized alcohol: linear amyl alcohol was preferred by Purolite C18-TLL and Purolite C18-EDA-VS-TLL-Gly, while higher activity was achieved utilizing isoamyl alcohol as nucleophile by Purolite C18-EDA-VS-TLL-Cys, Purolite C18-EDA-VS-TLL-Asp and IM-TLL as catalysts. All the results indicate the influence of the blocking step on the final biocatalyst features.

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.

Hypertriglyceridemia Results From an Impaired Catabolism of Triglyceride-Rich Lipoproteins in PLIN1-Related Lipodystrophy.

BACKGROUND: Pathogenic variants in PLIN1-encoding PLIN1 (perilipin-1) are responsible for an autosomal dominant form of familial partial lipodystrophy (FPL) associated with severe insulin resistance, hepatic steatosis, and important hypertriglyceridemia. This study aims to decipher the mechanisms of hypertriglyceridemia associated with PLIN1-related FPL. METHODS: We performed an in vivo lipoprotein kinetic study in 6 affected patients compared with 13 healthy controls and 8 patients with type 2 diabetes. Glucose and lipid parameters, including plasma LPL (lipoprotein lipase) mass, were measured. LPL mRNA and protein expression were evaluated in abdominal subcutaneous adipose tissue from patients with 5 PLIN1-mutated FPL and 3 controls. RESULTS: Patients with PLIN1-mutated FPL presented with decreased fat mass, insulin resistance, and diabetes (glycated hemoglobin A1c, 6.68±0.70% versus 7.48±1.63% in patients with type 2 diabetes; mean±SD; P=0.27). Their plasma triglycerides were higher (5.96±3.08 mmol/L) than in controls (0.76±0.27 mmol/L; P<0.0001) and patients with type 2 diabetes (2.94±1.46 mmol/L, P=0.006). Compared with controls, patients with PLIN1-related FPL had a significant reduction of the indirect fractional catabolic rate of VLDL (very-low-density lipoprotein)-apoB100 toward IDL (intermediate-density lipoprotein)/LDL (low-density lipoprotein; 1.79±1.38 versus 5.34±2.45 pool/d; P=0.003) and the indirect fractional catabolic rate of IDL-apoB100 toward LDL (2.14±1.44 versus 7.51±4.07 pool/d; P=0.005). VLDL-apoB100 production was not different between patients with PLIN1-related FPL and controls. Compared with patients with type 2 diabetes, patients with PLIN1-related FPL also showed a significant reduction of the catabolism of both VLDL-apoB100 (P=0.031) and IDL-apoB100 (P=0.031). Plasma LPL mass was significantly lower in patients with PLIN1-related FPL than in controls (21.03±10.08 versus 55.76±13.10 ng/mL; P<0.0001), although the LPL protein expression in adipose tissue was similar. VLDL-apoB100 and IDL-apoB100 indirect fractional catabolic rates were negatively correlated with plasma triglycerides and positively correlated with LPL mass. CONCLUSIONS: We show that hypertriglyceridemia associated with PLIN1-related FPL results from a marked decrease in the catabolism of triglyceride-rich lipoproteins (VLDL and IDL). This could be due to a pronounced reduction in LPL availability, related to the decreased adipose tissue mass.

Neonatal dysregulation of 2-arachidonoylglycerol induces impaired brain function in adult mice.

The endocannabinoid system (ECS) regulates neurotransmission linked to synaptic plasticity, cognition, and emotion. While it has been demonstrated that dysregulation of the ECS in adulthood is relevant not only to central nervous system (CNS) disorders such as autism spectrum disorder, cognitive dysfunction, and depression but also to brain function, there are few studies on how dysregulation of the ECS in the neonatal period affects the manifestation and pathophysiology of CNS disorders later in life. In this study, DO34, a diacylglycerol lipase alpha (DAGLα) inhibitor affecting endocannabinoid 2-AG production, was injected into C57BL/6N male mice from postnatal day (PND) 7 to PND 10, inducing dysregulation of the ECS in the neonatal period. Subsequently, we examined whether it affects neuronal function in adulthood through electrophysiological and behavioral evaluation. DO34-injected mice showed significantly decreased cognitive functions, attributed to impairment of hippocampal synaptic plasticity. The findings suggest that regulation of ECS activity in the neonatal period may induce enduring effects on adult brain function.

In silico mining and identification of a novel lipase from Paenibacillus larvae: Rational protein design for improving catalytic performance.

Lipases play a vital role in various biological processes, from lipid metabolism to industrial applications. However, the ever-evolving challenges and diverse substrates necessitate the continual exploration of novel high-performance lipases. In this study, we employed an in silico mining approach to search for lipases with potential high sn-1,3 selectivity and catalytic activity. The identified novel lipase, PLL, from Paenibacillus larvae subsp. larvae B-3650 exhibited a specific activity of 111.2 ± 5.5 U/mg towards the substrate p-nitrophenyl palmitate (pNPP) and 6.9 ± 0.8 U/mg towards the substrate olive oil when expressed in Escherichia coli (E. coli). Computational design of cysteine mutations was employed to enhance the catalytic performance of PLL. Superior stability was achieved with the mutant K7C/A386C/H159C/K108C (2M3/2M4), showing an increase in melting temperature (Tm) by 1.9°C, a 2.05-fold prolonged half-life at 45°C, and no decrease in enzyme activity. Another mutant, K7C/A386C/A174C/A243C (2M1/2M3), showed a 4.9-fold enhancement in specific activity without compromising stability. Molecular dynamics simulations were conducted to explore the mechanisms of these two mutants. Mutant 2M3/2M4 forms putative disulfide bonds in the loop region, connecting the N- and C-termini of PLL, thus enhancing overall structural rigidity without impacting catalytic activity. The cysteines introduced in mutant 2M1/2M3 not only form new intramolecular hydrogen bonds but also alter the polarity and volume of the substrate-binding pocket, facilitating the entry of large substrate pNPP. These results highlight an efficient in silico exploration approach for novel lipases, offering a rapid and efficient method for enhancing catalytic performance through rational protein design.

Chemical Profile and Healthy Properties of Sicilian Diplotaxis harra subsp. crassifolia (Raf.) Maire.

This study was aimed at investigating the phytochemical profile and bioactivity of Diplotaxis harra subsp. crassifolia (Brassicaceae), a species from central-southern Sicily (Italy), where it is consumed as a salad. For this purpose, LC-ESI/HRMSn analysis of the ethanolic extract was performed, highlighting the occurrence, along with flavonoids, hydroxycinnamic acid derivatives, and oxylipins, of sulfated secondary metabolites, including glucosinolates and various sulfooxy derivatives (e.g., C13 nor-isoprenoids, hydroxyphenyl, and hydroxybenzoic acid derivatives), most of which were never reported before in the Brassicaeae family or in the Diplotaxis genus. Following ethnomedicinal information regarding this species used for the treatment of various pathologies such as diabetes and hypercholesterolemia, D. harra ethanolic extract was evaluated for its antioxidant potential using different in vitro tests such as 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), Ferric Reducing Ability Power, and ß-carotene bleaching tests. The inhibitory activity of carbohydrate-hydrolyzing enzymes (α-amylase and α-glucosidase) and pancreatic lipase was also assessed. In the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid assay, an IC50 value comparable to the positive control ascorbic acid (2.87 vs. 1.70 µg/mL, respectively) was obtained. The wild-wall rocket salad extract showed a significant α-amylase inhibitory effect. Obtained results indicate that Sicilian wild-wall rocket contains phytochemicals that can prevent hyperglycemia, hyperlipidemia, and obesity.

Expression of microbial lipase in filamentous fungus Aspergillus niger: a review.

Lipase has high economic importance and is widely used in biodiesel, food, detergents, cosmetics, and pharmaceutical industries. The rapid development of synthetic biology and system biology has not only paved the way for comprehensively understanding the efficient operation mechanism of Aspergillus niger cell factories but also introduced a new technological system for creating and optimizing high-efficiency A. niger cell factories. In this review, all relevant data on microbial lipase enzyme sources and general properties are gathered and updated. The relationship between A. niger strain morphology and protein production is discussed. The safety of A. niger strain is investigated to ensure product safety. The biotechnologies and factors influencing lipase expression in A. niger are summarized. This review focuses on various strategies to improve lipase expression in A. niger. The summary of these methods and the application of the gene editing technology CRISPR/Cas9 system can further improve the efficiency of constructing the engineered lipase-producing A. niger.

A lipase from Lacticaseibacillus rhamnosus IDCC 3201 with thermostability and pH resistance for use as a detergent additive.

Lipases are important biocatalysts and ubiquitous in plants, animals, and microorganisms. The high growth rates of microorganisms with low production costs have enabled the wide application of microbial lipases in detergent, food, and cosmetic industries. Herein, a novel lipase from Lacticaseibacillus rhamnosus IDCC 3201 (Lac-Rh) was isolated and its activity analyzed under a range of reaction conditions to evaluate its potential industrial application. The isolated Lac-Rh showed a molecular weight of 24 kDa and a maximum activity of 3438.5 ± 1.8 U/mg protein at 60 °C and pH 8. Additionally, Lac-Rh retained activity in alkaline conditions and in 10% v/v concentrations of organic solvents, including glycerol and acetone. Interestingly, after pre-incubation in the presence of multiple commercial detergents, Lac-Rh maintained over 80% of its activity and the stains from cotton were successfully removed under a simulated laundry  setting. Overall, the purified lipase from L. rhamnosus IDCC 3201 has potential for use as a detergent in industrial applications. KEY POINTS: • A novel lipase (Lac-Rh) was isolated from Lacticaseibacillus rhamnosus IDCC 3201 • Purified Lac-Rh exhibited its highest activity at a temperature of 60 °C and a pH of 8, respectively • Lac-Rh remains stable in commercial laundry detergent and enhances washing performance.

Lipase-catalyzed interesterification of Sunite sheep tail fat and flaxseed oil provides a fat having a unique fatty acid content and favorable physicochemical and nutritional properties.

The aim of this study is to combine flaxseed oil (FO), rich in α-linolenic acid (ALA), with Sunite sheep tail fat (STF) through a lipase-catalyzed transesterification reaction, in order to produce an edible oil with a fatty acid ratio suitable for human needs. Initially, the optimal conditions for esterification were determined using the Box-Behnken design, with the measurement criterion being the content of ALA at the sn-2 position. The results indicated that the highest content of sn-2 ALA was obtained under the conditions of using 6.8 wt% Lipozyme®RMIM as the catalyst, a reaction temperature of 57°C, a reaction time of 3.3 h, and a substrate mass ratio of 5.6:4.4 for STF and FO. This led to the rapid breaking and recombining of molecular bonds, resulting in the interesterified fat (IF) with the highest content of ALA at the sn-2 position. Comparing STF and FO, IF exhibited excellent fatty acid composition and content. Furthermore, IF had a lower melting point and crystallization temperature compared to STF, and its solid fat content decreased with increasing temperature, completely melting at temperatures above 30°C. Thus, IF is a synthesized fat with excellent properties from both animal and vegetable sources.