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Safranal is a free radical scavenger and useful as an antioxidant molecule; however, its promotive role in soybean is not explored. Salt stress decreased soybean growth and safranal improved it even if under salt stress. To study the positive mechanism of safranal on soybean growth, a proteomic approach was used. According to functional categorization, oppositely changed proteins were further confirmed using biochemical techniques. Actin and calcium-dependent protein kinase decreased in soybean root and hypocotyl, respectively, under salt stress and increased with safranal application. Xyloglucan endotransglucosylase/ hydrolase increased in soybean root under salt stress but decreased with safranal application. Peroxidase increased under salt stress and further enhanced by safranal application in soybean root. Actin, RuvB-like helicase, and protein kinase domain-containing protein were upregulated under salt stress and further enhanced by safranal application under salt stress. Dynamin GTPase was downregulated under salt stress but recovered with safranal application under salt stress. Glutathione peroxidase and PfkB domain-containing protein were upregulated by safranal application under salt stress in soybean root. These results suggest that safranal improves soybean growth through the regulation of cell wall and nuclear proteins along with reactive‑oxygen species scavenging system. Furthermore, it might promote salt-stress tolerance through the regulation of membrane proteins involved in endocytosis and post-Golgi trafficking. SIGNIFICANCE: To study the positive mechanism of safranal on soybean growth, a proteomic approach was used. According to functional categorization, oppositely changed proteins were further confirmed using biochemical techniques. Actin and calcium-dependent protein kinase decreased in soybean root and hypocotyl, respectively, under salt stress and increased with safranal application. Xyloglucan endotransglucosylase/ hydrolase increased in soybean root under salt stress but decreased with safranal application. Peroxidase increased under salt stress and further enhanced by safranal application in soybean root. Actin, RuvB-like helicase, and protein kinase domain-containing protein were upregulated under salt stress and further enhanced by safranal application under salt stress. Dynamin GTPase was downregulated under salt stress but recovered with safranal application under salt stress. Glutathione peroxidase and PfkB domain-containing protein were upregulated by safranal application under salt stress in soybean root. These results suggest that safranal improves soybean growth through the regulation of cell wall and nuclear proteins along with reactive‑oxygen species scavenging system. Furthermore, it might promote salt-stress tolerance through the regulation of membrane proteins involved in endocytosis and post-Golgi trafficking., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Lysosome, an organelle which contains a number of hydrolases and hydrogen ions, plays a crucial role in cellular survival and apoptosis. If selectively destroy lysosomes membrane, inner hydrolases and hydrogen ions will leak and induce cell death. In this work, three lysosome-targeting fluorescent probes (HCL 1-3, heptamethine cyanine lysosomal-targeting probe) were designed, synthesized and developed for photodynamic therapy. Piperazine and N, N-dimethyl structures made HCL 1-3 have good lysosome targeting ability while Pearson's correlation coefficients reached 0.85, 0.87 and 0.78. It can be concluded from MTT test, HCL 1-3 have high photo cytotoxicity and low dark cytotoxicity from MTT test. Calcein/PI staining assays also supported cytotoxicity of HCL 1-3 under light conditions. In vivo experiments, HCL 2 accumulated in tumor and a strong fluorescence signal was observed at 12 h post injection. All results showed that our experiments provide help and new ideas for cyanine dyes in cancer treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

In this study, the complete proteome of goat ejaculated semen including spermatozoa and seminal plasma was established, applying a tandem mass tag (TMT) labeling together with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In seminal plasma, 2299 proteins were identified and 2098 proteins were quantified. The GO analysis demonstrated that 32% proteins were involved in metabolic activities. 46% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. Regarding molecular function, 40% proteins are engaged on protein binding, hydrolase activity, and ion binding. The KEGG analysis indicated a primary involvement of the identified proteins in protein processing in endoplasmic reticulum, lysosome, and proteome. In spermatozoa, 2491 proteins were identified and quantified. 39% proteins are involved in metabolic activities. 48% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. 38% proteins are engaged on protein binding, hydrolase activity, and ion binding. The KEGG analysis demonstrated their roles derived from the identified proteins in proteasome, glycolysis, pyruvate metabolism, and citrate cycle. Additionally, 1312 proteins were simultaneously presented in spermatozoa and seminal plasma. The involvement of 42% proteins in metabolic activities were observed. 47% proteins are located at intracellular region, intracellular organelle, and membrane-bounded organelle. The common proteins are mainly engaged on protein processing in endoplasmic reticulum, proteome, glycolysis, lysosome, and citrate cycle. Collectively, this study established the protein database of goat semen. More studies should be used to elucidate functionality of these identified proteins., (Copyright © 2022. Published by Elsevier Ltd.)

Novel chemical biology probes linking a serine hydrolase-directed fluorophosphonate warhead and cereblon-binding pomalidomide were assessed for the degradation of serine hydrolases. A quantitative proteomics approach to detect degraded proteins revealed that, despite the engagement of ∼40 serine hydrolases, degradation was achieved for only a single serine hydrolase, lysophospholipase II (LYPLA2)., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The gut microbiome is the resident microbial community of the gastrointestinal tract. This community is highly diverse, but how microbial diversity confers resistance or susceptibility to intestinal pathogens is poorly understood. Using transplantation of human microbiomes into several animal models of infection, we show that key microbiome species shape the chemical environment of the gut through the activity of the enzyme bile salt hydrolase. The activity of this enzyme reduced colonization by the major human diarrheal pathogen Vibrio cholerae by degrading the bile salt taurocholate that activates the expression of virulence genes. The absence of these functions and species permits increased infection loads on a personal microbiome-specific basis. These findings suggest new targets for individualized preventative strategies of V. cholerae infection through modulating the structure and function of the gut microbiome., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Lipid bilayer nanodiscs are an attractive tool to study membrane proteins in a detergent-free lipid-bilayer environment. In the case of NMR studies, a sequence-specific resonance assignment is required in order to gain structural and functional insights with atomic resolution. Although NMR backbone assignments of membrane proteins in detergents are available, they are largely absent for membrane proteins in nanodiscs due to unfavorable relaxation properties of the slowly tumbling membrane protein-nanodisc complex. The necessary residue-specific reassignment of resonances in nanodiscs is therefore extremely time and sample consuming and represents the fundamental bottleneck in the application of nanodiscs for NMR studies. Here we present an elegant and fast solution to the problem. We show that a resonance assignment in detergent micelles can be transferred to a spectrum recorded in nanodiscs via detergent titration. The procedure requires that lipid-detergent exchange kinetics are in the fast exchange regime in order to follow linear and nonlinear peak shift trajectories with increasing detergent concentration. We demonstrate the feasibility of the approach on the 148-residue membrane protein OmpX. The titration method is then applied to VDAC, a 19-stranded β-barrel with 283 residues, for which 67% of the detergent assignment could be transferred to the nanodisc spectrum. We furthermore show that this method also works for the largest currently assigned membrane protein, BamA with 398 residues. The method is applicable for backbone amide and side chain methyl groups and represents a time and cost-effective assignment method, for example, to investigate membrane protein allostery and drug binding in a more natural and detergent-free lipid bilayer.

Background: Mites of the genus Chorioptes are non-burrowing and cause mange in a wide range of domestic and wild animals including cattle, horses, sheep, goats, panda, moose, camelids, mydaus and alpacas. Molecular biology and host-parasite interactions of Chorioptes texanus are poorly understood, and only a few C. texanus genes and transcript sequences are available in public databases including the allergen genes., Methods: Chorioptes texanus RNA was isolated from mites, and the transcriptome of C. texanus was analyzed using bioinformatics tools. Chorioptes texanus unigenes were compared with the allergen protein sequences from the mite allergen database website to predict the potential allergens. Chorioptes texanus putative allergen unigenes were compared with hydrolase genes by building a C. texanus hydrolase gene library with the best match of the homologous sequences. Three allergen genes were cloned and expressed, their recombinant proteins were purified and their allergenic activities were preliminarily investigated., Results: Transcriptome sequencing (RNA-Seq) of C. texanus was analyzed and results demonstrated that 33,138 unigenes were assembled with an average length of 751 bp. A total of 15,130 unigenes were annotated and 5598 unigenes were enriched in 262 KEGG signaling pathways. We obtained 209 putative allergen genes and 34 putative allergen-hydrolase genes. Three recombinant allergen proteins were observed to induce different degrees of allergic reactions on rabbit skin., Conclusions: The present transcriptome data provide a useful basis for understanding the host-parasite interaction and molecular biology of the C. texanus mite. The allergenic activities of recombinant Euroglyphus maynei 1-like (Eur m 1-like) protein, Dermatophagoides ptreronyssinus 1-like (Der p 1-like) protein and Dermatophagoides ptreronyssinus 7-like (Der p 7-like) protein were preliminarily investigated by intradermal skin test. Meanwhile, differences in eosinophil counts were observed in different injected sites of the skin. The identification of putative allergen genes and hydrolase genes offers opportunities for the development of new diagnostic, prevention and treatment methods.

Serine hydrolases play crucial biological roles and are important therapeutic targets in many clinical applications. Activity-based protein profiling of serine hydrolases by using fluorophosphonate probes, pioneered by Cravatt and co-workers, has been a powerful tool for interrogating serine hydrolases in various biological systems. Herein, we present new phenyl phosphonate probes with an azide handle for click chemistry that offer remarkable improvements over the classical fluorophosphonate serine hydrolase activity-based probes including ease of preparation, excellent cell permeability, and distinct reactivity profiles, as controlled by the phenolate leaving group. Thus, these new activity-based serine hydrolase probes are valuable tools to further interrogate this important class of enzymes., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This paper aims to characterize halophilic bacteria inhabiting Algerian Saline Ecosystems (Sebkha and Chott) located in arid and semi-arid ecoclimate zones (Northeastern Algeria). In addition, screening of enzymatic activities, heavy metal tolerance and antagonistic potential against phytopathogenic fungi were tested. A total of 74 bacterial isolates were screened and phylogenetically characterized using 16S rRNA gene sequencing. The results showed a heterogeneous group of microorganisms falling within two major phyla, 52 strains belonging to Firmicutes (70.2%) and 22 strains (30.8%) of γ-Proteobacteria. In terms of main genera present, the isolates were belonging to Bacillus, Halobacillus, Lentibacillus, Oceanobacillus, Paraliobacillus, Planomicrobium, Salicola, Terribacillus, Thalassobacillus, Salibacterium, Salinicoccus, Virgibacillus, Halomonas, Halovibrio, and Idiomarina. Most of the enzymes producers were related to Bacillus, Halobacillus, and Virgibacillus genera and mainly active at 10% of growing salt concentrations. Furthermore, amylase, esterase, gelatinase, and nuclease activities ranked in the first place within the common hydrolytic enzymes. Overall, the isolates showed high minimal inhibitory concentration values (MIC) for Ni 2+ and Cu 2+ (0.625 to 5 mM) compared to Cd 2+ (0.1 to 2 mM) and Zn 2+ (0.156 to 2 mM). Moreover, ten isolated strains belonging to Bacillus, Virgibacillus and Halomonas genera, displayed high activity against the pathogenic fungi (Botrytis cinerea, Fusarium oxyporum, F. verticillioides and Phytophthora capsici). This study on halophilic bacteria of unexplored saline niches provides potential sources of biocatalysts and novel bioactive metabolites as well as promising candidates of biocontrol agents and eco-friendly tools for heavy metal bioremediation.

A cocktail of biomass hydrolytic enzymes was produced by solid-state fermentation (SSF) by the mutant strain Aspergillus niger 3T5B8, using as substrate a mixture of grape pomace and wheat bran, and compared to the production when wheat bran was used as the sole substrate. The two enzymatic cocktails were subsequently used for the extraction of bioactive compounds from grape pomace and the relationship between the activities of the cocktail and the release of phenolic compounds was evaluated. Although the wheat bran SSF process was more effective for enzyme production, the enzymatic cocktail produced by the grape pomace - wheat bran mixture was more effective for the extraction of compounds with higher proanthocyanidins content and higher antioxidant potential (p < 0.05). A significant correlation between the bioactive compounds and enzyme activity was observed., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The fungus Trichoderma reesei is employed in the production of most enzyme cocktails used by the lignocellulosic biofuels industry today. Despite significant improvements, the cost of the required enzyme preparations remains high, representing a major obstacle for the industrial production of these alternative fuels. In this study, a new Trichoderma erinaceum strain was isolated from decaying sugarcane straw. The enzyme cocktail secreted by the new isolate during growth in pretreated sugarcane straw-containing medium presented higher specific activities of β-glucosidase, endoxylanase, β-xylosidase and α-galactosidase than the cocktail of a wild T. reesei strain and yielded more glucose in the hydrolysis of pretreated sugarcane straw. A proteomic analysis of the two strains' secretomes identified a total of 86 proteins, of which 48 were exclusive to T. erinaceum, 35 were exclusive to T. reesei and only 3 were common to both strains. The secretome of T. erinaceum also displayed a higher number of carbohydrate-active enzymes than that of T. reesei (37 and 27 enzymes, respectively). Altogether, these results reveal the significant potential of the T. erinaceum species for the production of lignocellulases, both as a possible source of enzymes for the supplementation of industrial cocktails and as a candidate chassis for enzyme production., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Trimeresurus nebularis is a montane pit viper that causes bites and envenomation to various communities in the central highland region of Malaysia, in particular Cameron's Highlands. To unravel the venom composition of this species, the venom proteins were digested by trypsin and subjected to nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) for proteomic profiling. Snake venom metalloproteinases (SVMP) dominated the venom proteome by 48.42% of total venom proteins, with a characteristic distribution of P-III: P-II classes in a ratio of 2:1, while P-I class was undetected. Snaclecs constituted the second most venomous protein family (19.43%), followed by snake venom serine proteases (SVSP, 14.27%), phospholipases A₂ (5.40%), disintegrins (5.26%) and minor proteins including cysteine-rich secretory proteins, L-amino acid oxidases, phosphodiesterases, 5'-nucleotidases. The venomic profile correlates with local (painful progressive edema) and systemic (hemorrhage, coagulopathy, thrombocytopenia) manifestation of T. nebularis envenoming. As specific antivenom is unavailable for T. nebularis , the hetero-specific Thai Green Pit viper Monovalent Antivenom (GPVAV) was examined for immunological cross-reactivity. GPVAV exhibited good immunoreactivity to T. nebularis venom and the antivenom effectively cross-neutralized the hemotoxic and lethal effects of T. nebularis (lethality neutralizing potency = 1.6 mg venom per mL antivenom). The findings supported GPVAV use in treating T. nebularis envenoming.

Enzymes are a class of macromolecules that function as highly efficient and specific biological catalysts requiring only mild reaction conditions. Enzymes are essential to maintaining life activities, including promoting metabolism and homeostasis, and participating in a variety of physiological functions. Accordingly, enzymatic levels and activity are closely related to the health of the organism, where enzymatic dysfunctions often lead to corresponding diseases in the host. Due to this, diagnosis of certain diseases is based on the levels and activity of certain enzymes. Therefore, rapid real-time and accurate detection of enzymes in situ are important for diagnosis, monitoring, clinical treatment and pathological studies of disease. Fluorescent probes have unique advantages in terms of detecting enzymes, including being simple to use in highly sensitive and selective real-time rapid in-situ noninvasive and highly spatial resolution visual imaging. However, fluorescent probes are most commonly used to detect oxidoreductases, transferases and hydrolases due to the processes and types of enzyme reactions. This paper summarizes the application of fluorescent probes to detect these three types of enzymes over the past five years. In addition, we introduce the mechanisms underlying detection of these enzymes by their corresponding probes., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

We have performed a systematic study to analyze the effect of ssDNA length, nucleobase composition, and the type of two-dimensional nanoparticles (2D-nps) on the desorption response of 36 two-dimensional nanoassemblies (2D-NAs) against several proteins. The studies were performed using fluorescently labeled polyA, polyC, and polyT with 23, 18, 12, and 7 nucleotide-long sequences. The results suggest that the ssDNAs with polyC and longer sequences are more resistant to desorption, compared to their counterparts. In addition, 2D-NAs assembled using WS 2 were least susceptible to desorption by the proteins tested, whereas nGO 2D-NAs were the most susceptible nanoassemblies. Later, the results of these systematic studies were used to construct a sensor array for discrimination of seven model proteins (BSA, lipase, alkaline phosphatase, acid phosphatase, protease, β-galactosidase, and Cytochrome c). Neither the ssDNAs nor the 2D-nps have any specific interaction with the proteins tested. Only the displacement of the ssDNAs from the 2D-np surface was measured upon the disruption of the existing forces within 2D-NAs. A customized sensor array with five 2D-NAs was developed as a result of a careful screening/filtering process. The sensor array was tested against 200 nM of protein targets, and each protein was discriminated successfully. The results suggest that the systematic studies performed using various ssDNAs and 2D-nps enabled the construction of a sensor array without a bind-and-release sensing mechanism. The studies also demonstrate the significance of systematic investigations in the construction of two-dimensional DNA nanoassemblies for functional studies.

Bacterial fish pathogens are pervasive in aquaculture. Control of bacterial fish pathogen is a difficult task among aquaculture practitioners. A large number of antibiotics are used for the control of prevalent bacterial pathogens in aquaculture. This may lead to drug resistance among pathogens and further treatment will be ineffective. Here, we can use probiotic bacteria as a biocontrol agent in fish disease and it is a novel field. In this study, antimicrobial potential of the bacterium Bacillus coagulans (MTCC-9872) has been evaluated through in vitro antagonistic activity of cellular preparations/components against potent pathogens. The cellular preparations/components such as Ethyl acetate extract, whole-cell product, heat-killed whole-cell product, and filtered broth were exhibited bactericidal activity against the tested pathogens. Bactericidal activity varied among different cellular preparation/components. The tested bacterium effectively produced biofilm as significant as tested positive control in a microtitre plate and effectively adhered on to the glass slide. In addition, the bacterium was capable of producing extracellular enzymes necessary for the digestion of food materials and was capable to grow in fish mucus from Oreochromis niloticus. The bacterium tolerated bile juice secreted by the host. Moreover, intraperitoneal injection of the bacterium did not induce any pathological signs, symptoms or mortalities in Oreochromis niloticus and revealed the safety of this bacterium in the fish.

Bioturbation and bioirrigation induced by burrowing macrofauna are recognized as important processes in aquatic sediment since macrofaunal activities lead to the alteration of sediment characteristics. However, there is a lack of information on how macrofauna influence microbial abundance and extracellular enzyme activity in mangrove sediment. In this study, the environmental parameters, extracellular enzyme activities, and microbial abundance were determined and their relationships were explored. Sediment samples were taken from the surface (S) and lower layer (L) without burrow, as well as crab burrow wall (W) and bottom of crab burrow (B) located at the Mai Po Nature Reserve, Hong Kong. The results showed that the burrowing crabs could enhance the activities of oxidase and hydrolases. The highest activities of phenol oxidase and acid phosphatase were generally observed in B sediment, while the highest activity of N-acetyl-glucosaminidase was found in W sediment. The enzymatic stoichiometry indicated that the crab-affected sediment had similar microbial nitrogen (N) and phosphorous (P) availability relative to carbon (C), lower than S but higher than L sediment. Furthermore, it was found that the highest abundance of both bacteria and fungi was shown in S sediment, and B sediment presented the lowest abundance. Moreover, the concentrations of phosphorus and soluble phenolics in crab-affected sediment were almost higher than the non-affected sediment. The alterations of phenolics, C/P and N/P ratios as well as undetermined environmental factors by the activities of crabs might be the main reasons for the changes of enzyme activity and microbial abundance. Finally, due to the important role of phenol oxidase and hydrolases in sediment organic matter (SOM) decomposition, it is necessary to take macrofaunal activities into consideration when estimating the C budget in mangrove ecosystem in the future.

Background: Vulvovaginal candidiasis (VVC) is caused by overgrowth of Candida species in the female lower genital tract and most commonly caused by Candida albicans. The production of various virulence factors may attribute to their pathogenicity. Hence, this study was aimed to determine the production of various virulence factors of Candida spp. causing VVC., Materials and Methods: A total of 51 Candida spp. were isolated prospectively from 50 patients among 211 clinically suspected cases of VVC. The haemolytic activity, biofilm production, proteinase activity, phospholipase activity and esterase activity were detected by standard methods. Statistical analysis was performed using OpenEpi version 3.01., Results: Haemolytic activity was observed in 42 Candida isolates (82.4%), biofilm activity in 21 Candida isolates (41.2%), proteinase and esterase activity in 19 Candida isolates (37.3%) each and phospholipase activity in 15 Candida isolates (29.4%). Phospholipase activity was observed in all of the C. albicans strains, whereas all strains of Candida krusei were able to produce biofilm. All strains of Candida parapsilosis and 87% strains of Candida glabrata were haemolytic. Five of the eight C. glabrata strains were found to produce strong proteinase (Pr z score ≤0.63). About 30.4% strains of C. glabrata and 20% strains of C. krusei were found to be positive for esterase activity. This is one of the few studies which revealed esterase activity among C. glabrata and C. krusei strains., Conclusions: This study highlighted that there is a change in the virulence factors among the non-albicans Candida species, especially C. glabrata strains which were haemolytic and produce strong proteinase activity and esterase activity. It may be one of the explanation of the most common causative agent of VVC in our study. Multicentric studies from this area might be required to get a more generalised conclusion., Competing Interests: There are no conflicts of interest

Fluorine can flow into the environment after leakage or spill accidents and these excessive amounts can cause adverse effects on terrestrial ecosystems. Using three media (filter paper, soil, and filter-paper-on-soil), we investigated the toxic effects of fluorine on the germination and growth of crops (barley, mung bean, sorghum, and wheat), on the activities of soil exoenzymes (acid phosphatase, arylsulfatase, fluorescein diacetate hydrolase, and urease) and on the survival, abnormality, and cytotoxicity of Eisenia andrei earthworms. The germination and growth of crops were affected by fluorine as exposure concentration increased. The activities of the four enzymes after 0-, 3-, 10-, and 20-day periods varied as exposure concentration increased. According to in vivo and in vitro earthworm assays, E. andrei mortality, abnormality, and cytotoxicity increased with increasing fluorine concentration. Overall, fluorine significantly affected each tested species in the concentration ranges used in this study. The activities of soil exoenzymes were also affected by soil fluorine concentration, although in an inconsistent manner. Albeit the abnormally high concentrations of fluorine in soil compared to that observed under natural conditions, its toxicity was much restrained possibly due to the adsorption of fluorine on soil particles and its combination with soil cations., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The virulence properties of Candida spp. presents in landfill leachate still unknown until today and they constitutes a serious source of potential danger for humans. We investigate the antifungal susceptibility, production of hydrolytic enzymes and biofilm formation on polystyrene as well as glass in Candida spp. strains isolated from a landfill leachate treatment station in Borj Chakir (Tunisia). 37 yeast strains were isolated belonging to the following species: C. robusta, C. lusitaniae, C. tropicalis, C. krusei. Most isolated yeast strains were resistant to Amphotericin B, produced several hydrolytic enzymes (67.56% produced phospholipase, 86.04% protease, 64.86% esterase) and most of them are able to degrade hemoglobin. All assayed Candida strains have been able to form biofilm on polystyrene depending on the species and strain of Candida. Landfills receiving clinical waste are a potential source of Candida ssp. strains with several virulence properties which allow them to survive in different aquatic biotopes., (Copyright © 2018. Published by Elsevier Ltd.)

Background: Daqu is a fermentative saccharification agent that is used to initiate fermentation in the production of Chinese liquor and vinegar. This study investigated the differences of amylase, protease and esterase in dominance of different types of Daqu, which can be useful for quality control and flavor improvement of Daqu production by enzyme technology., Results: Hydrolase activities in different Daqu samples were compared by principal component analysis (PCA). Based on protein electrophoresis and 1 H NMR spectroscopy, the protein patterns and metabolites in Daqu were further analysed. The results indicated that the highest amylase activities and diversities were found in low/medium-temperature of Daqu which had light-flavour and strong-flavour. Proteases play a significant role in determining the quality of high-temperature Daqu samples which had a sauce-flavour. Furthermore, the contributions of esterase to both strong and sauce flavour development in high-temperature Daqu are similar., Conclusion: Results from the present work showed that differences in amylase, protease and esterase play a leading role in different types of Daqu, which can be useful for quality control and technology development of Daqu. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

The remediation of chlorinated solvent contaminated sites frequently involves bioaugmentation with mixed cultures containing Dehalococcoides mccartyi. Their activity is then examined by quantifying reductive dehalogenase (RDase) genes. Recently, we described a rapid, low cost approach, based on loop mediated isothermal amplification (LAMP), which allowed for the visual detection of RDase genes from groundwater. In that study, samples were concentrated (without DNA extraction), incubated in a water bath (avoiding the use of a thermal cycler) and amplification was visualized by the addition of SYBR green (post incubation). Despite having a detection limit less than the threshold recommended for effective remediation, the application of the assay was limited because of the semi-quantitative nature of the data. Moreover, the assay was prone to false positives due to the aerosolization of amplicons. In this study, deoxyuridine triphosphate (dUTP) and uracil DNA glycosylase (UNG) were incorporated into the assay to reduce the probability of false positives. Optimization experiments revealed a UNG concentration of 0.2units per reaction was adequate for degrading trace levels of AUGC based contamination (~1.4×10 4 gene copies/reaction) without significant changes to the detection limit (~100 gene copies/reaction). Additionally, the optimized assay was used with the most probable number (MPN) method to quantify RDase genes (vcrA and tceA) in multiple groundwater samples from a chlorinated solvent contaminated site. Using this approach, gene concentrations were significantly correlated to concentrations obtained using traditional methods (qPCR and DNA templates). Although the assay underestimated RDase genes concentrations, a strong correlation (R 2 =0.78 and 0.94) was observed between the two data sets. The regression equations obtained will be valuable to determine gene copies in groundwater using the newly developed, low cost and time saving method., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The production of enzymes in clinical isolates of Candida haemulonii (Ch, n = 5), Candida duobushaemulonii (Cd, n = 4) and Candida haemulonii var. vulnera (Chv, n = 3) was identified by agar plate. Aspartic protease, phytase, caseinolytic and hemolytic activities were detected in all the isolates. A distinct scenario was evidenced regarding the production of lipases. In this way, 80%, 50% and 100% of Ch, Cd and Chv, respectively, were phospholipase producers. Regarding esterase activity, 100%, 50% and 66.7% of Ch, Cd and Chv, respectively, were positive isolates. Esterase activity was significantly higher in isolates recovered from cutaneous candidiasis compared with those recovered from body fluids., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Yarrowia lipolytica is ubiquitous in the environment, opportunistic, and might be considered as one of the causative agents of catheter-related candidemia. Our work aimed to study some virulence factors of Y. lipolytica such as hydrolases production and biofilm formation with comparison to the most frequent Candida specie in human disease. In sum, 58 clinical isolates of Y. lipolytica, 16 C. glabrata, and 12 C. albicans were collected from Intensive care unit (ICU). All were tested for enzymatic production and biofilm formation. All tested isolates of C. albicans and C. glabrata were able to degrade casein, and 98.2% of Y. lipolytica showed caseinase activity but no gelatinase activity was detected in all isolates. Y. lipolytica strains showed significantly lower (3.4%) in vitro phospholipase activity than C. albicans and C. glabrata (P < .05). No significant differences of the hemolytic activity were detected between the three species (P > .05). Concerning biofilm formation, and unlike the results obtained on polystyrene plate, the number of adhered and biofilm cultivable cells obtained by Y. lipolytica after 168 hours of catheter subcutaneous implantation is significantly greater and tends to be more compact and structured hyphal layer. Although C. albicans remains the most pathogenic yeast, development of selective ability of Y. lipolytica to adhere, to form a biofilm on catheter medical devices, and to produce phospholipase and hemolytic enzyme is of particular interest, and it is strongly recommended to be vigilant in the use of medical implanted medical devices, particularly in ICU., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

It is essential to remediate or amend soils contaminated with various heavy metals or pollutants so that the soils may be used again safely. Verifying that the remediated or amended soils meet soil quality standards is an important part of the process. We estimated the activity levels of eight soil exoenzymes (acid phosphatase, arylsulfatase, catalase, dehydrogenase, fluorescein diacetate hydrolase, protease, urease, and ß-glucosidase) in contaminated and remediated soils from two sites near a non-ferrous metal smelter, using colorimetric and titrimetric determination methods. Our results provided the levels of activity of soil exoenzymes that indicate soil health. Most enzymes showed lower activity levels in remediated soils than in contaminated soils, with the exception of protease and urease, which showed higher activity after remediation in some soils, perhaps due to the limited nutrients available in remediated soils. Soil exoenzymes showed significantly higher activity in soils from one of the sites than from the other, due to improper conditions at the second site, including high pH, poor nutrient levels, and a high proportion of sand in the latter soil. Principal component analysis revealed that ß-glucosidase was the best indicator of soil ecosystem health, among the enzymes evaluated. We recommend using ß-glucosidase enzyme activity as a prior indicator in estimating soil ecosystem health., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We aimed to assess the feasibility of enhancing the intestinal development of weaned rats using glucagon-like peptide-2 (GLP-2)-expressing Saccharomyces cerevisiae (S. cerevisiae). GLP-2-expressing S. cerevisiae (GLP2-SC) was generated using a recombinant approach. The diet of weaned rats was supplemented with the GLP2-SC strain. The average daily gain (ADG), the intestinal morphology and the activities of the digestive enzymes in the jejunum were tested to assess the influence of the GLP2-SC strain on intestinal development. The proliferation of rat enterocytes was also assessed in vitro. The study revealed that the ADG of the weaned rats that received GLP2-SC was significantly greater than that of the controls fed a basal diet (Control) and S. cerevisiae harbouring an empty vector (EV-SC) (P < 0.05) but was equivalent to that of positive control rats fed recombinant human GLP-2 (rh-GLP2) (P > 0.05). Furthermore, GLP2-SC significantly increased villous height (P < 0.01) and digestive enzyme activity (P < 0.05) in the jejunum. Immunohistochemistry analysis further affirmed that enterocyte proliferation was stimulated in rats fed the GLP2-SC strain, as indicated by the greater number of enterocytes stained with proliferative cell nuclear antigen (P < 0.05). In vitro, the proliferation of rat enterocytes was also stimulated by GLP-2 expressed by the GLP2-SC strain (P < 0.01). Herein, the combination of the GLP-2 approach and probiotic delivery constitute a possible dietary supplement for animals after weaning., (© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Enzyme triggered probes with a self-immolative linker for rapid and sensitive hydrolase detection through a cascade reaction have been reported. Their utility was proved by the preparation of three model compounds and their evaluation as enzyme substrates and demonstration of their applicability as fluorogenic probes for screening lipase, esterase and protease activities. These probes represent a new class of fluorogenic compounds, are stable under aqueous conditions and not susceptible to nonspecific degradation. The utilization of the carbamate cleavable linkage in a probe structure allows moving away of the bulky fluorophore from the enzyme recognition unit and targets different classes of enzymes with the same substrate.

Bacteria utilize small-molecule iron chelators called siderophores to support growth in low-iron environments. The Escherichia coli catecholate siderophore enterobactin is synthesized in the cytoplasm upon iron starvation. Seven enzymes are required for enterobactin biosynthesis: EntA-F, H. Given that EntB-EntE and EntA-EntE interactions have been reported, we investigated a possible EntA-EntB-EntE interaction in E. coli cells. We subcloned the E. coli entA and entB genes into bacterial adenylate cylase two-hybrid (BACTH) vectors allowing for co-expression of EntA and EntB with N-terminal fusions to the adenylate cyclase fragments T18 or T25. BACTH constructs were functionally validated using the CAS assay and growth studies. Co-transformants expressing T18/T25-EntA and T25/T18-EntB exhibited positive two-hybrid signals indicative of an intracellular EntA-EntB interaction. To gain further insights into the interaction interface, we performed computational docking in which an experimentally validated EntA-EntE model was docked to the EntB crystal structure. The resulting model of the EntA-EntB-EntE ternary complex predicted that the IC domain of EntB forms direct contacts with both EntA and EntE. BACTH constructs that expressed the isolated EntB IC domain fused to T18/T25 were prepared in order to investigate interactions with T25/T18-EntA and T25/T18-EntE. CAS assays and growth studies demonstrated that T25-IC co-expressed with the EntB ArCP domain could complement the E. coli entB(-) phenotype. In agreement with the ternary complex model, BACTH assays demonstrated that the EntB IC domain interacts with both EntA and EntE., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The activities of seven hydrolytic enzymes (L-alanine aminopeptidase, esterase, α-and β-glucosidase, phosphomonoesterase, phosphodiesterase, sulfatase) were monitored during 1 year in parallel and serial treatment units of the biological stage of a communal wastewater treatment plant. The spatial homogeneity of enzyme activities was high (coefficients of variation <10 % for the entire treatment stage). A significant difference between aerated and stirred tanks was not observed. Temperature seemed not to exert a direct influence. Long periods with comparably constant activities were interrupted by a few strong, short-time rises. The mean enzyme activities followed the sequence sulfatase < α-glucosidase < phosphodiesterase ≈ β-glucosidase≈esterase < phosphomonoesterase < L-alanine aminopeptidase. The enzyme activities correlated among themselves at different levels. Very strong (r > 0.8) and highly significant (p < 0.01) correlations between the activities of both glucosidases, both phosphoesterases, and between phosphomonoesterase and both glucosidases were ascertained, pointing to the importance of substrate specificity and similarity of metabolic functions. Moderate and strong activity correlations with various wastewater constituents and with process parameters, e.g., concentrations, loads and eliminated amounts of phosphorous, TOC concentrations and loads of the plant effluent, dry matter content of activated sludge, and sludge volume, were found. The esterase activity was least correlated with other enzymes and often showed deviating dependencies on process parameters, raising questions concerning its appropriateness as a sum parameter for enzymatic and heterotrophic activity.

Anti-neutrophil cytoplasmic antibody (ANCA) is associated with small-vessel vasculitis particularly in the kidneys and can induce the formation of neutrophil extracellular traps (NETs) from primed neutrophils. Recently we have reported that the induction of NETs correlates with ANCA affinity for myeloperoxidase (MPO) and disease activity in patients with MPO-ANCA-associated microscopic polyangiitis. To investigate whether MPO-ANCA affinity is associated with the formation of NETs in vivo, we examined the occurrence of NETs in the renal tissues of patients with MPO-ANCA-associated microscopic polyangiitis and ANCA affinity by double immunofluorescence staining for NET components of citrullinated histone, MPO and PAD4 and by ELISA competition with MPO, respectively. We divided 30 MPO-ANCA-associated microscopic polyangiitis patients into 2 groups based on their ANCA affinity levels (IC50 for the high: 0.11 ± 0.04 µg/mL (Group1) and IC50 for the low: 0.66 ± 0.24 µg/mL (Group2)). Group1 showed a higher Birmingham vasculitis activity score (15.6 ± 5.7) and 73% of the patients presented clinically with rapidly progressive glomerulonephritis and histologically with focal/crescentic glomerulonephritis (GN). Group 2 showed a lower Birmingham vasculitis activity score (9.2 ± 4.9) and 73% of the patients presented clinically with chronic renal failure and histologically with mixed/sclerotic GN. Group 1 showed a much higher occurrence of NETs than Group 2. Our findings indicate that ANCA affinity was associated with the in vivo formation of NETs, which might be involved in the pathophysiology of patients with MPO-ANCA-associated microscopic polyangiitis., (© 2016 The Authors Nephrology published by John Wiley & Sons Australia, Ltd on behalf of Asian Pacific Society of Nephrology.)

The antagonistic potential of Trichoderma strains was assayed by studying the effect of their culture filtrate on the radial growth of Sclerotium rolfsii, the causal agent of chickpea collar rot. Trichoderma harzianum-1432 (42.2%) and Trichoderma atroviride (40.3%) were found to be strong antagonists. To enhance their antagonistic potential, mutagenesis of these two selected strains was performed. Two mutants, Th-m1 and T. atroviride m1, were found to be more effective than their parent strains. The enzymatic activities of the selected parent and mutant strains were assayed, and although both mutants were found to have enhanced enzymatic activities compared to their respective parent strains, Th-m1 possessed the maximum cellulase (5.69U/mL) and β-1,3-glucanase activity (61.9U/mL). Th-m1 also showed high competitive saprophytic ability (CSA) among all of the selected parent and mutant strains, and during field experiments, Th-m1 was found to successfully possess enhanced disease control (82.9%)., (Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Four strains of Hortaea werneckii were isolated from different substrates in Brazil (a salt marsh macrophyte, a bromeliad and a marine zoanthid) and had their identification confirmed by sequencing of the 26S rDNA D1/D2 domain or ITS region. Most of the strains were able to express amylase, lipase, esterase, pectinase and/or cellulase, enzymes that recognize components of plant cells as substrates, but did not express albuminase, keratinase, phospholipase and DNAse, whose substrates are animal-related. Urease production was positive for all isolates, while caseinase, gelatinase and laccase production were variable among the strains. All the strains grew in media containing up to 30% NaCl. We propose that the primary substrate associated with H. werneckii is plant-related, in special in saline environments, where the fungus may live as a saprophyte and decomposer. Infection of animal-associated substrates would be secondary, with the fungus acting as an opportunistic animal pathogen. All strains were resistant to fluconazole and presented high MIC for amphotericin B, while they were susceptible to all the other antifungal agents tested.

Candida bracarensis is an uncommon Candida species found during an epidemiological study of candidiasis performed in Braga, Portugal. Initially, it was identified as C. glabrata, but recently detailed analyses pointed out their differences. So, little information is still available about C. bracarensis virulence factors and antifungal susceptibilities. Therefore, the main goal of this work is to evaluate the ability of C. bracarensis to form biofilms, to produce hydrolytic enzymes (proteases, phospholipases and hemolysins), as well as its susceptibility to amphotericin B and fluconazole. It was shown, for the first time, that all C. bracarensis strains were able to form biofilms and display proteinase and hemolytic activities. Moreover, although planktonic cells presented antifungal susceptibility, amphotericin B and fluconazole were unable to inhibit biofilm formation and eradicate pre-formed biofilms. Due to the propensity of C. bracarensis to display antifungal resistance and virulence attributes, the control of these emerging pathogens is recommended.

The Candida parapsilosis complex has emerged as an important fungal pathogen. In spite of this, relatively little is known about its characteristics. Thus, the purposes of this study were (1) to determine by BanI-RFLP-assay the occurrence of C. parapsilosis complex species among 81 clinical isolates primarily identified as C. parapsilosis; (2) to evaluate their in vitro production of virulence factors; and (3) to compare their susceptibility profiles, grown as planktonic cells and biofilms, against amphotericin B, fluconazole, voriconazole, and caspofungin by following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Seventy-seven isolates (95%) were identified as C. parapsilosis sensu stricto, 2 (2.5%) as C. orthopsilosis, and 2 (2.5%) as C. metapsilosis. Protease activity was detected in 29 (37.7%) isolates of C. parapsilosis sensu stricto, whereas only 7 (9.1%) exhibited phospholipase activity. None of the C. metapsilosis or C. orthopsilosis was able to produce protease or phospholipase. Biofilm production was detected in 35 (43.2%) isolates, among which 33 were C. parapsilosis sensu stricto and 2 were C. orthopsilosis. Antifungal resistance was uncommon; only one C. metapsilosis was fluconazole resistant. However, biofilm-producing isolates showed a marked resistance to all antifungal agents tested, particularly to voriconazole. This knowledge could be of clinical relevance for guiding therapeutic decisions.

Based on natural selection and the survival of the fittest by evolutionary adaption, a smart high-throughput system was developed to select active haloalkane dehalogenase variants from a large mutant library. Only active enzyme variants can hydrolyse toxic halogenated alkanes to promote growth, whereas inactive mutants starve or die due to the toxic compound. With this powerful tool, huge enzyme mutant libraries can be screened within a few days. The selection is done without any artificial substrates that are hard to synthesize and they also resemble typical ones for haloalkane dehalogenases. Three saturation libraries, with a size of more than 10(6) cells, based on inactive variants of the haloalkane dehalogenases DhaA or DhlA were successfully screened to retrieve active enzymes. The enrichment of the active wild-type enzyme in contrast to the inactive variants was about 340-fold. In addition, this selection approach can be applied for continuous directed evolution experiments for the enrichment of cells expressing adapted haloalkane dehalogenases.

The post-transitional modification of peptidyl arginine to citrulline by PAD2 can affect the inherent biophysical properties of the citrullinated protein. Furthermore, dysregulation of PAD2 activity has been implicated in a number of human diseases. Inhibition of these enzymes by small molecules can serve as essential probes in establishing a link to pathogenesis. Herein, we describe a profluorescent substrate analog that reports on the activity and the inhibition of PAD2 in a robust assay. Most noteworthy, we expect future drug discovery efforts based on PAD2 inhibition can be pursued via this assay., (© 2015 John Wiley & Sons A/S.)

Unlabelled: The aim of this cross sectional study was to evaluate the ureolytic and arginolytic activities of saliva in children and associate them with their caries status., Study Design: 65, 8 year old children, were randomly selected. The ureolytic and arginolytic activity of non stimulated saliva was studied and associated with DMFT and dmft index. Saliva of children were sampled under fasting conditions; Children refrained from any oral hygiene procedures during the 12 hours that preceded the sample collection. Caries activity was scored and divided in 3 groups: Group A: Index zero: without lesions; Group B: Moderate Index: 1 to 3 enamel caries lesions; and Group C: High Index: more than 4 dentin caries lesions., Results: DMFT scores were moderate: 0.4(±0.79) and dmft: 2.78(±2.45). Results expressed in μmol/min/mg/protein, for urease activity were statistically significant (p=0.048): Group A= 0.69 (±0.7); Group B= 0.45 (±0.43); and Group C= 0.39 (±0.55). The arginine deiminase activity was not statistically significant (p=0.16): Group A= 2.53 (±1.42), Group B= 2.31 (±1.57) and Group C= 1.97 (±2.0)., Conclusion: Higher levels of ureolytic (statistically significant) and arginolytic activity (trend) in saliva were associated with lower DMFT/dmft scores in 8 year old children. There was a higher production of ammonia from the arginine deiminase system than the urease enzyme in saliva (p>0.05).

Halophilic microorganisms are able to grow in the presence of salt and are also excellent source of enzymes and biotechnological products, such as exopolysaccharides (EPSs) and polyhydroxyalkanoates (PHAs). Salt-tolerant bacteria were screened in the Organic Composting Production Unit (OCPU) of São Paulo Zoological Park Foundation, which processes 4 ton/day of organic residues including plant matter from the Atlantic Rain Forest, animal manure and carcasses and mud from water treatment. Among the screened microorganisms, eight halotolerant bacteria grew at NaCl concentrations up to 4 M. These cultures were classified based on phylogenetic characteristics and comparative partial 16S rRNA gene sequence analysis as belonging to the genera Staphylococcus, Bacillus and Brevibacterium. The results of this study describe the ability of these halotolerant bacteria to produce some classes of hydrolases, namely, lipases, proteases, amylases and cellulases, and biopolymers. The strain characterized as of Brevibacterium avium presented cellulase and amylase activities up to 4 M NaCl and also produced EPSs and PHAs. These results indicate the biotechnological potential of certain microorganisms recovered from the composting process, including halotolerant species, which have the ability to produce enzymes and biopolymers, offering new perspectives for environmental and industrial applications.

The filamentous bacteria Streptomyces spp. produces diverse extracellular enzymes and other secondary metabolites. Proteomic analysis of the secretome of holocellulolytic Streptomyces sp. ssr-198 was done by tandem mass spectrometry using an Orbitrap Velos hybrid mass spectrometer. A wide range of hydrolytic enzymes, including glycoside hydrolases (17), proteases (17), polysaccharide lyases (3), esterases (2), and hypothetical proteins (14) were detected in the secretome analyzed. Overall, the secretome composition constituted of 12.50% cellulases, 17.50% hemicellulases, 21.25% proteases, 17.50% hypothetical proteins, and 31.25% other proteins. Comprehensive analysis of secretome will be useful in gaining better understanding of the unique role of hydrolytic enzymes in lignocellulose hydrolysis and helps in determining the industrial applications of these potent enzymes., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Different strategies have been employed for selective isolation of Streptomycetes from 20 marine samples varied in their biological nature. The recovery of Streptomycetes isolates (112) was influenced preferentially by different strategies; sediment samples were the best source of potential candidate Streptomycetes. All isolates exhibited antimicrobial activities with variable spectrum; the most promising isolates (31) were phenotypically characterized and identified as Streptomyces sp.; these isolates exhibited variable capacity for secretion of numerous hydrolytic enzymes such as catalase, protease, amylase, lipase, lecithinase, asparaginase, chitinase and pectinase. All the strains resisted both penicillin and streptomycin, 29 were sensitive to neomycin; the majority of strains (25) showed multiple antibiotic resistance index greater than 0.2; 23, 22 and 13 degraded the shrimp shell, chicken feather and corn cob, respectively, producing bioactive substance(s) which indicates their diversity and their ecological role in the marine ecosystem. At least 28 strains exhibited nematicidal activity in vitro and in vivo against root-knot nematode and supported plant growth. In vitro, the assessed Streptomyces species exhibited the ability to produce gibberellic acid, indole acetic acid, abscisic acid, kinetin and benzyladenine. Except for indole acetic acid, this is the first report concerning the ability of marine Streptomyces to produce such phytohormones and the use of shrimp shell waste as a mono component medium for production of phytohormones. The study is efficacious in selecting effective biodiverse strains of marine Streptomyces that may work under diverse agro-ecological conditions as a useful element in plant nutrition and as biocontrol agents involved in integrated management programs., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Objectives: Smoking is a well-established risk factor in rheumatoid arthritis (RA), and citrullination of self-antigens plays a pathogenic role in the majority of patients. Increased numbers of peptidylarginine deiminase 2 (PAD2)-containing macrophages have been demonstrated in bronchoalveolar lavage (BAL) fluid from smokers, but intracellularly located PAD cannot be responsible for citrullination of extracellular self-antigens. We aimed to establish a link between smoking and extracellular PAD2 in the lungs., Methods: BAL fluid samples were obtained from 13 smokers and 11 nonsmoking controls. Total protein content and C-reactive protein (CRP) concentration were determined after separating cells from the samples. PAD2 content in cell-free BAL fluids was measured by means of a PAD2-specific sandwich ELISA., Results: Significantly increased levels of soluble PAD2 were detected in cell-free BAL fluids from smokers as compared to non-smokers (p=0.018). The PAD2 content correlated with the overall CRP levels (p=0.009) and cell count (p=0.016)., Conclusions: This first demonstration of increased levels of extracellular PAD2 in the lungs of smokers supports the hypothesis that smoking promotes extracellular citrullination of proteins. This may represent a pathological event upstream for the production of anti-citrullinated protein antibodies (ACPAs) among RA patients carrying HLA-molecules capable of binding citrullinated self-peptides.

Agmatine deiminases (AgDs) catalyze the hydrolytic conversion of agmatine (decarboxylated arginine) to N-carbamoylputrescine with concomitant release of ammonia. These enzymes, which are encoded by some pathogenic bacterial species, confer a competitive survival advantage by virtue of energy production and acid tolerance through agmatine catabolism. Herein we report the development of a clickable activity-based protein profiling (ABPP) probe that targets the AgD encoded by Streptococcus mutans with high selectivity and sensitivity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The development of alternative energy sources by applying lignocellulose-based biofuel technology is critically important because of the depletion of fossil fuel resources, rising fossil fuel prices, security issues regarding the fossil fuel supply, and environmental issues. White-rot fungi have received much attention in recent years for their valuable enzyme systems that effectively degrade lignocellulosic biomasses. These fungi have powerful extracellular oxidative and hydrolytic enzymes that degrade lignin and cellulose biopolymers, respectively. Lignocellulosic biomasses from either agricultural or forestry wastes are abundant, low-cost feedstock alternatives in nature but require hydrolysis into simple sugars for biofuel production. This review provides a complete overview of the different lignocellulose biomasses and their chemical compositions. In addition, a complete list of the white-rot fungi-derived lignocellulolytic enzymes that have been identified and their molecular structures, mechanism of action in lignocellulose hydrolysis, and biochemical properties is summarized in detail. These enzymes include ligninolytic enzymes (laccase, manganese peroxidase, lignin peroxidase, and versatile peroxidase) and cellulolytic enzymes (endo-glucanase, cellobiohydrolase, and beta-glucosidase). The use of these fungi for low-cost lignocellulolytic enzyme production might be attractive for biofuel production.

The present study determined the activity of hydrolytic enzymes and the gene expression during direct interaction of the novelTrichoderma asperellumstrains TC74 (high antagonistic capacity) and Th1 (low antagonistic capacity) with the plant pathogenic fungusPhymatotrichopsis omnivora. TheT. asperellumstrains produced the lytic enzymes endochitinase, N-acetylglucosaminidase and β-1,3-glucanase when grown on two different carbon sources. The response of strain TC74 was more rapid than that of strain Th1. When directly exposed toP. omnivora, theT. asperellumstrains expressed one endochitinase and one N-acetylglucosaminidase, as shown by RT-PCR experiments. The strains also expressed two β-1,3-exoglucanases (designated as exg290 and exg343). TC74 and Th1 were able to express their chitinases and β-1,3-exoglucanase activities when grown on the different carbon sources tested. When theT. asperellumstrains were grown in the presence ofP. omnivora, qRT-PCR experiments revealed that mycoparasitism-related genes were first expressed prior to contact between the antagonist and the pathogen's mycelium. As a general response, the transcription level of these genes was increased at the post-contact stage. Although TC74 and Th1 both express the assayed genes when grown in direct contact withP. omnivora, significant differences in the time, intensity and kinetics of the response were observed., (Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Paracoccidioides species are dimorphic fungi that initially infect the lungs but can also spread throughout the body. The spreading infection is most likely due to the formation of a biofilm that makes it difficult for the host to eliminate the infection. Biofilm formation is crucial for the development of infections and confines the pathogen to an extracellular matrix. Its presence is associated with antimicrobial resistance and avoidance of host defenses. This current study provides the first description of biofilm formation by Paracoccidioides brasiliensis (Pb18) and an analysis of gene expression, using real-time PCR, associated with 3 adhesins and 2 hydrolytic enzymes that could be associated with the virulence profile. Biofilm formation was analyzed using fluorescence microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Metabolic activity was determined using the XTT reduction assay. P. brasiliensis was able to form mature biofilm in 144 h with a thickness of 100 μm. The presence of a biofilm was found to be associated with an increase in the expression of adhesins and enzymes. GP43, enolase, GAPDH and aspartyl proteinase genes were over-expressed, whereas phospholipase was down-regulated in biofilm. The characterization of biofilm formed by P. brasiliensis may contribute to a better understanding of the pathogenesis of paracoccidioidomycosis as well as the search for new therapeutic alternatives; while improving the effectiveness of treatment.

S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH-CC with biotin used in conjunction with streptavidin-horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the Kd values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established Kd values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Trametes versicolor is a promising white-rot fungus for the biological pretreatment of lignocellulosic biomass. In the present work, T. versicolor ATCC 20869 was grown on Pinus taeda wood chips under solid-state fermentation conditions to examine the wood-degrading mechanisms employed by this fungus. Samples that were subjected to fungal pretreatment for one-, two- and four-week periods were investigated. The average mass loss ranged from 5 % to 8 % (m m(-)(1)). The polysaccharides were preferentially degraded: hemicellulose and glucan losses reached 13.4 % and 6.9 % (m m(-)(1)) after four weeks of cultivation, respectively. Crude enzyme extracts were obtained and assayed using specific substrates and their enzymatic activities were measured. Xylanases were the predominant enzymes, while cellobiohydrolase activities were marginally detected. Endoglucanase activity, β-glucosidase activity, and wood glucan losses increased up to the second week of biodegradation and remained constant after that time. Although no lignin-degrading enzyme activity was detected, the lignin loss reached 7.5 % (m m(-)(1)). Soluble oxalic acid was detected in trace quantities. After the first week of biodegradation, the Fe(3+)-reducing activity steadily increased with time, but the activity levels were always lower than those observed in the undecayed wood. The progressive wood polymer degradation appeared related to the secretion of hydrolytic enzymes, as well as to Fe(3+)-reducing activity, which was restored in the cultures after the first week of biodegradation., (Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Objectives: To assess the relationships among alkali production, diet, oral health behaviors, and oral hygiene., Methods: Data from 52 subjects including demographics, diet, and oral hygiene scores were analyzed against the level of arginine and urea enzymes in plaque and saliva samples. An oral habit survey was completed that included: use of tobacco (TB), alcohol (AH), sugary drinks (SD), and diet. Alkali production through arginine deiminase (ADS) and urease activities were measured in smooth-surface supragingival dental plaque and un stimulated saliva samples from all subjects. ADS and urease activities were measured by quantification of the ammonia generated from the incubation of plaque or saliva samples. Spearman correlations were used to compute all associations., Results: Participants in the lowest SES (Socio-economic status) group had the habit of consuming sugary drinks the most and had the highest rate of tobacco use. Males consumed significantly more alcohol than females. No significant relationship was found between age or gender and alkali production. Higher rates of sugary drink consumption and tobacco use were significantly related to lower alkali production., Conclusion: The study showed a relationship between alkali production and oral hygiene, diet, and certain oral health behaviors. Poor oral hygiene was significantly associated with age, lower SES, tobacco use, and alcohol, and sugary drinks consumption. Clinical relevance Certain oral health behaviors have an impact on oral hygiene and on alkali production; it is important to address these factors with patients as a strategy for caries control.

Winged males of leaf-cutting ants are considered an ephemeral reproductive caste only produced before the mating flight season. Although much is known about the yeast diversity found in fungus gardens of attine ants, no study has focused on the yeasts associated with males of leaf-cutting ants. Here, we surveyed the yeasts on the integuments of males of Atta sexdens rubropilosa and assessed their potential role in the attine ant-microbe symbiosis. Using culture-dependent techniques, we found yeasts to be abundant on the integuments of males (54.5 %, n = 200 alates). A total of 242 yeast strains were obtained representing six orders, ten genera and 25 species. Strains of Aureobasidium, Cryptococcus, Hannaella and Rhodotorula were prevalent on the integuments and likely originated from the fungus garden of the parental nest or from the soil. The majority of strains (87.1 %) produced at least one of the evaluated enzymes: pectinase, polygalacturonase, cellulase, xylanase, ligninases and lipase. Aureobasidium pullulans accounted for the highest number of strains that produced all enzymes. In addition, yeasts showed the ability to assimilate the resulting oligosaccharides, supporting observations of other studies that yeasts may be involved in the plant biomass metabolism in the fungus gardens. Because winged males harbor several yeasts with putative functional roles, these fungi may take part and be beneficial in the microbial consortia of the new incipient nest.