Characterization of Pseudomonas spp. contamination and in situ spoilage potential in pasteurized milk production process.

To investigate the prevalence of Pseudomonas in the pasteurized milk production process and its effect on milk quality, 106 strains of Pseudomonas were isolated from the pasteurized milk production process of a milk production plant in Shaanxi Province, China. The protease, lipase and biofilm-producing capacities of the 106 Pseudomonas strains were evaluated, and the spoilage enzyme activities of their metabolites were assessed by simulating temperature incubation in the refrigerated (7 °C) and transport environment (25 °C) segments and thermal treatments of pasteurization (75 °C, 5 min) and ultra-high temperature sterilization (121 °C, 15 s). A phylogenetic tree was drawn based on 16S rDNA gene sequencing and the top 5 strains were selected as representative strains to identify their in situ spoilage potential by examining their growth potential and ability to hydrolyze proteins and lipids in milk using growth curves, pH, whiteness, Zeta-potential, lipid oxidation, SDS-PAGE and volatile flavor compounds. The results showed that half and more of the isolated Pseudomonas had spoilage enzyme production and biofilm capacity, and the spoilage enzyme activity of metabolites was affected by the culture temperature and sterilization method, but ultra-high temperature sterilization could not completely eliminate the enzyme activity. The growth of Pseudomonas lundensis and Pseudomonas qingdaonensis was less affected by temperature and time, and the hydrolytic capacity of extracellular protease and lipase secreted by Pseudomonas lurida was the strongest, which had the greatest effect on milk quality. Therefore, it is crucial to identify the key contamination links of Pseudomonas, the main bacteria responsible for milk spoilage, and the influence of environmental factors on its deterioration.

Mitochondria targeted drug delivery system overcoming drug resistance in intrahepatic cholangiocarcinoma by reprogramming lipid metabolism.

The challenge of drug resistance in intrahepatic cholangiocarcinoma (ICC) is intricately linked with lipid metabolism reprogramming. The hepatic lipase (HL) and the membrane receptor CD36 are overexpressed in BGJ398-resistant ICC cells, while they are essential for lipid uptake, further enhancing lipid utilization in ICC. Herein, a metal-organic framework-based drug delivery system (OB@D-pMOF/CaP-AC, DDS), has been developed. The specifically designed DDS exhibits a successive targeting property, enabling it to precisely target ICC cells and their mitochondria. By specifically targeting the mitochondria, DDS produces reactive oxygen species (ROS) through its sonodynamic therapy effect, achieving a more potent reduction in ATP levels compared to non-targeted approaches, through the impairment of mitochondrial function. Additionally, the DDS strategically minimizes lipid uptake through the incorporation of the anti-HL drug, Orlistat, and anti-CD36 monoclonal antibody, reducing lipid-derived energy production. This dual-action strategy on both mitochondria and lipids can hinder energy utilization to restore drug sensitivity to BGJ398 in ICC. Moreover, an orthotopic mice model of drug-resistant ICC was developed, which serves as an exacting platform for evaluating the multifunction of designed DDS. Upon in vivo experiments with this model, the DDS demonstrated exceptional capabilities in suppressing tumor growth, reprogramming lipid metabolism and improving immune response, thereby overcoming drug resistance. These findings underscore the mitochondria-targeted DDS as a promising and innovative solution in ICC drug resistance.

Total Content and Composition of Phenolic Compounds from Filipendula Genus Plants and Their Potential Health-Promoting Properties.

This current article was dedicated to the determination of the composition of phenolic compounds in extracts of four species of the genus Filipendula in order to establish a connection between the composition of polyphenols and biological effects. A chemical analysis revealed that the composition of the extracts studied depended both on the plant species and its part (leaf or flower) and on the extractant used. All four species of Filipendula were rich sources of phenolic compounds and contained hydrolyzable tannins, condensed tannins, phenolic acids and their derivatives, and flavonoids. The activities included data on those that are most important for creating functional foods with Filipendula plant components: the influence on blood coagulation measured by prothrombin and activated partial thromboplastin time, and on the activity of the digestive enzymes (pancreatic amylase and lipase). It was established that plant species, their parts, and extraction methods contribute meaningfully to biological activity. The most prominent result is as follows: the plant organ determines the selective inhibition of either amylase or lipase; thus, the anticoagulant activities of F. camtschatica and F. stepposa hold promise for health-promoting food formulations associated with general metabolic disorders.

SLC25A28 Overexpression Promotes Adipogenesis by Reducing ATGL.

Adipose tissue dysfunction is seen among obese and type 2 diabetic individuals. Adipocyte proliferation and hypertrophy are the root causes of adipose tissue expansion. Solute carrier family 25 member 28 (SLC25A28) is an iron transporter in the inner mitochondrial membrane. This study is aimed at validating the involvement of SLC25A28 in adipose accumulation by tail vein injection of adenovirus (Ad)-SLC25A28 and Ad-green fluorescent protein viral particles into C57BL/6J mice. After 16 weeks, the body weight of the mice was measured. Subsequently, morphological analysis was performed to establish a high-fat diet (HFD)-induced model. SLC25A28 overexpression accelerated lipid accumulation in white and brown adipose tissue (BAT), enhanced body weight, reduced serum triglyceride (TG), and impaired serum glucose tolerance. The protein expression level of lipogenesis, lipolysis, and serum adipose secretion hormone was evaluated by western blotting. The results showed that adipose TG lipase (ATGL) protein expression was reduced significantly in white and BAT after overexpression SLC25A28 compared to the control group. Moreover, SLC25A28 overexpression inhibited the BAT formation by downregulating UCP-1 and the mitochondrial biosynthesis marker PGC-1α. Serum adiponectin protein expression was unregulated, which was consistent with the expression in inguinal white adipose tissue (iWAT). Remarkably, serum fibroblast growth factor (FGF21) protein expression was negatively related to the expansion of adipose tissue after administrated by Ad-SLC25A28. Data from the current study indicate that SLC25A28 overexpression promotes diet-induced obesity and accelerates lipid accumulation by regulating hormone secretion and inhibiting lipolysis in adipose tissue.

Inquiry lipaseoring the mechanism of pancreatic lipase inhibition by isovitexin based on multispectral method and enzyme inhibition assay.

Isovitexin is a main natural flavonoid component in various plants. Currently, the inhibitory effect of isovitexin on pancreatic lipase (PL) and its mechanism have not been elucidated yet. In the present study, we investigated the inhibitory effect of isovitexin on PL, as well as its interaction mechanism, using enzyme inhibition methods, spectroscopic analysis, and molecular simulations. Results showed that isovitexin possessed significant PL inhibitory activity, with IC50 values of 0.26 ± 0.02 mM. The interaction between isovitexin and PL was dominated by static quenching, and mainly through hydrogen bonding and hydrophobic interaction forces. Analysis of fluorescence spectroscopy confirmed that isovitexin binding altered the conformation of the PL. Circular dichroism (CD) spectrum indicated that isovitexin altered the secondary structure of PL by decreasing the α-helix content and increasing the ß-fold content. Molecular simulations further characterize the conformational changes produced by the interaction between isovitexin with PL. The performed study may provide a new insight into the inhibitory mechanism of isovitexin as a novel PL inhibitor.

Hypocholesterolemic potential of Bacillus amyloliquefaciens KAVK1 modulates lipid accumulation on 3T3-L1 adipose cells and high fat diet-induced obese rat model.

The significance of microorganisms occurring in foods is predominantly targeted due to their application for identifying a novel range of the bacterial spectrum. Diverse microbial species are capable of exhibiting potential pharmacological activities like antimicrobial and anticancer. Microbial strains capable of reducing obesity-related syndromes have also been reported. In the present study, the hypocholesterolemic efficacy of Bacillus amyloliquefaciens isolated from dairy products was scrutinised by in vitro (3T3-L1 adipose cells) and in vivo (high-fat diet-induced obese Wistar albino rats) methods. Potential cholesterol-lowering isolates were screened using a plate assay method and optimised by physical parameters. Molecular identification of the topmost five cholesterol-lowering isolates was acquired by amplification of the 16 S rRNA gene region. Bacillus amyloliquefaciens strain KAVK1, followed by strains KAVK2, KAVK3, KAVK4, and KAVK5 were molecularly determined. Further, cholesterol-lowering strains degraded the spectral patterns determined by the side chain of a cholesterol molecule. The anti-lipase activity was demonstrated using the porcine pancreatic lipase inhibitory method and compared with the reference compound Atorvastatin. Lyophilised strain KAVK1 revealed maximum pancreatic lipase inhibition. Strain KAVK1 attenuated lipid accumulation in 3T3-L1 adipose cell line predicted by Oil Red O staining method. Significant reduction of body weight and change in lipid profile was recognised after the supplement of KAVK1 to obese rats. Histopathological changes in organs were predominantly marked. The result of this study implies that the cholesterol-lowering B. amyloliquefaciens KAVK1 strain was used to treat hypercholesterolemia.

Vitamin B6 ameliorates acute pancreatitis by suppressing the caspase3 signaling pathway.

BACKGROUND: Acute pancreatitis (AP) is a prevalent exocrine inflammatory disorder of the pancreas characterized by pancreatic inflammation and injury to acinar cells. Vitamin B6 (VB6) is a vital nutrient that plays a significant role in preserving human health and has anti-inflammatory and anti-apoptotic effects. METHODS: This study aimed to explore the potential pancreatic protective effects of VB6 in mitigating pancreatic inflammation and apoptosis induced by taurocholate sodium (TLCS) in an AP model and to assess the underlying mechanism of action. AP was induced in Sprague‒Dawley (SD) rats through TLCS administration and lipopolysaccharide (LPS)-treated AR42J cells, followed by treatment with VB6. RESULTS: Various parameters associated with AP were assessed in both plasma and pancreatic tissues. VB6 has been shown to ameliorate the severity of AP through various mechanisms. It effectively reduces the levels of serum amylase, lipase, and inflammatory factors, thereby mitigating histological injury to the pancreas. Moreover, VB6 inhibited pancreatic apoptosis by downregulating bax expression and up-regulating Bcl2 expression in TLCS-treated rats. Additionally, VB6 suppressed the expression of caspase3. The anti-inflammatory and anti-apoptotic effects of VB6 observed in LPS-treated AR42J cells are consistent with those observed in a rat model of AP. CONCLUSIONS: These results suggest that VB6 exerts anti-inflammatory and anti-apoptotic effects through inhibition of the caspase3 signaling pathway and has a protective effect against AP.

A patient-based iPSC-derived hepatocyte model of alcohol-associated cirrhosis reveals bioenergetic insights into disease pathogenesis.

Only ~20% of heavy drinkers develop alcohol cirrhosis (AC). While differences in metabolism, inflammation, signaling, microbiome signatures and genetic variations have been tied to the pathogenesis of AC, the key underlying mechanisms for this interindividual variability, remain to be fully elucidated. Induced pluripotent stem cell-derived hepatocytes (iHLCs) from patients with AC and healthy controls differ transcriptomically, bioenergetically and histologically. They include a greater number of lipid droplets (LDs) and LD-associated mitochondria compared to control cells. These pre-pathologic indicators are effectively reversed by Aramchol, an inhibitor of stearoyl-CoA desaturase. Bioenergetically, AC iHLCs have lower spare capacity, slower ATP production and their mitochondrial fuel flexibility towards fatty acids and glutamate is weakened. MARC1 and PNPLA3, genes implicated by GWAS in alcohol cirrhosis, show to correlate with lipid droplet-associated and mitochondria-mediated oxidative damage in AC iHLCs. Knockdown of PNPLA3 expression exacerbates mitochondrial deficits and leads to lipid droplets alterations. These findings suggest that differences in mitochondrial bioenergetics and lipid droplet formation are intrinsic to AC hepatocytes and can play a role in its pathogenesis.

Chemo-profiling and exploring therapeutic potential of Momordica dioica Roxb. ex Willd. for managing metabolic related disorders: In-vitro studies, and docking based approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Momordica dioica Roxb. ex Willd. (M. dioica Roxb.) a nutritious and therapeutic property rich crop of Cucurbitaceae plant family. In various folklore medicine including Ayurveda fruits are used to treat several metabolic related disorders i.e., hyperglycemia, hyperlipidemia, diabetes, obesity etc. Furthermore, traditionally it is used to treat fever, inflammation, ulcer, skin diseases, haemorrhoids, hypertension and also employed as cardioprotective, hepatoprotective, analgesic, diuretic. AIM OF THE STUDY: This study focuses to explore the therapeutic potential of Momordica dioica Roxb. ex Willd. through in-vitro and in-silico approach for managing hyperlipidemia, hyperglycemia and related metabolic disorders along with its phytochemical profiling for quality evaluation and validation of traditional claim. MATERIALS AND METHODS: The present study was carried out on hydroalcohol extract of dried leaf and fruit of Momordica dioica. In-vitro antioxidant potential using DPPH and Nitric oxide scavenging assay along with in-vitro enzyme inhibitory potential against α-amylase, α-glucosidase, and pancreatic lipase enzymes was studied. The bioactive metabolites were identified from the most potent bioactive extract by analysis with LC-QTOF-MS and also studied their role to lessen the metabolic related disorder through in-silico approaches. RESULTS: The results confirmed that the fruit extract is more active to possess antioxidant and prominent enzyme inhibition potential compared to the leaf. Sixteen identified metabolites in M. dioica Roxb. fruits may be responsible for the therapeutic potential related to metabolic related disorder. The in-silico study of the identified phytomolecules against α-amylase, α-glucosidase and pancreatic lipase showed significant docking scores ranging from -9.8 to -5.5, -8.3 to -4.8 and -8.3 to -6 respectively. CONCLUSION: The current study illustrated that M. dioica Roxb., a traditionally important plant is potential against metabolic related disorders. Phytocomponents present in the fruit extract may be responsible for antioxidant as well as the enzymes' inhibitory potential. Thus, fruits of M. dioica Roxb. will be useful as alternative therapeutics for treatment of hyperlipidemia, hyperglycemia and related metabolic disorders.

A New Approach in Lipase-Octyl-Agarose Biocatalysis of 2-Arylpropionic Acid Derivatives.

The use of lipase immobilized on an octyl-agarose support to obtain the optically pure enantiomers of chiral drugs in reactions carried out in organic solvents is a great challenge for chemical and pharmaceutical sciences. Therefore, it is extremely important to develop optimal procedures to achieve a high enantioselectivity of the biocatalysts in the organic medium. Our paper describes a new approach to biocatalysis performed in an organic solvent with the use of CALB-octyl-agarose support including the application of a polypropylene reactor, an appropriate buffer for immobilization (Tris base-pH 9, 100 mM), a drying step, and then the storage of immobilized lipases in a climatic chamber or a refrigerator. An immobilized lipase B from Candida antarctica (CALB) was used in the kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification with methanol, reaching a high enantiomeric excess (eep = 89.6 ± 2.0%). As part of the immobilization optimization, the influence of different buffers was investigated. The effect of the reactor material and the reaction medium on the lipase activity was also studied. Moreover, the stability of the immobilized lipases: lipase from Candida rugosa (CRL) and CALB during storage in various temperature and humidity conditions (climatic chamber and refrigerator) was tested. The application of the immobilized CALB in a polypropylene reactor allowed for receiving over 9-fold higher conversion values compared to the results achieved when conducting the reaction in a glass reactor, as well as approximately 30-fold higher conversion values in comparison with free lipase. The good stability of the CALB-octyl-agarose support was demonstrated. After 7 days of storage in a climatic chamber or refrigerator (with protection from humidity) approximately 60% higher conversion values were obtained compared to the results observed for the immobilized form that had not been stored. The new approach involving the application of the CALB-octyl-agarose support for reactions performed in organic solvents indicates a significant role of the polymer reactor material being used in achieving high catalytic activity.

Functionalized Ionic Liquids-Modified Metal-Organic Framework Material Boosted the Enzymatic Performance of Lipase.

The development of immobilized enzymes with high activity and stability is critical. Metal-organic frameworks (MOFs) have attracted much academic and industrial interest in the field of enzyme immobilization due to their unique properties. In this study, the amino-functionalized ionic liquid (NIL)-modified metal-organic framework (UiO-66-NH2) was prepared to immobilize Candida rugosa lipase (CRL), using dialdehyde starch (DAS) as the cross-linker. The results of the Fourier transform infrared (FT-IR) spectra, X-ray powder diffraction (XRD), and scanning electronic microscopy (SEM) confirmed that the NIL was successfully grafted to UiO-66-NH2. The CRL immobilized on NIL-modified UiO-66-NH2 (UiO-66-NH2-NIL-DAS@CRL) exhibited satisfactory activity recovery (79.33%), stability, reusability, and excellent organic solvent tolerance. The research results indicated that ionic liquid-modified UiO-66-NH2 had practical potential for application in enzyme immobilization.

Exploring the inhibitory action of betulinic acid on key digestive enzymes linked to diabetes via in vitro and computational models: approaches to anti-diabetic mechanisms.

Phytochemicals are now increasingly exploited as remedial agents for the management of diabetes due to side effects attributable to commercial antidiabetic agents. This study investigated the structural and molecular mechanisms by which betulinic acid exhibits its antidiabetic effect via in vitro and computational techniques. In vitro antidiabetic potential was analysed via on α-amylase, α-glucosidase, pancreatic lipase and α-chymotrypsin inhibitory assays. Its structural and molecular inhibitory mechanisms were investigated using Density Functional Theory (DFT) analysis, molecular docking and molecular dynamics (MD) simulation. Betulinic acid significantly (p < 0.05) inhibited α-amylase, α-glucosidase, pancreatic lipase and α-chymotrypsin enzymes with IC50 of 70.02 µg/mL, 0.27 µg/mL, 1.70 µg/mL and 8.44 µg/mL, respectively. According to DFT studies, betulinic acid possesses similar reaction in gaseous phase and water due to close values observed for highest occupied molecular orbital (HOMO) and lowest occupied molecular orbital (LUMO) and the chemical descriptors. The dipole moment indicates that betulinic acid has high polarity. Molecular electrostatic potential surface revealed the electrophilic and nucleophilic attack-prone atoms of the molecule. Molecular dynamic studies revealed a stable complex between betulinic acid and α-amylase, α-glucosidase, pancreatic lipase and α-chymotrypsin. The study elucidated the potent antidiabetic properties of betulinic acid by revealing its conformational inhibitory mode of action on enzymes involved in the onset of diabetes.

Lipase domain effector AGLIP1 in Rhizoctonia solani triggers necrotic killing in plants.

KEY MESSAGE: We highlight the emerging role of the R. solani novel lipase domain effector AGLIP1 in suppressing pattern-triggered immunity and inducing plant cell death. The dynamic interplay between plants and Rhizoctonia solani constitutes a multifaceted struggle for survival and dominance. Within this complex dynamic, R. solani has evolved virulence mechanisms by secreting effectors that disrupt plants' first line of defense. A newly discovered effector, AGLIP1 in R. solani, plays a pivotal role in inducing plant cell death and subverting immune responses. AGLIP1, a protein containing a signal peptide and a lipase domain, involves complex formation in the intercellular space, followed by translocation to the plant cytoplasm, where it induces cell death (CD) and suppresses defense gene regulation. This study provides valuable insights into the intricate molecular interactions between plants and necrotrophic fungi, underscoring the imperative for further exploration in this field.

Alkaliphilic and thermostable lipase production by leaf litter fungus Leptosphaerulina trifolii A SMR-2011.

Fungi that inhabit fire-prone forests have to be adapted to harsh conditions and fungi affiliated to Ascomycota recovered from foliar litter samples were used for bioprospecting of molecules such as enzymes. Agni's fungi isolated from leaf litter, whose spores are capable of tolerating 110 oC were screened for thermostable lipases. One of the isolates, Leptosphaerulina trifolii A SMR-2011 exhibited high positive lipase activity than other isolates while screening through agar plate assay using Tween 20 in the medium. Maximum lipase activity (173.2 U/mg) of L. trifolii was observed at six days of inoculation and decreased thereafter. Among different oils used, the maximum lipase activity was attained by soybean oil (940.1 U/mg) followed by sunflower oil (917.1 U/mg), and then by mustard oil (884.8 U/mg), showing its specificity towards unsaturated fatty acids. Among the various organic nitrogen sources tested, soybean meal showed maximum lipase activity (985.4 U/mg). The partially purified enzyme was active over a wide range of pH from 8 to 12 with a pH optimum of 11.0 (728.1 U/mg) and a temperature range of 60-80 oC with an optimal temperature of 70 oC (779.1 U/mg). The results showed that lipase produced by L. trifolii is alkali stable and retained 85% of its activity at pH 11.0. This enzyme also showed high thermal stability retaining more than 50% of activity when incubated at 60 oC to 90 °C for 2 h. The ions Ca2+ and Mn2+ induced the lipase activity, while Cu2+ and Zn2+ ions lowered the activity compared to control. These results suggests that the leaf litter fungus L. trifolii serves as a potential source for the production of alkali-tolerant and thermostable lipase.

The bile salt/phospholipid ratio determines the extent of in vitro intestinal lipolysis of triglycerides: Interfacial and emulsion studies.

This study focused on the protein-stabilised triglyceride (TG)/water interfaces and oil-in-water emulsions, and explored the influence of varying molar ratios of bile salts (BSs) and phospholipids (PLs) on the intestinal lipolysis of TGs. The presence of these two major groups of biosurfactants delivered with human bile to the physiological environment of intestinal digestion was replicated in our experiments by using mixtures of individual BSs and PLs under in vitro small intestinal lipolysis conditions. Conducted initially, retrospective analysis of available scientific literature revealed that an average molar ratio of 9:4 for BSs to PLs (BS/PL) can be considered physiological in the postprandial adult human small intestine. Our experimental data showed that combining BSs and PLs synergistically enhanced interfacial activity, substantially reducing oil-water interfacial tension (IFT) during interfacial lipolysis experiments with pancreatic lipase, especially at the BS/PL-9:4 ratio. Other BS/PL molar proportions (BS/PL-6.5:6.5 and BS/PL-4:9) and an equimolar amount of BSs (BS-13) followed in IFT reduction efficiency, while using PLs alone as biosurfactants was the least efficient. In the following emulsion lipolysis experiments, BS/PL-9:4 outperformed other BS/PL mixtures in terms of enhancing the TG digestion extent. The degree of TG conversion and the desorption efficiency of interfacial material post-lipolysis correlated directly with the BS/PL ratio, decreasing as the PL proportion increased. In conclusion, this study highlights the crucial role of biliary PLs, alongside BSs, in replicating the physiological function of bile in intestinal lipolysis of emulsified TGs. Our results showed different contributions of PLs and BSs to lipolysis, strongly suggesting that any future in vitro studies aiming to simulate the human digestion conditions should take into account the impact of biliary PLs - not just BSs - to accurately mimic the physiological role of bile in intestinal lipolysis. This is particularly crucial given the fact that existing in vitro digestion protocols typically focus solely on applying specific concentrations and/or compositions of BSs to simulate the action of human bile during intestinal digestion, while overlooking the presence and concentration of biliary PLs under physiological gut conditions.

Lipase-mediated detoxification of host-derived antimicrobial fatty acids by Staphylococcus aureus.

Long-chain fatty acids with antimicrobial properties are abundant on the skin and mucosal surfaces, where they are essential to restrict the proliferation of opportunistic pathogens such as Staphylococcus aureus. These antimicrobial fatty acids (AFAs) elicit bacterial adaptation strategies, which have yet to be fully elucidated. Characterizing the pervasive mechanisms used by S. aureus to resist AFAs could open new avenues to prevent pathogen colonization. Here, we identify the S. aureus lipase Lip2 as a novel resistance factor against AFAs. Lip2 detoxifies AFAs via esterification with cholesterol. This is reminiscent of the activity of the fatty acid-modifying enzyme (FAME), whose identity has remained elusive for over three decades. In vitro, Lip2-dependent AFA-detoxification was apparent during planktonic growth and biofilm formation. Our genomic analysis revealed that prophage-mediated inactivation of Lip2 was rare in blood, nose, and skin strains, suggesting a particularly important role of Lip2 for host - microbe interactions. In a mouse model of S. aureus skin colonization, bacteria were protected from sapienic acid (a human-specific AFA) in a cholesterol- and lipase-dependent manner. These results suggest Lip2 is the long-sought FAME that exquisitely manipulates environmental lipids to promote bacterial growth in otherwise inhospitable niches.

A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization.

BACKGROUND: Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. RESULTS: The sfGFP(-15) mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose (DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn't improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80 °C. Therefore, D101 was chosen as the supporting material of the target protein. CONCLUSION: The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.

An important role for triglyceride in regulating spermatogenesis.

Drosophila is a powerful model to study how lipids affect spermatogenesis. Yet, the contribution of neutral lipids, a major lipid group which resides in organelles called lipid droplets (LD), to sperm development is largely unknown. Emerging evidence suggests LD are present in the testis and that loss of neutral lipid- and LD-associated genes causes subfertility; however, key regulators of testis neutral lipids and LD remain unclear. Here, we show LD are present in early-stage somatic and germline cells within the Drosophila testis. We identified a role for triglyceride lipase brummer (bmm) in regulating testis LD, and found that whole-body loss of bmm leads to defects in sperm development. Importantly, these represent cell-autonomous roles for bmm in regulating testis LD and spermatogenesis. Because lipidomic analysis of bmm mutants revealed excess triglyceride accumulation, and spermatogenic defects in bmm mutants were rescued by genetically blocking triglyceride synthesis, our data suggest that bmm-mediated regulation of triglyceride influences sperm development. This identifies triglyceride as an important neutral lipid that contributes to Drosophila sperm development, and reveals a key role for bmm in regulating testis triglyceride levels during spermatogenesis.

Antioxidant, anti-amylase, anti-lipase, and efficiency of Satureja fatty acid on the anti-inflammatory parameters in lipopolysaccharide-stimulated macrophage through Nrf2/NF-kB/NADH oxidase pathway.

Satureja is an aromatic plant that is used for flavoring, perfume, and food manufacturing due to its pleasant essential oil. Modern medicine research revealed several biological activities of Satureja essential oil, including antifungal, antibacterial, antiviral, antioxidant, anticancer, and anti-inflammatory. However, the functional properties of Satureja fatty acid have not been explored. This study examined the fatty acid profile, lipid nutritional quality, antioxidant, anti-amylase, and anti-lipase capacities of Satureja. The efficiency of Satureja fatty acid on the anti-oxidative and anti-inflammatory parameters in LPS-induced macrophage through the Nrf2/NF-kB/NADH oxidase pathway was examined. The whole lipid extract was prepared with chloroform/methanol/water solution. Fatty acids methyl ester from whole lipid extract were prepared with methanol/sulfuric acid reagent. The fatty acid profile was analyzed using gas chromatography-mass spectrometry. Total antioxidant was determined by ABTS decolorization. Lipase and amylase activities were determined by monitoring the decomposition of p-nitrophenyl butyrate and starch. The macrophage cell line was grown in DMEM media in the presence of fatty acid. The hydrogen peroxide production in treated cells was monitored using the FOX reagent. NADH oxidase activity was measured by monitoring NADH breakdown. The expression of NOX, NF-kB, and NRF2, were tested in the treated cells by real-time PCR. The main components of the Satureja fatty acid were linolenic acid (24.67-37.32%), palmitic acid (10.65-20.29%), linoleic acid (8.31-13.39%), oleic acid (4.42-14.35%), stearic acid (2.76-8.77%) and palmitoleic acid (1.77-4.95%). Given the nutritional quality, omega-3 PUFA (23.58-37.32%), SFA (21.53-26.70%), omega-6 PUFA (10.86-16.14%), omega-9 MUFA (4.42-14.35%), and omega-7 MUFA (1.77-4.95%) comprise the majority of fatty acids. Satureja fatty acid has a promising unsaturation index (120.77-164.27), PUFA/MUFA (2.07-6.41), hypocholesterolemic index (2.44-3.47), health-promoting index (2.03-2.42), PUFA/SFA (1.37-1.94), nutritive value index (0.53-1.71), MUFA/SFA (0.30-0.80) omega-6/omega-3 (0.34-0.65), atherogenicity index (0.41-0.49), and thrombogenicity index (0.17-0.27). Satureja fatty acid displayed strong antioxidant capacity (with IC50 ranging from 354 to 428 µg/mL), anti-lipase capacity (with IC50 ranging from 354 to 428 µg/mL), and anti-amylase capacity (with IC50 ranging from 370 to 390 µg/mL). LPS induced the expression of NOX, NRF2, and NF-kB and the synthesis of hydrogen peroxide in macrophage cells. In LPS-stimulated macrophages, Satureja fatty acid reduced NOX expression, hydrogen peroxide, and NF-kB expression and increased NRF2 at 0.04 mg/mL. In conclusion, Satureja fatty acids have potent antioxidant, anti-amylase, anti-lipase, and anti-inflammatory activities. The mechanisms in lowering oxidative stress markers depended on down-regulating superoxide-producing enzymes at gene and protein levels. Satureja polyunsaturated omega-3 fatty acids could be recommended for healthy products combined with dietary therapy to treat obesity, diabetes, and oxidative stress.

Engineered polymer nanoparticles as artificial chaperones facilitating the selective refolding of denatured enzymes.

Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these designs often have a limited range of substrates they can work with. In this paper, we present molecularly imprinted flexible polymer nanoparticles (nanoMIPs) designed as customizable biomimetic chaperones. We used model proteins such as cytochrome c, laccase, and lipase to screen polymeric monomers and identify the most effective formulations, offering tunable charge and hydrophobic properties. Utilizing a dispersed phase imprinting approach, we employed magnetic beads modified with destabilized whole-protein as solid-phase templates. This process involves medium exchange facilitated by magnetic pulldowns, resulting in the synthesis of nanoMIPs featuring imprinted sites that effectively mimic chaperone cavities. These nanoMIPs were able to selectively refold denatured enzymes, achieving up to 86.7% recovery of their activity, significantly outperforming control samples. Mechanistic studies confirmed that nanoMIPs preferentially bind denatured rather than native enzymes, mimicking natural chaperone interactions. Multifaceted analyses support the functionality of nanoMIPs, which emulate the protective roles of chaperones by selectively engaging with denatured proteins to inhibit aggregation and facilitate refolding. This approach shows promise for widespread use in protein recovery within biocatalysis and biomedicine.