Esterification promotes the intracellular accumulation of roxadustat, an activator of hypoxia-inducible factors, to extend its effective duration.

Kidney injury often causes anemia due to a lack of production of the erythroid growth factor erythropoietin (EPO) in the kidneys. Roxadustat is one of the first oral medicines inducing EPO production in patients with renal anemia by activating hypoxia-inducible factors (HIFs), which are activators of EPO gene expression. In this study, to develop prodrugs of roxadustat with improved permeability through cell membrane, we investigated the effects of 8 types of esterification on the pharmacokinetics and bioactivity of roxadustat using Hep3B hepatoma cells that HIF-dependently produce EPO. Mass spectrometry of cells incubated with the esterified roxadustat derivatives revealed that the designed compounds were deesterified after being taken up by cells and showed low cytotoxicity compared to the original compound. Esterification prolonged the effective duration of roxadustat with respect to EPO gene induction and HIF activation in cells transiently exposed to the compounds. In the kidneys and livers of mice, both of which are unique sites of EPO production, a majority of the methyl-esterified roxadustat was deesterified within 6 h after drug administration. The deesterified roxadustat derivative was continuously detectable in plasma and urine for at least 48 h after administration, while the administered compound became undetectable 24 h after administration. Additionally, we confirmed that methyl-esterified roxadustat activated erythropoiesis in mice by inducing Epo mRNA expression exclusively in renal interstitial cells, which have intrinsic EPO-producing potential. These data suggest that esterification could lead to the development of roxadustat prodrugs with improvements in cell membrane permeability, effective duration and cytotoxicity.

Pharmacologic and genetic inhibition of cholesterol esterification enzymes reduces tumour burden: A systematic review and meta-analysis of preclinical models.

Cholesterol esterification proteins Sterol-O acyltransferases (SOAT) 1 and 2 are emerging prognostic markers in many cancers. These enzymes utilise fatty acids conjugated to coenzyme A to esterify cholesterol. Cholesterol esterification is tightly regulated and enables formation of lipid droplets that act as storage organelles for lipid soluble vitamins and minerals, and as cholesterol reservoirs. In cancer, this provides rapid access to cholesterol to maintain continual synthesis of the plasma membrane. In this systematic review and meta-analysis, we summarise the current depth of understanding of the role of this metabolic pathway in pan-cancer development. A systematic search of PubMed, Scopus, Web of Science, and Cochrane Library for preclinical studies identified eight studies where cholesteryl ester concentrations were compared between tumour and adjacent-normal tissue, and 24 studies where cholesterol esterification was blocked by pharmacological or genetic approaches. Tumour tissue had a significantly greater concentration of cholesteryl esters than non-tumour tissue (p < 0.0001). Pharmacological or genetic inhibition of SOAT was associated with significantly smaller tumours of all types (p ≤ 0.002). SOAT inhibition increased tumour apoptosis (p = 0.007), CD8 + lymphocyte infiltration and cytotoxicity (p ≤ 0.05), and reduced proliferation (p = 0.0003) and metastasis (p < 0.0001). Significant risk of publication bias was found and may have contributed to a 32% overestimation of the meta-analysed effect size. Avasimibe, the most frequently used SOAT inhibitor, was effective at doses equivalent to those previously reported to be safe and tolerable in humans. This work indicates that SOAT inhibition should be explored in clinical trials as an adjunct to existing anti-neoplastic agents.

Preparation of highly diffusible porous cross-linked lipase B from Candida antarctica conjugates: Advances in mass transfer and application in transesterification of 5-Hydroxymethylfurfural.

The present work pronounces the three phase partitioning (TPP)-facilitated preparation of porous cross-linked Candida antarctica lipase B (CaLB) aggregates (pCLEAs) for 5-Hydroxymethylfurfural (HMF) esters synthesis. CLEAs and pCLEAs of CaLB were prepared with eupergit as the support under the optimized conditions of pH 8.0, eupergit/protein ratio of 3.0:1.0, 50 mM cross-linker concentration and 3.3 mg/mL BSA concentration in 4 h. The optimum starch concentration for pCLEAs was 0.20%, m/v. The maximum biocatalytic load was 650 U/g (CLEAs) and 721 U/g (pCLEAs), and the immobilized biocatalysts were stable over a pH range of 6.0-9.0 and temperature range of (40-60)°C. The BET surface area of CLEAs and pCLEAs were 21.3 and 29.1 m2/g, respectively, and the catalytic efficiency of pCLEAs was 2.2-fold higher than that of CLEAs. Subsequently, the pCLEAs of CaLB were utilized for the manufacturing of industrially significant HMF esters. Under the optimized transesterification conditions, HMF conversion with pCLEAs CaLB was 1.41- and 1.25-fold higher than with free and CLEAs CaLB, respectively. The pCLEAs were reused upto 8 consecutive transesterification cycles and the produced HMF esters reduced the surface tension of water from 72 mN/m to 32.6 mN/m, proving its potential application as surface-active compounds.

Omega-3 rich oils from microalgae: A chitosan mediated in situ transesterification method.

Microalgae are a precious source of polyunsaturated fatty acids (PUFA), however extraction is difficult due to the peculiar microalgae cell structure. Here we describe a new method based on the application of chitosan nanoaggregates as CO2 responsive emulsifier, used to promote the swelling of algae cell wall and the formation of a large oil - ethanol interphase area during the ethanolysis. Tests were carried out with Pseudokirchneriella subcapitata and Nannochloropsis sp. at different biomass/ethanol/chitosan ratios. CO2 was added to trigger demulsification to promote an easy recovery of the lipid fraction. The highest yields in PUFA were obtained with Nannochloropsis sp. (207.9 mg/g of oil) using 0.4% wt of chitosan and 1:10 biomass:ethanol ratio; 43.6 mg/g of linolenic acid were obtained from Pseudokirchneriella subcapitata. Overall, because the method employs ethanol, a generally recognized as safe (GRAS) solvent, and food grade additives, it is suited for the preparation of PUFA supplements.

Improvement of biodiesel production from palm oil by co-immobilization of Thermomyces lanuginosa lipase and Candida antarctica lipase B: Optimization using response surface methodology.

Candida antarctica lipase B (CALB) and Thermomyces lanuginose lipase (TLL) were co-immobilized on epoxy functionalized silica gel via an isocyanide-based multicomponent reaction. The immobilization process was carried out in water (pH 7) at 25 °C, rapidly (3 h) resulting in high immobilization yields (100%) with a loading of 10 mg enzyme/g support. The immobilized preparations were used to produce biodiesel by transesterification of palm oil. In an optimization study, response surface methodology (RSM) and central composite rotatable design (CCRD) methods were used to study the effect of five independent factors including temperature, methanol to oil ratio, t-butanol concentration and CALB:TLL ratio on the yield of biodiesel production. The optimum combinations for the reaction were CALB:TLL ratio (2.1:1), t-butanol (45 wt%), temperature (47 °C), methanol: oil ratio (2.3). This resulted in a FAME yield of 94%, very close to the predicted value of 98%.

Cholesterol metabolism: New functions and therapeutic approaches in cancer.

Cholesterol and its metabolites (precursors and derivatives) play an important role in cancer. In recent years, numerous studies have reported the functions of cholesterol metabolism in the regulation of tumor biological processes, especially oncogenic signaling pathways, ferroptosis, and tumor microenvironment. Preclinical studies have over the years indicated the inhibitory effects of blocking cholesterol synthesis and uptake on tumor formation and growth. Besides, some new cholesterol metabolic molecules such as SOAT1, SQLE, and NPC1 have recently emerged as promising drug targets for cancer treatment. Here, we systematically review the roles of cholesterol and its metabolites, and the latest advances in cancer therapy targeting cholesterol metabolism.

Impact of Extracellular Fatty Acids and Oxygen Tension on Lipid Synthesis and Assembly in Pancreatic Cancer Cells.

Lipid oxidation and biosynthesis are crucial for cell survival, especially for rapidly proliferating cancer cells in a heterogeneous metabolic environment. The storage of high-energy lipid reservoirs competitively advantages the cancer cell over non-neoplastic tissue. Disrupting lipid biosynthetic processes, through modulation of fatty acid (FA) esterification or de novo lipogenesis (DNL), is of interest in drug discovery. Mimicking the in vivo environment in vitro is also vital for testing the efficacy of potential drug compounds. We present here a stable isotope tracer-based approach for examining the impact of exogenous FA and oxygen tension on the pathways that affect lipid biosynthesis, including the rates of metabolic flux. By applying tandem mass spectrometry (MS/MS) analyses to studies using parallel tracers, we characterized the impact of FA bioavailability on the positional enrichment within specific lipids. Our observations suggest that adding bioavailable FA as a carbon source preferentially biases the cellular metabolism away from DNL and toward esterification of free fatty acid pools. Additionally, we have found that this FA addition, under hypoxic conditions, led to a biased increase in the total triglyceride pool (nearly 5-fold, as compared to phospholipids), regardless of the isotope tracer utilized. We discuss the implications of this metabolic flexibility on studies that aim to characterize apparent drug efficacy.

Optimization, characterization and rheological behavior study of pectin extracted from chayote (Sechium edule) using ultrasound assisted method.

Pectin is one of high-value functional food ingredients. Better knowledge of properties of chayote pectin would help to promote the application of the chayote as a pectin source. In this study, ultrasound-assisted extraction (UAE) was used to extract chayote pectin. The extraction parameters were optimized by a Box-Behnken response surface design. The highest yield (6.19%) was obtained at a liquid/solid ratio of 50 mL/g, ultrasonic temperature of 70 °C and ultrasonic time of 40 min as optimized extraction conditions. The chemical properties, spectral information and rheological properties of pectin extracted with UAE under the optimum conditions (PEUO) were measured and the results indicated that the PEUO exhibited a low degree of esterification, high molar mass (2.47 × 106 g/mol) and suitable foaming capacity. The PEUO featured a stronger antioxidant capacity compared to commercially apple pectin and the PEUO solutions (1%, 2%, 3% and 5%) showed a non-Newtonian behavior. Hence, PEUO may provide beneficial characteristics to find use in food industry.

High-methylated pectin from walnut processing wastes as a potential resource: Ultrasound assisted extraction and physicochemical, structural and functional analysis.

The ultrasound-assisted extraction of pectin from walnut processing waste was optimized by Box-Behnken design. The highest extraction yield (12.78 ± 0.83%) was obtained at ultrasound power of 200 W, sonication time of 10 min, pH of 1.5 and LS ratio of 15 v/w. The resulting pectin in these conditions was rich in galacturonic acid (69.44%) and was high in degree of esterification (59.21%) which was confirmed by NMR and FTIR spectra. In addition, the molecular weight distribution analysis showed that the obtained pectin had a heterogeneous natural with low molecular weight (6.30-158.48 kDa). The XRD spectrum of the walnut pectin showed an amorphous structure with few crystalline portions. Furthermore, walnut green husk pectin had good emulsifying properties, water and oil holding capacities, and radical-scavenging activity. Given that the obtained result, the extraction of pectin from this by-product in addition to being able to reduce environmental problems, it can also provide financial benefits for the walnut production sector.

Disrupting cholesterol esterification by bitter melon suppresses triple-negative breast cancer cell growth.

Triple negative breast cancer (TNBC) is aggressive with a worse prognosis. We have recently shown that bitter melon extract (BME) treatment was more effective in inhibition of TNBC tumor growth in mouse models as compared to ER positive breast tumor growth. Aberrant dysregulation of lipid metabolism is associated with breast cancer progression, however, anti-cancer mechanism of BME linking lipid metabolism in breast cancer growth remains unexplored. Here, we observed that accumulation of esterified cholesterol was reduced in BME treated TNBC cell lines as compared to control cells. We next evaluated expression levels of acyl-CoA: cholesterol acyltransferase 1 (ACAT-1) in TNBC cells treated with BME. Our results demonstrated that BME treatment inhibited ACAT-1 expression in TNBC cells. Subsequently, we found that sterol regulatory element-binding proteins-1 and -2, and FASN was significantly reduced in BME treated TNBC cell lines. Low-density lipoprotein receptor was also downregulated in BME treated TNBC cells as compared to control cells. We further demonstrated that BME feeding reduced tumor growth in TNBC mammospheres implanted into NSG mice, and inhibits ACAT-1 expression. To our knowledge, this is the first report demonstrating BME suppresses TNBC cell growth through ACAT-1 inhibition, and have potential for additional therapeutic regimen against human breast cancer.

Cholesterol esterification inhibition and gemcitabine synergistically suppress pancreatic ductal adenocarcinoma proliferation.

Recent advances have recognized metabolic reprogramming as an underlying mechanism for cancer drug resistance. However, the role of cholesterol metabolism in drug resistance remain elusive. Herein, we report an increased accumulation of cholesteryl ester in gemcitabine-resistant pancreatic ductal adenocarcinoma (PDAC) cells. A potent inhibitor of acyl-CoA cholesterol acyltransferase-1 (ACAT-1), avasimibe, effectively suppressed proliferation of gemcitabine-resistant PDAC cells. Combination of avasimibe and gemcitabine showed strong synergistic effect in suppressing PDAC cell viability in vitro and tumor growth in vivo. Immunoblotting analysis suggests downregulation of Akt by avasimibe is likely to contribute to the synergism. Collectively, our study demonstrates a new combinational therapeutic strategy to overcome gemcitabine resistance for PDAC treatment.

Cell wall pectin methyl-esterification and organic acids of root tips involve in aluminum tolerance in Camellia sinensis.

Tea plant (Camellia sinensis (O.) Kuntze) can survive from high levels of aluminum (Al) in strongly acidic soils. However, the mechanism driving its tolerance to Al, the predominant factor limiting plant growth in acid condition, is still not fully understood. Here, two-year-old rooted cuttings of C. sinensis cultivar 'Longjingchangye' were used for Al resistance experiments. We found that the tea plants grew better in the presence of 0.4 mM Al than those grew under lower concentration of Al treatments (0 and 0.1 mM) as well as higher levels treatment (2 and 4 mM), confirming that appropriate Al increased tea plant growth. Hematoxylin staining assay showed that the apical region was the main accumulator in tea plant root. Subsequently, immunolocalization of pectins in the root tip cell wall showed a rise in low-methyl-ester pectin levels and a reduction of high-methyl-ester pectin content with the increasing Al concentration of treatments. Furthermore, we observed the increased expressions of C. sinensis pectin methylesterase (CsPME) genes along with the increasing de-esterified pectin levels during response to Al treatments. Additionally, the levels of organic acids increased steadily after treatment with 0.1, 0.4 or 2 mM Al, while they dropped after treatment with 4 mM Al. The organic acids secretion from root followed a similar trend. Similarly, a gradual increase in malate dehydrogenase (MDH), citrate synthase (CS) and glycolate oxidase (GO) enzyme activities and relevant metabolic genes expression were detected after the treatment of 0.1, 0.4 or 2 mM Al, while a sharp decrease was resulted from treatment with 4 mM Al. These results confirm that both pectin methylesterases and organic acids contribute to Al tolerance in C. sinensis.

Cholesterol esterification inhibition and imatinib treatment synergistically inhibit growth of BCR-ABL mutation-independent resistant chronic myelogenous leukemia.

Since the advent of tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, and dasatinib, chronic myelogenous leukemia (CML) prognosis has improved greatly. However, ~30-40% of patients develop resistance to imatinib therapy. Although most resistance is caused by mutations in the BCR-ABL kinase domain, 50-85% of these patients develop resistance in the absence of new mutations. In these cases, targeting other pathways may be needed to regain clinical response. Using label-free Raman spectromicroscopy, we evaluated a number of leukemia cell lines and discovered an aberrant accumulation of cholesteryl ester (CE) in CML, which was found to be a result of BCR-ABL kinase activity. CE accumulation in CML was found to be a cancer-specific phenomenon as untransformed cells did not accumulate CE. Blocking cholesterol esterification with avasimibe, a potent inhibitor of acyl-CoA cholesterol acyltransferase 1 (ACAT-1), significantly suppressed CML cell proliferation in Ba/F3 cells with the BCR-ABLT315I mutation and in K562 cells rendered imatinib resistant without mutations in the BCR-ABL kinase domain (K562R cells). Furthermore, the combination of avasimibe and imatinib caused a profound synergistic inhibition of cell proliferation in K562R cells, but not in Ba/F3T315I. This synergistic effect was confirmed in a K562R xenograft mouse model. Analysis of primary cells from a BCR-ABL mutation-independent imatinib resistant patient by mass cytometry suggested that the synergy may be due to downregulation of the MAPK pathway by avasimibe, which sensitized the CML cells to imatinib treatment. Collectively, these data demonstrate a novel strategy for overcoming BCR-ABL mutation-independent TKI resistance in CML.

Effects of exogenous isoprenoid diphosphates on in vivo attachment to bacteriochlorophyllide c in the green sulfur photosynthetic bacterium Chlorobaculum tepidum.

Metabolic substitution of the esterifying chain in bacteriochlorophyll (BChl) c in green photosynthetic bacteria grown by supplementation of exogenous alcohols has attracted attentions to study supramolecular structures and biogenesis of major antenna complexes chlorosomes in these bacteria as well as BChl pigment biosynthesis. Actual substrates in the enzymatic attachment of the esterifying moieties to the precursor of BChl c, namely bacteriochlorophyllide (BChlide) c, in these bacteria are believed to be diphosphate esters of alcoholic substrates, although only intact alcohols have so far been supplemented in the bacterial cultures. We report herein BChl c compositions in the green sulfur photosynthetic bacterium Chlorobaculum tepidum by supplementation with geranyl and geranylgeranyl diphosphates. The supplementation of these diphosphates hardly produced BChl c derivatives esterified with geraniol and geranylgeraniol in Cba. tepidum, whereas these BChl c derivatives were accumulated by supplementation of intact geraniol and geranylgeraniol. The sharp contrast of the incorporation efficiency of the supplemental isoprenoid moieties in BChl c using the isoprenoid diphosphates to that by the isoprenoid alcohols was mainly ascribable to less penetration abilities of the diphosphate substrates into Cba. tepidum cells because of their anionic and polar diphosphate moiety.

Toxicity of clove essential oil and its ester eugenyl acetate against Artemia salina / Toxicidade do óleo essencial de cravo e seu éster acetato de eugenila contra Artemia salina

Abstract The production of compounds via enzymatic esterification has great scientific and technological interest due to the several inconveniences related to acid catalysis, mainly by these systems do not fit to the concept of “green chemistry”. Besides, natural products as clove oil present compounds with excellent biological potential. Bioactives compounds are often toxic at high doses. The evaluation of lethality in a less complex animal organism can be used to a monitoring simple and rapid, helping the identification of compounds with potential insecticide activity against larvae of insect vector of diseases. In this sense, the toxicity against Artemia salina of clove essential oil and its derivative eugenyl acetate obtained by enzymatic esterification using Novozym 435 as biocatalyst was evaluated. The conversion of eugenyl acetate synthesis was 95.6%. The results about the evaluation of toxicity against the microcrustacean Artemia salina demonstrated that both oil (LC50= 0.5993 µg.mL–1) and ester (LC50= 0.1178 µg.mL–1) presented high toxic potential, being the eugenyl acetate almost 5 times more toxic than clove essential oil. The results reported here shows the potential of employing clove oil and eugenyl acetate in insecticide formulations.

Resumo A produção de compostos via esterificação enzimática possui grande interesse científico e tecnológico devido às inúmeras inconveniências relacionadas com a catálise ácida, principalmente por estes sitemas não se adequarem ao atual termo “tecnologias limpas”. Além disso, produtos naturais como o óleo de cravo, apresentam compostos com excelentes potenciais biológicos. Compostos bioativos são quase sempre tóxicos em altas doses. A avaliação da letalidade em um organismo animal menos complexo pode ser usada para um monitoramento simples e rápido, servindo também para a identificação de compostos com potencial atividade inseticida contra larvas de insetos vetores de doenças. Neste sentido, foi determinada a toxicidade frente a Artemia salina do óleo essencial de cravo e do seu derivado acetato de eugenila obtido por esterificação enzimática com lipase Novozym 435. A conversão da reação de síntese de acetato de eugenila foi de 95,6%. Os resultados referentes à avaliação da toxicidade frente ao microcrustáceo Artemia salina demonstraram que tanto o óleo (LC50= 0,5993 µg.mL–1) quanto o éster (LC50= 0,1178 µg.mL–1) apresentam elevado potencial toxicológico, sendo que o éster apresenta aproximadamente 5 vezes mais toxicidade em relação ao óleo. Estes resultados demonstram o potencial emprego do óleo de cravo e de acetato de eugenila em formulações de inseticidas.

Evaluating the effectiveness of various biochars as porous media for biodiesel synthesis via pseudo-catalytic transesterification.

This study focuses on investigating the optimized chemical composition of biochar used as porous material for biodiesel synthesis via pseudo-catalytic transesterification. To this end, six biochars from different sources were prepared and biodiesel yield obtained from pseudo-catalytic transesterification of waste cooking oil using six biochars were measured. Biodiesel yield and optimal reaction temperature for pseudo-catalytic transesterification were strongly dependent on the raw material of biochar. For example, biochar generated from maize residue exhibited the best performance, which yield was reached ∼90% at 300°C; however, the maximum biodiesel yield with pine cone biochar was 43% at 380°C. The maximum achievable yield of biodiesel was sensitive to the lignin content of biomass source of biochar but not sensitive to the cellulose and hemicellulose content. This study provides an insight for screening the most effective biochar as pseudo-catalytic porous material, thereby helping develop more sustainable and economically viable biodiesel synthesis process.

Wet in situ transesterification of microalgae using ethyl acetate as a co-solvent and reactant.

This study addresses wet in situ transesterification of microalgae for the production of biodiesel by introducing ethyl acetate as both reactant and co-solvent. Ethyl acetate and acid catalyst are mixed with wet microalgae in one pot and the mixture is heated for simultaneous lipid extraction and transesterification. As a single reactant and co-solvent, ethyl acetate can provide higher FAEE yield and more saccharification of carbohydrates than the case of binary ethanol and chloroform as a reactant and a co-solvent. The optimal yield was 97.8wt% at 114°C and 4.06M catalyst with 6.67mlEtOAC/g dried algae based on experimental results and response surface methodology (RSM). This wet in situ transesterification of microalgae using ethyl acetate doesn't require an additional co-solvent and it also promises more economic benefit as combining extraction and transesterification in a single process.

Glucose-dependent insulinotropic polypeptide promotes lipid deposition in subcutaneous adipocytes in obese type 2 diabetes patients: a maladaptive response.

Glucose-dependent insulinotropic polypeptide (GIP) beyond its insulinotropic effects may regulate postprandial lipid metabolism. Whereas the insulinotropic action of GIP is known to be impaired in type 2 diabetes mellitus (T2DM), its adipogenic effect is unknown. We hypothesized that GIP is anabolic in human subcutaneous adipose tissue (SAT) promoting triacylglycerol (TAG) deposition through reesterification of nonesterified fatty acids (NEFA), and this effect may differ according to obesity status or glucose tolerance. Twenty-three subjects categorized into four groups, normoglycemic lean (n = 6), normoglycemic obese (n = 6), obese with impaired glucose regulation (IGR; n = 6), and obese T2DM (n = 5), participated in a double-blind, randomized, crossover study involving a hyperglycemic clamp with a 240-min GIP infusion (2 pmol·kg-1·min-1) or normal saline. Insulin, NEFA, SAT-TAG content, and gene expression of key lipogenic enzymes were determined before and immediately after GIP/saline infusions. GIP lowered NEFA concentrations in the obese T2DM group despite diminished insulinotropic activity (mean NEFA AUC0-4 h ± SE, 41,992 ± 9,843 µmol·l-1·min-1 vs. 71,468 ± 13,605 with placebo, P = 0.039, 95% CI: 0.31-0.95). Additionally, GIP increased SAT-TAG in obese T2DM (1.78 ± 0.4 vs 0.86 ± 0.1-fold with placebo, P = 0.043, 95% CI: 0.1-1.8). Such effect with GIP was not observed in other three groups despite greater insulinotropic activity. Reduction in NEFA concentration with GIP correlated with adipose tissue insulin resistance for all subjects (Pearson, r = 0.56, P = 0.005). There were no significant gene expression changes in key SAT lipid metabolism enzymes. In conclusion, GIP appears to promote fat accretion and thus may exacerbate obesity and insulin resistance in T2DM.

Toxicity of clove essential oil and its ester eugenyl acetate against Artemia salina.

The production of compounds via enzymatic esterification has great scientific and technological interest due to the several inconveniences related to acid catalysis, mainly by these systems do not fit to the concept of "green chemistry". Besides, natural products as clove oil present compounds with excellent biological potential. Bioactives compounds are often toxic at high doses. The evaluation of lethality in a less complex animal organism can be used to a monitoring simple and rapid, helping the identification of compounds with potential insecticide activity against larvae of insect vector of diseases. In this sense, the toxicity against Artemia salina of clove essential oil and its derivative eugenyl acetate obtained by enzymatic esterification using Novozym 435 as biocatalyst was evaluated. The conversion of eugenyl acetate synthesis was 95.6%. The results about the evaluation of toxicity against the microcrustacean Artemia salina demonstrated that both oil (LC50= 0.5993 µg.mL-1) and ester (LC50= 0.1178 µg.mL-1) presented high toxic potential, being the eugenyl acetate almost 5 times more toxic than clove essential oil. The results reported here shows the potential of employing clove oil and eugenyl acetate in insecticide formulations.

Coating polypropylene surfaces with protease weakens the adhesion and increases the dispersion of Candida albicans cells.

OBJECTIVES: To investigate the ability of the proteases, subtilisin and α-chymotrypsin (aCT), to inhibit the adhesion of Candida albicans biofilm to a polypropylene surface. RESULTS: The proteases were immobilized on plasma-treated polypropylene by covalently linking them with either glutaraldehyde (GA) or N'-diisopropylcarbodiimide (DIC) and N-hydroxysuccinimide (NHS). The immobilization did not negatively affect the enzyme activity and in the case of subtilisin, the activity was up to 640% higher than that of the free enzyme when using N-acetyl phenylalanine ethyl ester as the substrate. The efficacies against biofilm dispersal for the GA-linked SubC and aCT coatings were 41 and 55% higher than the control (polypropylene coated with only GA), respectively, whereas no effect was observed with enzymes immobilized with DIC and NHS. The higher dispersion efficacy observed for the proteases immobilized with GA could be both steric (proper orientation of the active site) and dynamic (higher protein mobility/flexibility). CONCLUSIONS: Proteases immobilized on a polypropylene surface reduced the adhesion of C. albicans biofilms and therefore may be useful in developing anti-biofilm surfaces based on non-toxic molecules and sustainable strategies.