Enhancing the Enantioselectivity and Catalytic Efficiency of Esterase from Bacillus subtilis for Kinetic Resolution of l-Menthol through Semirational Design.

Enzymatic kinetic resolution is a promising way to produce l-menthol. However, the properties of the reported biocatalysts are still unsatisfactory and far from being ready for industrial application. Herein, a para-nitrobenzylesterase (pnbA) gene from Bacillus subtilis was cloned and expressed to produce l-menthol from d,l-menthyl acetate. The highest enantiomeric excess (ee) value of the product generated by pnbA was only approximately 80%, with a high conversion rate (47.8%) of d,l-menthyl acetate with the help of a cosolvent, indicating high catalytic activity but low enantioselectivity (E = 19.95). To enhance the enantioselectivity and catalytic efficiency of pnbA to d,l-menthyl acetate in an organic solvent-free system, site-directed mutagenesis was performed based on the results of molecular docking. The F314E/F315T mutant showed the best catalytic properties (E = 36.25) for d,l-menthyl acetate, with 92.11% ee and 30.58% conversion of d,l-menthyl acetate. To further improve the properties of pnbA, additional mutants were constructed based on the structure-guided triple-code saturation mutagenesis strategy. Finally, four mutants were screened for the best enantioselectivity (ee > 99%, E > 300) and catalytic efficiency at a high substrate concentration (200 g/L) without a cosolvent. This work provides several generally applicable biocatalysts for the industrial production of l-menthol.

An overview of microbial enzymatic approaches for pectin degradation.

Pectin, a complex natural macromolecule present in primary cell walls, exhibits high structural diversity. Pectin is composed of a main chain, which contains a high amount of partly methyl-esterified galacturonic acid (GalA), and numerous types of side chains that contain almost 17 different monosaccharides and over 20 different linkages. Due to this peculiar structure, pectin exhibits special physicochemical properties and a variety of bioactivities. For example, pectin exhibits strong bioactivity only in a low molecular weight range. Many different degrading enzymes, including hydrolases, lyases and esterases, are needed to depolymerize pectin due to its structural complexity. Pectin degradation involves polygalacturonases/rhamnogalacturonases and pectate/pectin lyases, which attack the linkages in the backbone via hydrolytic and ß-elimination modes, respectively. Pectin methyl/acetyl esterases involved in the de-esterification of pectin also play crucial roles. Many α-L-rhamnohydrolases, unsaturated rhamnogalacturonyl hydrolases, arabinanases and galactanases also contribute to heterogeneous pectin degradation. Although numerous microbial pectin-degrading enzymes have been described, the mechanisms involved in the coordinated degradation of pectin through these enzymes remain unclear. In recent years, the degradation of pectin by Bacteroides has received increasing attention, as Bacteroides species contain a unique genetic structure, polysaccharide utilization loci (PULs). The specific PULs of pectin degradation in Bacteroides species are a new field to study pectin metabolism in gut microbiota. This paper reviews the scientific information available on pectin structural characteristics, pectin-degrading enzymes, and PULs for the specific degradation of pectin.

Isolation and Characterization of the Zearalenone-Degrading Strain, Bacillus spizizenii B73, Inspired by Esterase Activity.

Zearalenone (ZEN) is a widespread mycotoxin found in grain and feed, presenting a serious threat to animal and human health. This study investigated the ability of the novel strain B73, isolated from petroleum-contaminated soil, to detoxify ZEN. B73 was identified as Bacillus spizizenii through physiological and biochemical tests, and further confirmed based on the 16S rRNA gene sequence and the complete genome sequence. B. spizizenii B73 was capable of degrading up to 99.3% of ZEN at a concentration of 10 µg/mL in a minimal medium (pH = 7.0) within 8 h at 37 °C via HPLC-UV. In addition, B. spizizenii B73 was used to treat ZEN-contaminated wheat bran, dried distillers grains (DDGS), and corn meal, whereby the respective degradation rates reached 96.32%, 98.73%, and 80.31% after 36 h of treatment. HPLC-Q-Exactive-MS/MS analysis revealed one of the degradation products to have the formula C17H24O4. B. spizizenii B73 is a novel strain isolated from petroleum-contaminated soil, and the extracellular enzymes secreted by this strain show a remarkable ability to degrade ZEN.

Investigation of pectolytic and PR genes expression, quality and phytochemical contents in organic and non-organic table grapes at harvest and during storage.

The demand for organic table grapes is increasing worldwide. However, comprehensive information of quality parameters and phytochemical compounds in organically grown fruit remain unclear. Furthermore, table grapes are perishable and postharvest quality retention and waste prevention is very important. In this study we have compared the differences between organic and non-organic table grapes in terms of phytochemical compounds and quality parameters as well as the changes in the expression levels of pathogen related and lytic genes during storage. Organic fruit showed higher levels of phenolics, flavonoids, caffeic acid, hydrogen peroxide, protein content, antioxidant and anti-stress enzymes and total antioxidant activities at harvest and during storage. Although, the expression levels of polygalactronases, pectin methyl esterase, chitinase and glucanase genes was lower in organically grown table grapes at harvest, but the expression of all these genes was significantly increased during cold storage. After 60 days of cold storage the expression levels of pectin methyl esterase, chitinase and glucanase genes was significantly higher than the conventionally grown grape berries in organic ones. The highest expression of polygalacturonase was recorded in organic samples after 30 days of storage. There was no significant difference between the two types of table grapes for decay extension and tissue deterioration rate. The results of this study indicate that due to higher levels of phytochemicals and antioxidant compounds the organic table grapes have a higher nutritional quality. Furthermore, the increase in PR and pectolytic genes expression levels is enough for decreasing the fruit susceptibility to decay pathogens and enhancing the postharvest life of organic grapes.

Effects of Phenanthrene Exposure on the B-esterases Activities of Octopus maya (Voss and Solís Ramírez, 1996) Embryos.

No ecotoxicological information exists on phenanthrene (Phe) exposure in cephalopods, animals of commercial and ecological importance. This study investigated the effect of Phe on two B-esterases, Acetylcholinesterase (AChE) and Carboxylesterases (CbE), in Octopus maya embryos. Octopus embryos were exposed to different treatments: control (seawater), solvent control (seawater and DMSO 0.01%), 10 and 100 µg/L of Phe. AChE and CbE activities were measured at different developmental stages (blastula, organogenesis, and growth). B-esterase activities increased in control and solvent control as the embryos developed, showing no statistically significant differences between them. On the other hand, the embryos exposed to Phe had significant differences from controls, and between the high and low concentrations. Our results indicate that B-esterases are sensitive biomarkers of exposure to Phe in O. maya. Still, complementary studies are needed to unravel the toxicodynamics of Phe and the implications of the found inhibitory effect in hatched organisms.

Bacteriocin production and technological properties of Enterococcus mundtii and Enterococcus faecium strains isolated from sheep and goat colostrum.

Enterococci are lactic acid bacteria (LAB) that play a role in the aroma formation, maturation, and sensory development of fermented foods such as meat and dairy products. They also contribute to the improvement of the extended shelf life of fermented foods by producing bacteriocin. The aim of this study was to isolate bacteriocin-producing LAB from sheep and goat colostrum, to characterize the bacteriocin-producing strains, and determine the technological properties of the strains. A total of 13 bacteriocin-producing LAB was isolated and identified as 11 Enterococcus mundtii and two Enterococcus faecium. The strains were found to be genetically different from each other by phylogenetic analysis of 16S rRNA gene sequences and random amplified polymorphic-DNA (RAPD-PCR). It has been determined that bacteriocins show activity in a wide pH range and are resistant to heat, lose their activity with proteolytic enzymes and α-amylase, but are resistant to detergents. While the presence of the munKS gene was detected in all of the strains, it was determined that E. faecium HC121.4, HC161.1, E. mundtii HC147.1, HC166.5, and HC166.8 strains contained multiple enterocin genes. Trisin-SDS-PAGE analysis revealed two active protein bands of approximately 5.1 and 5.5 kDa in E. faecium HC121.4 and one active protein band with a weight of approximately 4.96 kDa in other strains. E. mundtii strains and E. faecium HC161.1 were identified as mundticin KS producers, and E. faecium HC121.4 was defined as an enterocin A and B producer. Except for E. mundtii HC166.8, acid production of strains was found to be slow at 6 h and moderate at 24 h. None of them showed extracellular proteolytic and lipolytic activities. It was found that the strains had esterase, esterase lipase, leucine arylamidase, acid phosphatase, and naphthol-AS-Bl-phosphohydrolase activities, while protease activities were low and peptidase activities were high. In conclusion, bacteriocin producer 13 Enterococcus strains isolated from sheep and goat colostrum were found to have the potential to be included in starter culture combinations.

Excavating phytochemicals from plants possessing antiviral activities for identifying SARS-CoV hemagglutinin-esterase inhibitors by diligent computational workflow.

Coronaviruses (CoVs) belong to a group of RNA viruses that cause diseases in vertebrates including. Newer and deadlier than SARS CoV-2 are sought to appear in future for which the scientific community must be prepared with the strategies for their control. Spike protein (S-protein) of all the CoVs require angiotensin-converting enzyme2 (ACE2), while CoVs also require hemagglutinin-acetylesterase (HE) glycoprotein receptor to simultaneously interact with O-acetylated sialic acids on host cells, both these interactions enable viral particle to enter host cell leading to its infection. Target inhibition of viral S-protein and HE glycoprotein receptor can lead to a development of therapy against the SARS CoV-2. The proposition is to recognize molecules from the bundle of phytochemicals of medicinal plants known to possess antiviral potentials as a lead that could interact and mask the active site of, HE glycoprotein which would ideally bind to O-acetylated sialic acids on human host cells. Such molecules can be addressed as 'HE glycoprotein blockers'. A library of 110 phytochemicals from Withania somnifera, Asparagus racemosus, Zinziber officinalis, Allium sativum, Curcuma longa and Adhatoda vasica was constructed and was used under present study. In silico analysis was employed with plant-derived phytochemicals. The molecular docking, molecular dynamics simulations over the scale of 1000 ns (1 µs) and ADMET prediction revealed that the Withania somnifera (ashwagandha) and Asparagus racemosus (shatavari) plants possessed various steroidal saponins and alkaloids which could potentially inhibit the COVID-19 virus and even other CoVs targeted HE glycoprotein receptor.Communicated by Ramaswamy H. Sarma.

Eco-friendly biosynthesis of TiO2 nanoparticles using Desmostachya bipinnata extract: Larvicidal and pupicidal potential against Aedes aegypti and Spodoptera litura and acute toxicity in non-target organisms.

The resistance to insecticides among insects, including mosquitoes and agricultural pests, and the impact of these compounds' environmental risks and health issues have motivated the proposition of eco-friendly alternatives. Thus, we aimed to explore the potential use of Desmostachya bipinnata for the biosynthesis of TiO2NPs and evaluate their larvicidal and pupicidal activity of target (Aedes aegypti and Spodoptera litura) and acute toxicity in non-target organisms (Toxorhynchites splendens and Eisenia fetida), at distinct concentrations, after 24 h of exposure. The characterization of the biosynthesized TiO2NPs was carried out by FT-IR, XRD, SEM, and EDX analysis. Under the UV-vis spectrum analysis, a sharp peak was recorded at 200 to 800 nm, which indicated the production of TiO2NPs by the plant extract. The SEM analysis revealed that the synthesized TiO2NPs were spherical with a diameter of 36.4 nm and were detected in the XRD spectrum analysis related to the TiO2NPs. The highest percentage of mortality recorded at 900 µg/mL was 96 % and 94 % in the 2nd instar of A. aegypti and S. litura larvae, respectively, and exhibited the LC50 and LC90 values 5 of 458.79 and 531.01 µg/mL, respectively. The biosynthesized TiO2NPs showed concentration-dependent increased pupal lethality for both A. aegypti and S. litura. We also observed increased detoxification enzyme activity (α esterase, ß esterase, and glutathione-S-transferase) of A. aegypti and S. litura exposed to different concentrations of biosynthesized TiO2NPs as histopathological changes in the midgut region of these animals. On the other hand, the mortality rate of non-target organisms (T. splendens and E. fetida) was lower when exposed to TiO2NPs, compared to the high lethality induced by synthetic pesticides (cypermethrin and monocrotophos for E. fetida; and cypermethrin and temphos for T. splendens). Thus, our study provides pioneering evidence on the potential use of D. bipinnata-mediated TiO2NPs for controlling mosquito vectors and agricultural pest management.

An esterase-activatable curcumin prodrug for tumor-targeting therapy.

A tumor-targeting therapy strategy is urgently needed to increase the accumulation of drugs in tumors and reduce the side effects in normal tissues. Herein, we developed an esterase-activatable curcumin prodrug Cur-RGD for tumor-targeting therapy. Armed with the tumor-targeting RGD peptide and in situ esterase-triggered drug release, this prodrug Cur-RGD can efficiently improve the therapeutic effect of curcumin in tumors.

The Potential Neuroprotective Effect of Cyperus esculentus L. Extract in Scopolamine-Induced Cognitive Impairment in Rats: Extensive Biological and Metabolomics Approaches.

The aim of the present study is to investigate the phytochemical composition of tiger nut (TN) (Cyperus esculentus L.) and its neuroprotective potential in scopolamine (Scop)-induced cognitive impairment in rats. The UHPLC-ESI-QTOF-MS analysis enabled the putative annotation of 88 metabolites, such as saccharides, amino acids, organic acids, fatty acids, phenolic compounds and flavonoids. Treatment with TN extract restored Scop-induced learning and memory impairments. In parallel, TN extract succeeded in lowering amyloid beta, ß-secretase protein expression and acetylcholine esterase (AChE) activity in the hippocampus of rats. TN extract decreased malondialdehyde levels, restored antioxidant levels and reduced proinflammatory cytokines as well as the Bax/Bcl2 ratio. Histopathological analysis demonstrated marked neuroprotection in TN-treated groups. In conclusion, the present study reveals that TN extract attenuates Scop-induced memory impairments by diminishing amyloid beta aggregates, as well as its anti-inflammatory, antioxidant, anti-apoptotic and anti-AChE activities.

Ammonium improved cell wall and cell membrane P reutilization and external P uptake in a putrescine and ethylene dependent pathway.

Ammonium promotes rice P uptake and reutilization better than nitrate, under P starvation conditions; however, the underlying mechanism remains unclear. In this study, ammonium treatment significantly increased putrescine and ethylene content in rice roots under P deficient conditions, by increasing the protein content of ornithine decarboxylase and 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase compared with nitrate treatment. Ammonium treatment increased rice root cell wall P release by increasing pectin content and pectin methyl esterase (PME) activity, increased rice shoot cell membrane P release by decreasing phosphorus-containing lipid components, and maintained internal P homeostasis by increasing OsPT2/6/8 expression compared with nitrate treatment. Ammonium also improved external P uptake by regulating root morphology and increased rice grain yield by increasing the panicle number compared with nitrate treatment. The application of putrescine and ethylene synthesis precursor ACC further improved the above process. Our results demonstrate for the first time that ammonium increases rice P acquisition, reutilization, and homeostasis, and rice grain yield, in a putrescine- and ethylene-dependent manner, better than nitrate, under P starvation conditions.

Anti-inflammatory, cytotoxicity and antilipidemic properties: novel bioactivities of true cinnamon (Cinnamomum zeylanicum Blume) leaf.

BACKGROUND: The leaf of Ceylon cinnamon (true cinnamon) is traditionally claimed for a variety of health benefits. However, reported scientific information is scanty and needs urgent attention for value addition. METHODS: Ethanolic (95%) and Dichloromethane:Methanol (DM, 1:1 v/v) leaf extracts of Ceylon cinnamon were evaluated for a range of medically important bioactivities namely anti-inflammatory [nitric oxide scavenging activity (NOSA), superoxide scavenging activity (SCA), COX1 and COX2 inhibition], growth inhibition & cytotoxicity against MCF7, HePG2 and AN3CA carcinoma cell lines, glutathionase-S-transferase (GST) inhibition and antilipidemic (anti-HMG-CoA reductase, anti-lipase, anti-cholesterol esterase, and cholesterol micellization inhibition) properties in vitro (n = 3). Further, a range of bioactive compounds in both leaf extracts was also quantified (n = 3). RESULTS: Both leaf extracts had all the investigated bioactive compounds and possessed moderately potent bioactivities compared to the reference drugs used in the study. Ethanolic leaf extract (ELE) exhibited the highest activities (IC50: µg/mL) for NOSA (40.26 ± 0.52), SCA (696.24 ± 40.02), cholesterol esterase inhibition (110.19 ± 1.55), cholesterol micellization inhibition (616.69 ± 7.09), GST inhibition (403.78 ± 2.70) and growth inhibition (GI50: 144.84 ± 1.59-269.00 ± 0.51) & cytotoxicity (LC50: 355.44 ± 9.38-717.71 ± 23.69) against studied cancer cell lines. In contrast, COX1 & COX2 (IC50: 6.62 ± 0.85 and 44.91 ± 3.06 µg/mL) and HMG-CoA reductase & lipase inhibitory activities (36.72 ± 4.74 and 19.71 ± 0.97% inhibition at 200 and 600 µg/mL) were highest in DM extract. ELE also showed the highest quantities (0.81 ± 0.06-104.38 ± 1.79) of tested compounds (mg/g extract) where eugenol was the highest and gallic acid was the lowest among quantified. CONCLUSION: Both leaf extracts of Ceylon cinnamon had all the tested bioactive compounds and possess all the investigated bioactivities. This is the 1st study to report all the investigated bioactivities of the leaf of Ceylon Cinnamon.

Metal Ions and Chemical Modification Reagents Inhibit the Enzymatic Activity of Lecithin-Dependent Hemolysin from Vibrio parahaemolyticus.

Lecithin-dependent thermolabile hemolysin (LDH) is a virulence factor excreted by Vibrio parahaemolyticus, a marine bacterium that causes important losses in shrimp farming. In this study, the function of LDH was investigated through its inhibition by metal ions (Mg2+, Ca2+, Mn2+, Co2+, Ni2+ and Cu2+) and chemical modification reagents: ß-mercaptoethanol (ßME), phenylmethylsulfonyl fluoride (PMSF) and diethyl pyrocarbonate (DEPC). LDH was expressed in the Escherichia coli strain BL-21, purified under denaturing conditions, and the enzymatic activity was evaluated. Cu2+, Ni2+, Co2+ and Ca2+ at 1 mmol/L inhibited the LDH esterase activity by 20−95%, while Mg2+ and Mn2+ slightly increased its activity. Additionally, PMSF and DEPC at 1 mmol/L inhibited the enzymatic activity by 40% and 80%, respectively. Dose-response analysis showed that DEPC was the best-evaluated inhibitor (IC50 = 0.082 mmol/L), followed by Cu2+ > Co2+ > Ni2+ and PMSF (IC50 = 0.146−1.5 mmol/L). Multiple sequence alignment of LDH of V. parahaemolyticus against other Vibrio species showed that LDH has well-conserved GDSL and SGNH motifs, characteristic of the hydrolase/esterase superfamily. Additionally, the homology model showed that the conserved catalytic triad His-Ser-Asp was in the LDH active site. Our results showed that the enzymatic activity of LDH from V. parahaemolyticus was modulated by metal ions and chemical modification, which could be related to the interaction with catalytic amino acid residues such as Ser153 and/or His 393.

Evaluation of Pure Bile Salts in Place of Bile Extract in the Standardized INFOGEST Digestion Protocol for Quantification of Sterol Bioaccessibility.

Improving standardized in vitro digestion protocols for phytosterols (PSs) is critical for understanding their bioaccessibility (BA) in food products and supplements. In this study, in vitro BA of phytosterol esters (PSEs) and free cholesterol (Ch) was compared under modified digestion conditions. The addition of Ch esterase (CE) to the INFOGEST model containing bovine bile resulted in a 70% increase in PS BA and an 18.5% reduction in Ch micellarization. Relative to the standardized INFOGEST model, substitution of pure bile salts (PBSs) did not significantly change PS BA or Ch micellarization. In the presence of CE, the substitution resulted in a 49.9% reduction in PS BA and a 13% increase in Ch micellarization. The differing results may be due to inhibitory effects of PBSs on the activity of intestinal enzymes, including CE. These results suggest that the INFOGEST model should include Ch esterase and the continued use of bile extract to evaluate PS BA.

Larvicidal, antioxidant and biotoxicity assessment of (2-(((2-ethyl-2 methylhexyl)oxy)carbonyl)benzoic acid isolated from Bacillus pumilus against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus.

Mosquitoes are a vector for many dreadful diseases known for their public health concern. The continued use of synthetic insecticides against vector control has led to serious environmental impacts, human health problems, and the development of insect resistance. Hence, alternative mosquito control methods are needed to protect the environment and human health. In the present study, the bioefficacy of (2-(((2-ethyl-2 methylhexyl)oxy)carbonyl) benzoic acid isolated from Bacillus pumilus were tested against Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi. The isolated bioactive compound was characterized through thin layer chromatography (TLC), UV-visible spectroscopy (UV), Fourier-transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, and gas chromatography-mass spectrometry analysis. The pure compound caused a high percent mortality rate in a dose-dependent manner, the obtained values were 96, 82, 69, 50 and 34%; 86, 72, 56, 43, and 44%; 100, 90, 83, 70 and 56% against Ae. aegypti, Cx. quinquefasciatus, and An. stephensi respectively. The effective lethal concentration values (LC50) were 13.65, 14.90 and 9.64 ppm against Ae. aegypti, Cx. quinquefasciatus, An. Stephensi, respectively. The effect of (2-(((2-ethyl-2 methylhexyl)oxy)carbonyl) benzoic acid significantly increased the superoxide dismutase, catalase, α, ß esterase and Glutathione-S-transferase level after 24 h of the treatment period. The comet assay confirmed that isolated compound causes DNA damage in all tested insects. Histopathological examinations of treated larvae showed shrunken body posture, damaged epithelial cells and microvillus as compared to control organisms. The biosafety of the isolated compound was assessed against G. affinis and did not produce mortality which confirmed that the activity of the isolated compound is species specific. The current study concludes that the critical success factors of new insecticidal agent development are based on the eco-compatibility and alternative tools for the pesticide producing industry.

Continuous treatment of diethyl hexyl and dibutyl phthalates by fixed-bed reactor: Comparison of two esterase bionanocomposites.

The removal of Diethyl hexyl phthalate (DEHP) and Dibutyl phthalate (DBP) is of great importance due to their potential adverse effects on the environment and human health. In this study, two bionanocomposites prepared by immobilization of Bacillus subtilis esterase by crosslinking to halloysite and supported in chitosan and alginate beads were studied and proposed as a green approach. The esterase immobilization was confirmed by physical-chemical characterization. Bionanocomposite using chitosan showed the best degradation levels in batch tests attaining complete degradation of DBP and around 90% of DEHP. To determine the operational stability and efficiency of the system, two fixed bed reactors filled with both bionanocomposites were carried out operating in continuous mode. Chitosan based bionanocomposite showed the best performance being able to completely remove DBP and more than 85% of DEHP at the different flowrates. These results proved the potential of these synthesized bionanocomposites to effectively remove Phthalic Acid Esters.

Sweetpotato chip texture and fat content: Effects of enzymatic modification of cell wall polymers.

Impacts of cell wall polymers on sweetpotato chip texture and fat content were investigated through enzymatic modification. Covington sweetpotato slices were treated with cellulase, hemicellulase, pectinase, pectin methyl esterase, protease, the enzyme blend Viscozyme, or no enzymes (control) at 40-45°C for 0.5-2 h. Treated slices were blanched, dried, and fried in triplicate per experimental condition. Breaking forces of 20 chips per frying replicate were measured followed by chip fat, moisture, sugar, alcohol insoluble solids, glass transition temperature, and color analyses. Untreated slices from each batch (daily check) were fried and analyzed to account for starting material variability. Viscozyme and protease-treated chips had the greatest reduction in breaking force from untreated chips (-30.9% and -23.7%, respectively), while pectin methyl esterase-treated chips had the lowest reduction in breaking force (-9.0%). Chips treated with Viscozyme for 2 h were 6.7-6.3 percentiles lower in fat than the control. Principal component analysis elucidated that chip breaking force was associated with unfried slice puncture force, alcohol insoluble solids, and chip color, and chip fat content was inversely associated with maltose content and glass transition temperature. Breaking down multiple cell wall polysaccharides or structural proteins weakened chip textures, while strengthening the pectic fraction resulted in harder chips. Chip fat reduction also occurred when multiple cell wall polysaccharides were broken down. Therefore, cell wall polymers impact sweetpotato chip texture and fat contents, and their attributes should be considered when selecting cultivars and processes for sweetpotato chips. PRACTICAL APPLICATION: Sweetpotato chips are an increasingly popular snack, but there is little understanding how cell wall polymers impact chip textures and fat contents. Raw sweetpotato slices were enzymatically treated to selectively modify cell wall polymers before frying. Chip breaking forces were lowered by protease or Viscozyme (cell wall enzyme blend) treatments, while breaking forces were increased with pectin methyl esterase. In addition, chip fat contents were reduced by the Viscozyme treatment. Since cell wall modifications could impact chip texture and fat content, cell wall polymer attributes should be considered in selection and processing of sweetpotatoes for chip manufacturing.

Metabolic Activation of Militarine In Vitro and In Vivo.

Bletilla striata is consumed as food and herbal medicine. Militarine (MLT) is a major ingredient in B. striata. Previous studies demonstrated that MLT showed teratogenic toxicity to zebrafish embryos. The present study aimed to identify reactive metabolites possibly involved in the cytotoxicity of MLT and determine the metabolic pathways involved. MLT was found to be hydrolyzed to p-hydroxybenzyl alcohol (HBA) by ß-glucosidase and esterases. The resulting HBA further underwent spontaneous dehydration to form quinone methide. HBA was also metabolized to the corresponding sulfate, followed by departure of the sulfate to generate a quinone methide. The resultant quinone methide reacted with hepatic glutathione (GSH) and protein to form the corresponding GSH conjugate and protein adduction. Additionally, inhibition of sulfotransferases (SULTs) attenuated the susceptibility of hepatocytes to the toxicity of MLT. This study provides that the hydrolytic enzymes ß-glucosidase, esterases, and SULTs participate in the metabolic activation of MLT.

Virulence and Reproductive Fitness of Meloidogyne paranaensis Field Populations in Coffee Genotypes.

The aim of this study was to characterize Meloidogyne paranaensis populations collected from infested coffee crops. Methodologies used to characterize the 11 studied populations from municipalities in Paraná and Minas Gerais States involved the morphological analysis of perineal patterns, biochemical analysis by isozyme electrophoresis, sequencing of internal transcribes spacer 1 (ITS-1) and D2/D3 ribosomal DNA (rDNA) regions, reproductive fitness, and virulence characterization in coffee genotypes. Morphological evaluations showed the existence of variation between populations, although the majority of them showed typical perineal patterns. The biochemical identification was based on α-esterase isozyme analyses and resulted in the appearance of three distinct profiles: P1 (typical), P2 (atypical), and a nondescribed profile, P2b. BLAST of the ITS-1 and D2/D3 rDNA regions indicated homology (>95%) with other sequences deposited in GenBank. For reproductive fitness and virulence characterization, 13 coffee genotypes (5 Coffea arabica and 8 C. canephora) were inoculated with 11 M. paranaensis populations. Variation in the reproductive fitness of populations was observed for cultivar Mundo Novo, a genotype without resistance genes, and variation in the virulence of populations was observed in genotypes carrying resistance genes. Three populations exhibited virulence combined with high reproductive fitness, while one showed virulence with low reproductive fitness. Some hosts were resistant to 11 populations, while one of the hosts was resistant to only one population, indicating the presence of different resistance genes. Nevertheless, no relationship was observed between the origin of population and their variations in perineal patterns, esterase profiles, phylogeny, or reproductive fitness in coffee genotypes, or between the different characterizations, although differences were observed within each characteristic.

The yin and yang of intracellular delivery of amphipathic optical probes using n-butyl charge masking.

Monitoring and manipulation of ionized intracellular calcium concentrations within intact, living cells using optical probes with organic chromophores is a core method for cell physiology. Since all these probes have multiple negative charges, they must be smuggled through the plasma membrane in a transiently neutral form, with intracellular esterases used to deprotect the masked anions. Here we explore the ability of the synthetically easily accessible n-butyl ester protecting group to deliver amphipathic cargoes to the cytosol. We show that the size of the caging chromophore conditions the ability of intracellular probe delivery and esterase charge unmasking.