Lipolytic Enzymes Research Articles

Narirutin-Rich Celluclast Extract from Mandarin (Citrus unshiu) Peel Alleviates High-Fat Diet-Induced Obesity and Promotes Energy Metabolism in C57BL/6 Mice.

Mandarin peel, a main by-product from the processing of citrus juice, has been highlighted for its various bioactivities and functional ingredients. Our previous study proved the inhibitory effects of Celluclast extract from mandarin peel (MPCE) on lipid accumulation and differentiation in 3T3-L1 adipocytes. Therefore, the current study aimed to evaluate the anti-obesity effect of MPCE in high-fat diet (HFD)-induced obese mice. The high-performance liquid chromatography (HPLC) analysis exhibited that narirutin and hesperidin are the main active components of MPCE. Our current results showed that MPCE supplementation decreased adiposity by reducing body and organ weights in HFD-induced obese mice. MPCE also reduced triglyceride (TG), alanine transaminase (ALT), aspartate transaminase (AST), and leptin contents in the serum of HFD-fed mice. Moreover, MPCE significantly inhibited hepatic lipid accumulation by regulating the expression levels of proteins associated with lipid metabolism, including sterol regulatory element-binding protein (SREBP1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). Furthermore, MPCE administration significantly inhibited both adipogenesis and lipogenesis, with modulation of energy metabolism by activating 5' adenosine monophosphate-activated protein kinase (AMPK) and lipolytic enzymes such as hormone-sensitive lipase (HSL) in the white adipose tissue (WAT). Altogether, our findings indicate that MPCE improves HFD-induced obesity and can be used as a curative agent in pharmaceuticals and nutraceuticals to alleviate obesity and related disorders.

Identification and Characterization of a Novel Thermostable GDSL Lipase LipGt6 from Geobacillus thermoleovorans H9

Lipases are versatile biocatalysts for various biological reactions. In the detergent industry, lipases must exhibit high activity in environments with high temperature, high pH values, metal ions, and organic solvents. Therefore, researchers are intensively searching for more stable and efficient lipases. A new thermophilic lipase, LipGt6, was identified in Geobacillus thermoleovorans H9, a new thermophilic strain isolated from ultrahigh-temperature compost. A structural model of LipGt6 was constructed using an esterase from Geobacillus thermodenitrificans as a template, and site-directed mutagenesis confirmed the predicted active site residues. LipGt6 exhibited the highest activity towards medium- and long-chain fatty acids (C8–C14), and the optimum temperature and pH were 50 °C and 9.0, respectively. LipGt6 was found to be thermostable up to 70 °C. In the presence of 1% H2O2 and sodium deoxycholate, LipGt6 retained 70 to 75% relative activity. These findings reveal that LipGt6 is potentially useful for the industrial production of detergent. Based on comparison of the amino acid sequences, the enzyme belongs to a new subfamily called lipolytic enzyme family II. The catalytic residues Ser and His were more critical than Asp, and the Asp221 catalytic residue is not likely critical for the lipolytic reaction of LipGt6.

New Bioremediation by Lipase Purified from Ralstonia mannitolilytica for Petroleum Hydrocarbons

A growing issue of contaminated soil caused by oil derivers in Iraq, requires detection of new methods for bioremediation. The present work aims to purify of extracellular lipase enzyme that can eliminate oil derivers. Seven strains of lipolytic bacteria were collected from Al- Dora electricity station soil Baghdad/ Iraq that polluted with oil derivers. Ralstonia mannitolilytica considered higher ability in lipolytic enzymes production. Crude lipase activity gave (1.66 U/ml). The purification procedure resulted in 8.9 fold with a 23.2% final yield. The purity of lipase and molecular weight confirmed by SDS-PAGE. The efficiency of ten aliphatic hydrocarbon compounds in the soil contaminated with diesel decreases after treatment with purified enzyme (7.6 U/ml) for 24 hr. at natural environment to mimic the nature compared with control. The results concluded the degradation of aliphatic hydrocarbon compounds by lipase enzyme that purified from local Ralstonia mannitolilytica isolates was an efficient (biological) treatment. Although there are studies related to bioremediation used different bacterial isolates types and enzymes such as Pseudomonas sp. none of it used Ralstonia mannitolilytica isolate and lipase enzyme as bioremediation of oil spills.

Mutational scanning pinpoints distinct binding sites of key ATGL regulators in lipolysis

ATGL is a key enzyme in intracellular lipolysis and plays an important role in metabolic and cardiovascular diseases. ATGL is tightly regulated by a known set of protein-protein interaction partners with activating or inhibiting functions in the control of lipolysis. Here, we use deep mutational protein interaction perturbation scanning and generate comprehensive profiles of single amino acid variants that affect the interactions of ATGL with its regulatory partners: CGI-58, G0S2, PLIN1, PLIN5 and CIDEC. Twenty-three ATGL amino acid variants yield a specific interaction perturbation pattern when validated in co-immunoprecipitation experiments in mammalian cells. We identify and characterize eleven highly selective ATGL switch mutations which affect the interaction of one of the five partners without affecting the others. Switch mutations thus provide distinct interaction determinants for ATGL’s key regulatory proteins at an amino acid resolution. When we test triglyceride hydrolase activity in vitro and lipolysis in cells, the activity patterns of the ATGL switch variants trace to their protein interaction profile. In the context of structural data, the integration of variant binding and activity profiles provides insights into the regulation of lipolysis and the impact of mutations in human disease.

Modulation of Phospholipase A2 Membrane Activity by Anti-inflammatory Drugs.

The phospholipase A2 (PLA2) superfamily consists of lipolytic enzymes that hydrolyze specific cell membrane phospholipids and have long been considered a central hub of biosynthetic pathways, where their lipid metabolites exert a variety of physiological roles. A misregulated PLA2 activity is associated with mainly inflammatory-derived pathologies and thus has shown relevant therapeutic potential. Many natural and synthetic anti-inflammatory drugs (AIDs) have been proposed as direct modulators of PLA2 activity. However, despite the specific chemical properties that these drugs share in common, little is known about the indirect modulation able to finely tune membrane structural changes at the precise lipid-binding site. Here, we use a novel experimental strategy based on differential scanning calorimetry to systematically study the structural properties of lipid membrane systems during PLA2 cleavage and under the influence of several AIDs. For a better understanding of the AIDs-membrane interaction, we present a comprehensive and comparative set of molecular dynamics (MD) simulations. Our thermodynamic results clearly demonstrate that PLA2 cleavage is hindered by those AIDs that significantly reduce the lipid membrane cooperativity, while the rest of the AIDs oppositely tend to catalyze PLA2 activity to different extents. On the other hand, our MD simulations support experimental results by providing atomistic details on the binding, insertion, and dynamics of each AID on a pure lipid system; the drug efficacy to impact membrane cooperativity is related to the lipid order perturbation. This work suggests a membrane-based mechanism of action for diverse AIDs against PLA2 activity and provides relevant clues that must be considered in its modulation.

Albumin is an important factor in the control of serum free fatty acid flux in both male and female mice.

Albumin knockout (Alb-/-) mice exhibit a low plasma free fatty acid (FFA) concentration, but it was not known if the suppressed concentration reflects a lower rate of appearance (Ra) of FFA in the circulation (i.e., lower FFA flux) or if the absence of albumin alters the relationship between FFA flux and concentration. For understanding the role of albumin in FFA transport through the bloodstream, it is not sufficient to rely on FFA concentration data alone. Therefore, we developed a method to study FFA kinetics in Alb-/- mice. Using an albumin-free formulation of [U-13C]palmitate tracer, serum FFA kinetics were tested in Alb-/- and wild-type (WT) mice. Results indicate that the flux of FFA in serum of Alb-/- mice was significantly lower than in WT mice (P < 0.05), while albumin deficiency did not alter the relationship between FFA flux and concentration. Next, to test if suppressed lipolysis might have also been involved in the suppressed FFA kinetics, gene expression of a lipolytic enzyme (adipose triglyceride lipase, Atgl) and a marker of lipolysis (phosphorylation of hormone-sensitive lipase, p-HSL) were measured in adipose tissue. In contrast to the low FFA flux in Alb-/-, both Atgl gene expression and p-HSL protein were significantly higher in adipose tissue of Alb-/- than in WT mice (P < 0.05). Thus, the low FFA flux in Alb-/- appeared to be driven by the absence of albumin's FFA binding functions rather than through regulation of lipolysis, indicating that albumin is an important factor in determining the flux of FFA in circulation.NEW & NOTEWORTHY To improve understanding of the albumin protein's function in vivo, we tested plasma free fatty acid kinetics in albumin knockout mice compared with wild-type mice. Using a new tracer formulation strategy, it was discovered that the appearance rate of free fatty acids in serum is lower in albumin knockout mice than in wild-type mice. The results indicate that albumin is a major controller of free fatty acid kinetics.

Screening and characterization of Enterococcus durans isolates from raw organic milk in Southern Brazil: Assessing technological potential

This study assessed 34 strains of Enterococcus durans isolated from 8 samples of raw organic milk collected in both winter and summer seasons, with the aim of evaluating their technological capability. To achieve this, the following traits were tested: diacetyl production capability, autolysis capacity, organic acid production, expression of proteolytic and lipolytic enzymes, antagonistic capacity against pathogenic bacteria, as well as resistance to different types of antibiotics, hemolysin production, gelatinase production, and biofilm formation ability, as virulence factors. It was observed that 2.9% of the isolates exhibited the ability to produce diacetyl and showed proteolytic and autolytic capacity. Additionally, 8.8% displayed autolytic capacity, and 5.9% exhibited antagonistic capacity against Escherichia coli and Listeria monocytogenes, demonstrating that there are isolates with technological use potential.

Secondary Metabolites from Symbiotic Bacteria of Seagrass and Molluscs as A Reference for Natural Food Preservatives

Symbiotic bacteria from seagrass and molluscs have the potential to be a source of secondary metabolites. Compounds from these secondary metabolites, such as natural food preservatives, can be used in the bioindustry sector. This research aims to identify the potential secondary metabolites from symbiotic bacteria in molluscs and seagrass that can support the Sustainable Development Goals (SDG) public health program. The samples used in this study were symbiotic bacteria of mollusc from seagrass environments, symbiotic bacteria from seagrass, and seagrass samples. The seagrass samples were analysed phytochemically, while the symbiotic bacteria samples from molluscs and seagrass were tested for their antibacterial activity against food spoilage pathogens such as Staphylococcus aureus and Escherichia coli using the disc diffusion method. Bacteria that showed potential antibacterial activity were identified using molecular methods, and the content of active compounds was analysed using gas chromatography-mass spectrometry. The phytochemical analysis revealed that seagrass contains flavones, tannins, steroids, and compounds with potential antibacterial properties. GC-MS analysis indicated that seagrass is predominantly composed of unsaturated fatty acids, which have antimicrobial effects by damaging bacterial cell membranes and disrupting the function of enzymes essential for growth and reproduction. Ten symbiotic bacteria isolates demonstrated proteolytic, cellulolytic, and lipolytic enzyme activity, suggesting their potential as antibacterial agents due to their ability to break down cell walls. Molecular identification of selected bacteria revealed the presence of mollusc symbionts, including Vibrio owensii, Bacillus paramycoides, and Pseudoalteromonas flavipulchra. The symbiotic bacteria found in seagrass in this study included Bacillus velezensis, Bacillus paramycoides, Nocardiopsis alba, Nocardiopsis alba, Bacillus tropicus, Lysinibacillus fusiformis, and Bacillus tropicus. GC-MS analysis of selected symbiotic bacteria from seagrass showed that the dominant compound components were phthalic acid, bis(7-methyl octyl) ester (25.18, 43.84, 35.55 %), and trimethoxy-3-piperidyl-2,2'-binaphthalene-1,1',4,4'-tetrone (36.36, 47.71, 64.48 %). In conclusion, seagrass and its symbiotic bacteria, as well as symbiotic bacteria from molluscs living in seagrass, have the potential to serve as antibacterial agents and can be used as references for food preservatives. HIGHLIGHTS Seagrass and mollusc symbiotic bacteria produce secondary metabolites that have antimicrobial properties, making them suitable for food preservation The component that acts as an antibacterial agent is found in seagrass: Essential fatty acids, which can have antibacterial properties Symbiotic bacteria are microorganisms that live in mutualistic symbiosis with other organisms. The enzymatic activities of symbiotic bacteria can involve the production of enzymes that break down complex molecules into simpler molecules In food preservation, symbiotic bacteria, with their enzymatic activity, can be used as natural preservatives Using natural food preservatives derived from seagrass and mollusc symbiotic bacteria can contribute to a more sustainable and environmentally friendly approach to food preservation GRAPHICAL ABSTRACT

Extrusion inhibits lipolytic enzymes in pearl millet (Pennisetum glaucum) flour

The present study was undertaken to evaluate the effect of extrusion technology on anti-nutritional factors and lipolytic enzymes in pearl millet. The effect of process parameters such as die temperature (140–160 °C), screw speed (150–190 rpm), and feed moisture content (24–30%) was evaluated using response surface methodology. The extrudates so obtained were evaluated for their physico-chemical and functional properties. Optimized extrudate that showed maximum inhibition of anti-nutritional factors and lipolytic enzymes was evaluated for extent of starch gelatinization by differential scanning calorimetry, structural properties by FTIR, morphology by scanning electron microscopy, and crystallinity by X-ray diffractometry. Extrusion at 140 °C/150 rpm/30% moisture content could decrease trypsin inhibitors (∼100%), phytic acid (up to 89%) and condensed tannin (up to 85%), and inhibited lipase and lipoxygenase activities by 79.2 and 77.9%, respectively. The results so obtained are promising for extending the shelf life of pearl millet flour and utilizing it in food premixes. Industrial relevance textThe presence of lipase and lipoxygenase enzymes in pearl millet limits its shelf life due to generation of free fatty acids which results in bitter taste and off flavour. The presence of anti-nutrients such as phytic acid, trypsin inhibitor and condensed tannins restricts its digestibility, and poses challenges to its commercial as well as domestic use. Extrusion has attracted attention in millet processing due to its feasibility for implementation in the food industry without major additional costs. The extrusion processing parameters, mainly temperature, screw speed and moisture content 140 °C/150 rpm/30% decreased trypsin inhibitors (∼100%), phytic acid (up to 89%) and condensed tannin (up to 85%), and inhibited lipase and lipoxygenase activities by 79.2 and 77.9%, respectively. These results on inhibition of lipolytic enzymes through extrusion could help in extending the shelf life of pearl millet flour. It is anticipated that extruded pearl millet flour could form a base for premixes based thereon.

Thermal Stabilization of Lipases Bound to Solid-Phase Triazine-Scaffolded Biomimetic Ligands: A Preliminary Assessment

Biomimetic ligands are synthetic compounds that mimic the structure and binding properties of natural biological ligands. The first uses of textile dyes as pseudo-affinity ligands paved the way for the rational design and de novo synthesis of low-cost, non-toxic and highly stable triazine-scaffolded affinity ligands. A novel method to assess and enhance protein stability, employing triazine-based biomimetic ligands and using cutinase from Fusarium solani pisi as a protein model, has been previously reported. This innovative approach combined the concepts of molecular modeling and solid-phase combinatorial chemistry to design, synthesize and screen biomimetic compounds able to bind cutinase through complementary affinity-like interactions while maintaining its biological functionality. The screening of a 36-member biased combinatorial library enabled the identification of promising lead ligands. The immobilization/adsorption of cutinase onto a particular lead (ligand 3′/11) led to a noteworthy enhancement in thermal stability within the temperature range of 60–80 °C. In the present study, similar triazine-based compounds, sourced from the same combinatorial library and mimicking dipeptides of diverse amino acids, were selected and studied to determine their effectiveness in binding and/or improving the thermal stability of several lipases, enzymes which are closely related in function to cutinases. Three ligands with different compositions were screened for their potential thermostabilizing effect on different lipolytic enzymes at 60 °C. An entirely distinct enzyme, invertase from Saccharomyces cerevisiae, was also assessed for binding to the same ligands and functioned as a ‘control’ for the experiments with lipases. The high binding yield of ligand 3′/11 [4-({4-chloro-6-[(2-methylbutyl)amino]-1,3,5-triazin-2-yl}amino)benzoic acid] to cutinase was confirmed, and the same ligand was tested for its ability to bind lipases from Aspergillus oryzae (AOL), Candida rugosa (CRL), Chromobacterium viscosum (CVL), Rhizomucor miehei (RML) and Rhizopus niveus (RNL). The enzymes CRL, CVL, RNL and invertase showed significant adsorption yields to ligand 3′/11—32, 29, 36 and 94%, respectively, and the thermal stability at 60 °C of free and adsorbed enzymes was studied. CVL and RNL were also stabilized by adsorption to ligand 3′/11. In the case of CRL and invertase, which bound but were not stabilized by ligand (3′/11), other ligands from the original combinatorial library were tested. Between the two alternative ligands, one was effective at stabilizing C. rugosa lipase, while none stabilized invertase.

Roles of Lipolytic enzymes in Mycobacterium tuberculosis pathogenesis.

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that can endure for long periods in an infected patient, without causing disease. There are a number of virulence factors that increase its ability to invade the host. One of these factors is lipolytic enzymes, which play an important role in the pathogenic mechanism of Mtb. Bacterial lipolytic enzymes hydrolyze lipids in host cells, thereby releasing free fatty acids that are used as energy sources and building blocks for the synthesis of cell envelopes, in addition to regulating host immune responses. This review summarizes the relevant recent studies that used in vitro and in vivo models of infection, with particular emphasis on the virulence profile of lipolytic enzymes in Mtb. A better understanding of these enzymes will aid the development of new treatment strategies for TB. The recent work done that explored mycobacterial lipolytic enzymes and their involvement in virulence and pathogenicity was highlighted in this study. Lipolytic enzymes are expected to control Mtb and other intracellular pathogenic bacteria by targeting lipid metabolism. They are also potential candidates for the development of novel therapeutic agents.

A Comprehensive Study on the Influence of Superheated Steam Treatment on Lipolytic Enzymes, Physicochemical Characteristics, and Volatile Composition of Lightly Milled Rice.

Enzyme inactivation is crucial for enhancing the shelf life of lightly milled rice (LMR), yet the impact of diverse superheated steam (SS) treatment conditions on lipolytic enzyme efficiency, physicochemical properties, and volatile profiles of LMR remains unclear. This study investigated varying SS conditions, employing temperatures of 120 °C, 140 °C, and 160 °C and exposure times of 2, 4, 6, and 8 min. The research aimed to discern the influence of these conditions on enzyme activities, physicochemical characteristics, and quality attributes of LMR. Results indicated a significant rise in the inactivation rate with increased treatment temperature or duration, achieving a notable 70% reduction in enzyme activities at 120 °C for 6 min. Prolonged exposure to higher temperatures also induced pronounced fissures on LMR surfaces. Furthermore, intensive SS treatment led to a noteworthy 5.52% reduction in the relative crystallinity of LMR starch. GC/MS analysis revealed a consequential decrease, ranging from 44.7% to 65.7%, in undesirable odor ketones post-SS treatment. These findings underscore the potential of SS treatment in enhancing the commercial attributes of LMR.

AMWEst, a new thermostable and detergent-tolerant esterase retrieved from the Albian aquifer

A fosmid library was constructed with the metagenomic DNA from the high-temperature sediment-rich water of the Albian aquifer (Algeria). Functional screening of this library was subsequently done looking for genes encoding lipolytic enzymes. We identified a novel gene named AMWEst (1209 base pairs) encoding a protein of 402 amino acids with a predicted molecular weight of 43.44 kDa and conferring esterase activity. AMWEst was successfully overexpressed in the yeast mesophilic host Saccharomyces cerevisiae, and the expression system used proved to be efficient and produced sufficient activity for its biochemical characterization. Multiple sequence alignment indicated that AMWEst contained a conserved pentapeptide motif (Gly120-His121-Ser122-Gln123-Gly124). The optimum pH and temperature of the recombinant esterase AMWEst were 8 and 80 °C, respectively. Additionally, AMWEst showed higher activity towards short carbon substrates and showed maximum activity for p-nitrophenyl hexanoate (C6). Notably, AMWEst has a remarkable thermostability, and the enzyme retains almost maximum activity at 70 °C after incubation for 1 h. Moreover, enzyme activity was enhanced by high concentrations of SDS and Triton X-100 detergents.Key points• A novel thermostable esterase has been retrieved through functional metagenomics• The esterase is detergent-tolerant, which is attractive for some applications• The esterase can be expressed in a yeast mesophilic host to enhance its yield

In vitro gastrointestinal digestion of marine oil emulsions and liposomal solutions: fate of LC-PUFAs upon lipolysis.

The bioaccessibility and bioavailability of dietary fatty acids depend on the lipid to which they are esterified, the organisation of theses lipids in water and their recognition by lipolytic enzymes. In this work, we studied the release of marine long-chain polyunsaturated fatty acids (LC-PUFA), depending on their presentation either in the form of phospholipids (PL) or triacylglycerol (TAG). Two formulations based on marine PL or TAG extracted from salmon heads (Salmo salar) were prepared. Lipolysis was first tested in vitro by using individual gastrointestinal lipases and phospholipases to identify the enzymes involved in the digestion. Second, the lipolysis of the prepared formulations by a combination of enzymes was tested under in vitro conditions mimicking the physiological conditions found in the GI tract, both in the stomach and in the upper small intestine, in order to evaluate digestibility of TAG and LC-PUFA-containing liposomes. The in vitro results showed that TAG emulsion was hydrolyzed by porcine pancreatic extracts (PPE) and pure pancreatic lipase (PPL) with its cofactor, colipase, and to a lesser extent by pancreatic-lipase-related protein 2 (PLRP2) and a gastric extract (RGE) containing gastric lipase while no hydrolysis was observed with purified pancreatic phospholipase A2 (PLA2) and carboxyl ester hydrolase (CEH). The PL substrate was found to be hydrolysed by PLA2, PPE and PLRP2. Their phospholipase activities on liposomes formulation was dependent on the presence of bile salts. Using a two-step in vitro digestion model, we measured the kinetics of fatty acid release from TAG and PL during the gastric and intestinal phases of digestion. The highest overall lipolysis level was obtained with liposomes (around 75%) during the intestinal phase while they were preserved during the gastric phase. The overall lipolysis level of TAG emulsion was lower (around 33%), while it started already in the gastric phase. In conclusion, liposomes appear as a better delivery system for intestinal absorption of LC-PUFA than TAG. In addition, their resistance to lipolysis under gastric condition can protect LC-PUFA and provide a gastric stable delivery system for other molecules.

Use of thistle seeds of modified composition in chocolate mass technology

The object of the study is the analytical values of lipids of milk thistle seeds of a modified composition, namely acid and peroxide values, as well as the induction period of lipids of chocolate mass with the addition of milk thistle seeds. The paper substantiates rational parameters of thistle seed processing for the inactivation of lipases and lipoxygenases. Approximate dependences of the acid and peroxide values of thistle seed lipids on the pH of the wetting solution and the wetting degree of seeds were obtained. This makes it possible to substantiate such rational processing parameters of thistle seeds that inhibit the accumulation of free fatty acids and primary oxidation products in seeds during storage. An increase in the induction period of the oxidation of the lipid component of the chocolate mass with the use of thistle seeds of a modified composition was proven, which is 2.5 times higher than the induction period of the chocolate mass sample with thistle seeds with a native enzyme complex. The research results make it possible to develop a technology of healthy chocolate mass using thistle seeds of a modified composition, the lipid component of such mass is stable to oxidation and hydrolysis. The data obtained in the work are explained by an increase in the ability to inactivate lipolytic and lipoxygenase enzymes of milk thistle at high humidity in an acidic environment under the influence of microwave radiation. The advantage of the obtained results is the possibility of not violating the integrity of thistle seeds during the inactivation of the enzyme complex, which allows extending the shelf life of this raw material. An applied aspect of using the scientific result is the possibility of expanding the range of healthy chocolate masses with the use of thistle seeds

Lipase and esterase activity in soils from ancient burial vessels

The activity of lipolytic enzymes was assessed using the microplate method using heteromolecular exchange in soils from the bottom of burial vessels, followed by cluster analysis. Groups with a contrasting ratio of lipases C12 - C18 and esterases C4 - C8 were identified in a sample that included 57 burial pots aged two to three thousand years. The total lipolytic activity of soil samples from dishes with the presumptive initial presence of fat-containing food products was above 6% of the maximum value within the sample. Inhibition of esterases in the presence of high concentrations of fatty acids is reflected in the extremely low relative values of esterase activity of the soil microbial community against the background of maximum activity levels of C18 and C12 lipases in two groups of pots. In three groups, esterase activity was observed against the background of high relative activity of C16 lipases, average relative activity of C18 lipases and extremely low activity of true lipases. In the group of pots with high total lipolytic activity (with the exception of certain extreme values of this indicator), the proportion of true lipase activity was 1.7-1.8 times higher compared to the proportion of esterase activity. The grouping of burial vessels by the ratio of lipase and esterase activity reflects the characteristics of the funeral food, while the absolute values of enzyme activity among selected vessels with the highest total lipolytic activity are grouped according to the type of burial.

Discovery and Development of Quinazolinones and Quinazolinediones for Ameliorating Nonalcoholic Fatty Liver Disease (NAFLD) by Modulating COP1-ATGL Axis.

E3 ubiquitin ligase, Constitutive Photomorphogenic 1 (COP1) regulates turnover of Adipose Triglyceride Lipase (ATGL), the rate-limiting lipolytic enzyme. Genetic perturbation in the COP1-ATGL axis disrupts lipid homeostasis, leading to liver steatosis. Using drug development strategies, we herein report quinazolinone and quinazolinedione based modulators for COP1-ATGL axis. Systematic SAR studies and subsequent optimization were performed by incorporating relevant functional groups at the N1, N3, C5, and C6 positions of both scaffolds. Compounds' efficacy was evaluated by multiple biological assays and ADME profiling. The lead compound 86 could increase ATGL protein expression, reduce ATGL ubiquitination and COP1 autoubiquitination, and diminish lipid accumulation in hepatocytes in the nanomolar range. Oral administration of 86 abrogated triglyceride accumulation and resolved fibrosis in preclinical Nonalcoholic Fatty Liver Disease (NAFLD) model. The study thus establishes quinazolinedione as a viable chemotype to therapeutically modulate the activity of COP1 and ATGL in relevant clinical contexts.

Роль липолитических ферментов в улучшении органолептических показателей низкожирных сыров

Lipolytic enzymes can improve the sensory profile of low-fat cheeses. In this study, bovine lipase improved the flavor of semi-hard cheese with a 30.0% fat mass fraction.

Genome-wide analysis of lipolytic enzymes and characterization of a high-tolerant carboxylesterase from Sorangium cellulosum.

Microorganisms are important sources of lipolytic enzymes with characteristics for wide promising usages in the specific industrial biotechnology. The cellulolytic myxobacterium Sorangium cellulosum is rich of lipolytic enzymes in the genome, but little has been investigated. Here, we discerned 406 potential lipolytic enzymes in 13 sequenced S. cellulosum genomes. These lipolytic enzymes belonged to 12 families, and most are novel with low identities (14-37%) to those reported. We characterized a new carboxylesterase, LipB, from the alkaline-adaptive So0157-2. This enzyme, belonging to family VIII, hydrolyzed glyceryl tributyrate and p-nitrophenyl esters with short chain fatty acids (≤C12), and exhibited the highest activity against p-nitrophenyl butyrate. It retained over 50% of the activities in a broad temperature range (from 20°C to 60°C), alkaline conditions (pH 8.0-9.5), and the enzymatic activity was stable with methanol, ethanol and isopropanol, and stimulated significantly in the presence of 5 mM Ni2+. LipB also exhibited β-lactamase activity on nitrocefin, but not ampicillin, cefotaxime and imipenem. The bioinformatic analysis and specific enzymatic characteristics indicate that S. cellulosum is a promising resource to explore lipolytic enzymes for industrial adaptations.

Development of a rapid assay for β-etherase activity using a novel chromogenic substrate

Biocatalytic processes play a crucial role in the valorization of lignin; therefore, methods enabling the monitoring of enzymes such as β-etherases, capable of breaking β-O-4 aryl-ether bonds, are of significant biotechnological interest. A novel method for quantifying β-etherase activity was developed based on the β-ester bond formation between a chromophore and acetovainillone. The chromogenic substrate β-(ρ-nitrophenoxy)-α-acetovanillone (PNPAV), was chemically synthesized. Kintetic monitoring of ρ-nitrophenolate release at 410 nm over 10 min, using recombinant LigF from Sphingobium sp SYK-6, LigF-AB and LigE-AB from Althererytrobacter sp B11, yielded enzimatic activities of 404. 3 mU/mg, 72 mU/mg, and 50 mU/mg, respectively. This method is applicable in a pH range of 7.0–9.0, with a sensitivity of up to 50 ng of enzyme, exhibiting no interference with lipolytic, glycolytic, proteolytic, and oxidoreductase enzymes.