Enzyme Activity Profiles Research Articles

Integrative Biomarker Indices in a Benthic Indicator Species Modiolus Modiolus (L.) Under a Simulated Oil Spill

The horse mussel Modiolus modiolus (L.) is a sentinel bivalve species used in monitoring programs to assess potential biological exposure to anthropogenic contaminants, including oil hydrocarbons, in benthic environments. In an aquarium based experiment, these mussels were exposed to a simulated oil spill where crude oil at realistic concentrations (from low to high) interacted with an inert environment (seawater, gravel surface, etc.) and the biota. Using a combination of endpoints that included tissue contaminant load, protease activity, antioxidant enzyme activity, and low-molecular antioxidant profiles, we characterized M. modiolus responses to this simulated crude oil spill. Significant differences were observed in tissue protein reserves, protease activity, and oxidative stress markers including glutathione content and glutathione-S-transferase activity in the hepatopancreas and gill tissues of the bivalves treated with oil. Total concentrations of oil-derived polycyclic aromatic hydrocarbons in M. modiolus tissues were generally highest under the highest ambient oil concentration with much lower levels at the lowest. This general pattern does match the activity of protein quality control proteases but not the antioxidant enzyme activity profiles. Glutathione-S-transferase activity in bivalves showed decreased activity under the high compared to the lower oil load and oil-free animals, while glutathione content and calpain activity were positively correlated with oil uptake. The data suggest that these benthic organisms were exhibiting biological responses to the oil polycyclic aromatic hydrocarbons and support the bioindicator value of the species.
 Keywords: M. modiolus, oil spill, PAHs, integrative biomarker approach, protein quality control, GSH, GST

Cloning and functional analysis of the phenylalaninammo-nialyase gene from Rhododendron fortunei

Phenylalaninammo-nialyase (PAL) is a key enzyme in the synthesis of methyl benzoate - a plant aroma compound. In order to understand the function of this enzyme in the formation of fragrance in the scented Rhododendron species-Rhododendron fortunei, we cloned a gene encoding this enzyme and subsequently examined the gene expression patterns and the profile of enzyme activity during development in various tissues. The full length of RhPAL gene was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The expression levels of RhPAL gene were measured by real-time quantitative reverse transcription PCR (qRT-PCR) and the amount of phenylalanine and cinnamic acid were assayed with LC-MS. The results showed that the ORF sequence of RhPAL gene amplified from the cDNA templates of flower buds had 2 145 bp, encoding 715 amino acids, and shared 90% homology to the PAL amino acid sequences from other species. qRT-PCR analysis showed that the expression of RhPAL in petals during flowering kept in rising even until the flowers wilted. The expression of RhPAL in pistil was much higher than that in stamen, while the expression in the younger leaves was higher than in old leaves. However, the expression level was relatively lower in petal and stamen compared to that in leaves. We also measured the PAL activity by Enzyme-linked immuno sorbent assay in the petals of flowers at different flowering stages. The results showed that PAL activity reached the highest at the bud stage and then decreased gradually to the lowest when the flowers wilted, which followed a similar trend in the emission of the flower fragrance. The phenylalanine and cinnamic acid contents measured by LC-MS were highly correlated to the expression level of RhPAL in various tissues and at different flowering stages, implying that RhPAL plays an important role in the formation of the flower fragrance. This work may facilitate the breeding and improvement of new fragrant Rhododendron cultivars.

Phenotypic characterization of bacterial isolates from marine waters and plastisphere communities of the Ross Sea (Antarctica)

Background: In aquatic environments, microbial biofilms are hot spots of microbial diversity, as well as a substrate for larval settlement of many invertebrate species. Examining the functional diversity of microorganisms in polar regions is a new and still unknown field of aquatic microbiology, that is attracting increasing interest for its significance in both scientific research and resources exploitation. The context and purpose of the study: In the framework of research aimed at studying microbial colonization occurring at Terra Nova Bay, culturable heterotrophic bacteria were quantified and characterized phenotypically at two study areas (Road Bay and Tethys Bay) with different environmental characteristics: the first one was close to Mario Zucchelli research station and impacted by anthropogenic pressure due to sewage wastes, while the second was exposed to salinity gradients due to glacier melting. In this context, artificial structures hosting plastic (polyvinyl chloride, PVC and polyethylene, PE) panels were deployed at -5 and -20m depths at each of the stations (one Impact and one Control) chosen per each study area. Water samples were contextually collected at the time of recovery of the plastic panels, namely after 12 months of deployment; at Road Bay, at the -5 m depth, a short-term experiment was also performed, with the recovery of the panels after 2.5 and 9 months of deployment. Bacterial strains were isolated in axenic culture both from water and biofilm matrices, and examined for their main phenotypical traits and functional diversity, through Gram staining, oxidase production, glucose fermentation and screened for enzymatic activity profiles (proteolytic, glycolytic and phosphatasic activities) using specific fluorogenic substrates. Results: Most of the bacterial isolates were Gram-negative, oxidase-positive and glucose-fermenting strains. Higher enzyme diversification was found in Road Bay at the site impacted by the sewage wastes from the research station, compared to the control site. A higher abundance of heterotrophic bacteria was detected on PVC compared to PE. Several pigmented strains, ascribable to Flavobacterium sp., were isolated from biofilm with respect to the ones isolated from the pelagic environment. Main findings: The screening of bacterial isolates for the production of hydrolytic enzymes revealed that proteolytic and phosphatase activities were a common metabolic trait of both water and biofilm-derived isolated. Microbes inhabiting Antarctica represent a very important and not completely known source of bioactive molecules with possibly multiple applications, from biomedicine to pharmaceutical, cosmetics, biodegradation, and so on. Conclusions: This is the first contribution to characterize phenotypically the bacteria colonizing the Ross Sea seabed in comparison with pelagic heterotrophic bacteria and to explore their metabolic abilities and the biotechnological potential of these microorganisms. The preliminary data regarding the metabolic profiles and composition of the microbial community have highlighted the plasticity of the microbial community, whose metabolism is modulated by the organic matter supplies found near the Mario Zucchelli Station. Brief summary: Heterotrophic bacteria in the waters and in the biofilm covering the surface of settlement panels in plastics deployed at sea for different time periods were studied in two bays of the Ross Sea, Road Bay and Tethys Bay. The main phenotypical characteristics of the bacterial isolates were examined. The production of hydrolytic enzymes makes microbial biofilms interesting sources of molecules with a potential applicative interest. Any potential implications: Bioprospecting research on polar microorganisms represents an attractive field of microbiology that is supported by increasing attention towards the discovery of hydrolytic enzymes as new active compounds having unexplored properties and applications.

Low Inclusion Levels of Tenebrio Molitor Larvae Meal in Laying Japanese Quail (Coturnix Japonica, Gould, 1837) Diet Improve the Intestinal Morphometry, Enzymatic Activity and Caecal Short Chain Fatty Acids Profile

Low Inclusion Levels of Tenebrio Molitor Larvae Meal in Laying Japanese Quail (Coturnix Japonica, Gould, 1837) Diet Improve the Intestinal Morphometry, Enzymatic Activity and Caecal Short Chain Fatty Acids Profile

Effects of Salt Treatment Time on the Metabolites, Microbial Composition, and Quality Characteristics of the Soy Sauce Moromi Extract.

Salt is one of the most important factors for fermented foods, but the effect of salt treatment time on the quality of fermented foods has rarely been studied. In this study, the effect of different salt treatment times (0, 48, and 96 h) after the start of fermentation on the quality of the soy sauce moromi extract (SSME) was investigated. As the salt treatment time was delayed, the population of Aspergillus oryzae, Lactobacillaceae, and Enterococcaecea in SSME increased, whereas the population of Staphylococcaceae and Bacillaceae decreased, leading to changes in the enzymatic activity and metabolite profiles. In particular, the contents of amino acids, peptides, volatile compounds, acidic compounds, sugars, and secondary metabolites were significantly affected by the salt treatment time, resulting in changes in the sensory quality and appearance of SSME. The correlation data showed that metabolites, bacterial population, and sensory parameters had strong positive or negative correlations with each other. Moreover, based on metabolomics analysis, the salt treatment-time-related SSME metabolomic pathway was proposed. Although further studies are needed to elucidate the salt treatment mechanism in fermented foods, our data can be useful to better understand the effect of salt treatment time on the quality of fermented foods.

Quantifying the immediate response of the soil microbial community to different grazing intensities on irrigated pastures

Grazing is known to affect soil microbial communities, nutrient cycling, and forage quantity and quality over time. However, a paucity of information exists for the immediate changes in the soil physicochemical and microbial environment in response to different grazing strategies. Soil microbes drive nutrient cycling and are involved in plant-soil-microbe relationships, making them potentially vulnerable to plant-driven changes in the soil environment caused by grazing. To test the hypothesis that variable grazing intensities modulate immediate effects on the soil microbial community, we conducted a grazing trial of three management approaches; high-intensity, short-duration grazing (HDG), low-intensity, medium-duration grazing (LDG), and no grazing (NG). Soil and vegetation samples were collected before grazing and 24h, 1 week, and 4 weeks after HDG grazing ended. Soil labile carbon (C) and nitrogen (N) pools, vegetation biomass, and soil microbial diversity and functional traits were determined, including extracellular enzymatic assays and high-throughput sequencing of the bacterial 16S rRNA and fungal ITS2 regions. We found that labile soil C and inorganic N increased following LDG grazing while C-cycling extracellular enzymatic activities increased in response to HDG grazing but both total extracellular enzymatic activity profiles and soil abiotic profiles were mostly affected by temporal fluxes. The soil fungal community composition was strongly affected by the interaction of sampling time and grazing treatment, while the soil bacterial community composition was largely affected by sampling time with a lesser impact from grazing treatment. We identified several key fungal taxa that may influence immediate responses to grazing and modulate plant-soil-microbe interactions. There was strong evidence of temporal influences on soil biogeochemical variables and the soil microbiome, even within our narrow sampling scheme. Our results indicate that the soil ecosystem is dynamic and responsive to different grazing strategies within very short time scales, showing the need for further research to understand plant-soil-microbe interactions and how these feedback mechanisms can inform sustainable land management.

Inoculation of tomato (Solanum lycopersicum) roots with growth promoting Pseudomonas strains induces distinct local and systemic metabolic biosignatures

The genus Pseudomonas harbours numerous strains that positively affect plant growth and defence through diverse mechanisms such as nutrient solubilisation and production of phytohormones or secondary metabolites. The aim of this study was to compare the impact of six plant-beneficial Pseudomonas strains on tomato (Solanum lycopersicum) growth and holobiont physiology. The physiological impact was determined by profiling the activities of key enzymes in the central carbohydrate and antioxidant metabolism. Root inoculation of tomato seedlings with Pseudomonas strains in a greenhouse experiment induced plant growth, measured as biomass and plant height promotion. The bacterial strains also increased leaf chlorophyll content and caused distinct carbohydrate and antioxidative metabolism enzyme activity profiles in leaf and root tissue respectively. For the carbohydrate metabolism, the activities of several key enzymes involved in assimilate partitioning from source to sink and processing of the transport sugar sucrose for catabolism and anabolism were stimulated. For the antioxidative metabolism, both enzymes involved in detoxification of reactive oxygen species and redox buffering were increased. These increased enzyme activities in response to bacterial inoculation could contribute to balancing plant growth and defence. Importantly, positive correlations between plant growth parameters and distinct enzyme activities suggest that host plant biosignatures may be predicting bacteria with plant growth-promoting potential. These findings offer new perspectives for integrating physiological fingerprinting in the screening of microbes during early developmental stages of the host plant. In addition, determining plant metabolic biosignatures could be a rapid tool for predicting the potential and improvement of stress resiliency.

Bioprospecting of xylanase producing fungal strains: Multilocus phylogenetic analysis and enzyme activity profiling.

The study aims to explore potential xylanase-producing indigenous fungi isolated from soil and vegetable wastes containing plant degraded matter, reporting multilocus phylogenetic analysis and xylanase enzyme activity from selective strains. Four potential xylanolytic fungi were identified through distinct primary and secondary screening of 294 isolates obtained from the samples. Morphological characterization and multigene analysis (ITS rDNA, 18S rDNA, LSU rDNA, β-tubulin, and actin gene) confirmed them as Aspergillus sp. AUMS56, Aspergillus tubingensis AUMS60 and AUMS64, and Aspergillus fumigatus AUKEMS24; achieving crude xylanase activities (through submerged fermentation using corn cobs) of 18.9, 32.29, 30.68, and 15.82 U ml-1 , respectively. AUMS60 and AUMS64 (forming lineage with A. tubingensis and Aspergillusniger in the same phylogroup with 100% Bayesian posterior probability support) secreted single xylanase (Xyn60; 36 kDa) and multiple xylanases (Xyn64A and Xyn64B; 33.4 and 19.8 kDa) respectively, having pH optima of 6.0 and exhibiting maximal activity at 60°C. These enzymes were highly stable at 40°C (120 h) and retained more than 70% activity at 50°C and at pH 5-6 (upon 72 h incubation). Our analysis suggested these enzymes to be endoxylanases demonstrating substrate hydrolysis within 15 min of reaction and maximum efficiency of xylanases from AUMS60 and AUMS64 achieving 51.1% (13 h) and 52.2% (24 h) saccharification, respectively. They also showed enhanced catalytic activity with various cations. Based on our investigation on xylan hydrolysis, we believe that these xylanases may find significant industrial applications as they have a real potential of working as a bio-catalytic cocktail (patent file number: IN E1/38213/2020-DEL) for the enhanced saccharification of lignocelluloses.

Phylogenetic and Functional Diversity of Saprolegniales and Fungi Isolated from Temperate Lakes in Northeast Germany.

The contribution of fungi to the degradation of plant litter and transformation of dissolved organic matter (humic substances, in particular) in freshwater ecosystems has received increasing attention recently. However, the role of Saprolegniales as one of the most common eukaryotic organisms is rarely studied. In this study, we isolated and phylogenetically placed 51 fungal and 62 Saprolegniales strains from 12 German lakes. We studied the cellulo-, lignino-, and chitinolytic activity of the strains using plate assays. Furthermore, we determined the capacity of 10 selected strains to utilize 95 different labile compounds, using Biolog FF MicroPlates™. Finally, the ability of three selected strains to utilize maltose and degrade/produce humic substances was measured. Cladosporium and Penicillium were amongst the most prevalent fungal strains, while Saprolegnia, Achlya, and Leptolegnia were the most frequent Saprolegniales strains. Although the isolated strains assigned to genera were phylogenetically similar, their enzymatic activity and physiological profiling were quite diverse. Our results indicate that Saprolegniales, in contrast to fungi, lack ligninolytic activity and are not involved in the production/transformation of humic substances. We hypothesize that Saprolegniales and fungi might have complementary roles in interacting with dissolved organic matter, which has ecological implications for carbon cycling in freshwater ecosystems.

Ipa1 improves rice drought tolerance at seedling stage mainly through activating abscisic acid pathway.

KEY MESSAGE: ipa1 enhances rice drought tolerance mainly through activating the ABA pathway. It endows rice seedlings with a more developed root system, smaller leaf stomata aperture, and enhanced carbon metabolism. Drought is a major abiotic stress to crop production. IPA1 (IDEAL PLANT ARCHITECTURE 1)/OsSPL14 encodes a transcription factor and has been reported to function in both rice ideal plant architecture and biotic resistance. Here, with a pair of IPA1 and ipa1-NILs (Near Iso-genic Lines), we found that ipa1 could significantly improve rice drought tolerance at seedling stage. The ipa1 plants had a better-developed root system and smaller leaf stomatal aperture. Analysis of carbon-nitrogen metabolism-associated enzyme activity, gene expression, and metabolic profile indicated that ipa1 could tip the carbon-nitrogen metabolism balance towards an increased carbon metabolism pattern. In both the control and PEG-treated conditions, ABA content in the ipa1 seedlings was significantly higher than that in the IPA1 seedlings. Expression of the ABA biosynthesis genes was detected to be up-regulated, whereas the expression of ABA catabolism genes was down-regulated in the ipa1 seedlings. In addition, based on yeast one-hybrid assay and dual-luciferase assay, IPA1 was found to directly activate the promoter activity of OsHOX12, a transcription factor promoting ABA biosynthesis, and OsNAC52, a positive regulator of the ABA pathway. The expression of OsHOX12 and OsNAC52 was significantly up-regulated in the ipa1 plants. Combined with the previous studies, our results suggested that ipa1 could improve rice seedling drought tolerance mainly through activating the ABA pathway and that regulation of the ipa1-mediated ABA pathway will be an important strategy for improving drought resistance of rice.

Vaccination of Gilthead Seabream After Continuous Xenoestrogen Oral Exposure Enhances the Gut Endobolome and Immune Status via GPER1.

In fish culture settings, the exogenous input of steroids is a matter of concern. Recently, we unveiled that in the gilthead seabream (Sparus aurata), the G protein-coupled estrogen receptor agonist G-1 (G1) and the endocrine disruptor 17α-ethinylestradiol (EE2) are potent modulators in polyreactive antibody production. However, the integral role of the microbiota upon immunity and antibody processing in response to the effect of EE2 remains largely unexplored. Here, juvenile seabreams continuously exposed for 84 days to oral G1 or EE2 mixed in the fish food were intraperitoneally (i.p.) immune primed on day 42 with the model antigen keyhole limpet hemocyanin (KLH). A critical panel of systemic and mucosal immune markers, serum VTG, and humoral, enzymatic, and bacteriolytic activities were recorded and correlated with gut bacterial metagenomic analysis 1 day post-priming (dpp). Besides, at 15 dpp, animals received a boost to investigate the possible generation of specific anti-KLH antibodies at the systemic and mucosal interphases by the end of the trial. On day 43, EE2 but not G1 induced a significant shift in the serum VTG level of naive fish. Simultaneously, significant changes in some immune enzymatic activities in the serum and gut mucus of the EE2-treated group were recorded. In comparison, the vaccine priming immunization resulted in an attenuated profile of most enzymatic activities in the same group. The gut genes qPCR analysis exhibited a related pattern, only emphasized by a significant shift in the EE2 group’s il1b expression. The gut bacterial microbiome status underwent 16S rRNA dynamic changes in alpha diversity indices, only with the exposure to oral G1, supporting functional alterations on cellular processes, signaling, and lipid metabolism in the microbiota. By the same token, the immunization elevated the relative abundance of Fusobacteria only in the control group, while this phylum was depleted in both the treated groups. Remarkably, the immunization also promoted changes in the bacterial class Betaproteobacteria and the estrogen-associated genus Novosphingobium. Furthermore, systemic and mucosal KLH-specific immunoglobulin (Ig)M and IgT levels in the fully vaccinated fish showed only slight changes 84 days post-estrogenic oral administration. In summary, our results highlight the intrinsic relationship among estrogens, their associated receptors, and immunization in the ubiquitous fish immune regulation and the subtle but significant crosstalk with the gut endobolome.

The association between PON1 gene polymorphisms (Q192R and L55M) and nephrotic syndrome in Iraqi children

Background: The role of paraoxonase 1 enzyme (PON1) and its single nucleotide polymorphisms (SNPs) in children with nephrotic syndrome (NS) has been reported previously in different ethnic and racial groups with divergent results. The human PON1 gene contains two coding region polymorphisms leading to two different PON1 isoforms. Objectives: The aim of the present study was to find out the association between the PON1 (Q192R and L55M) polymorphisms and their relation with serum PON1 activity as well as lipid profile tests (total cholesterol, TC; triglycerides, TG; high-density lipoprotein cholesterol, HDL-c; and low-density lipoprotein cholesterol, LDL-c) in children with NS. Methods: This study included a total of 80 participants (40 with NS in the age group of 2-14 years and 40 age and sex-matched healthy controls). The PON1 enzyme activity and lipid profile tests were measured in serum samples of all included participants. The PON1 genotype was determined by PCR-restriction enzyme fragment length polymorphism (PCR-RFLP) for both PON1 alleles (192 and 55) SNPs. Results: Our findings showed that the mean levels of lipid profile tests (TC, TG, LDL-c) were significantly increased in patients when compared with healthy controls (p<0.05), while the HDL-c concentration was significantly decreased in patients than that of controls. Also, the patients had significantly lower concentrations of PON1 when compared with the controls regardless of the genotype Q192R and L55M polymorphisms. Moreover, the homozygous RR genotype for PON1 SNP 192 and MM homozygous genotype for PON1 SNP 55 were significantly frequent in patients when compared with the controls. Conclusions: Our results support that the presence of the homozygous RR genotype for PON1 SNP 192 and MM homozygous genotype for PON1 SNP 55 were significantly higher in patients compared with the controls.

Investigation of proline in superficial scald development during low temperature storage of ‘Dangshansuli’ pear fruit

The role of proline in superficial scald development in pear fruit was investigated in this study. During low temperature storage of ‘Dangshansuli’ pear fruit, superficial scald incidence and index accumulated in association with the alternation of α-farnesene and conjugated trienols (CTols). 14 out of 17 free amino acids were identified; proline content gradually decreased, which was consistent with the up-regulation of PbrP5CS activity and down-regulation of PbrProDH activity in its metabolic pathway. A total of 14 genes involved in proline metabolism were identified based on transcriptome annotation with diverse expression profiles. Results from correlation analysis among proline content further indicate that enzyme activity and gene expression profile, PbrProDH2, PbrProDH4, PbrProDH5 &PbrP5CS5 might play a critical role in proline metabolism during low temperature storage of ‘Dangshansuli’ fruit; thus, influencing superficial scald development which was then functionally validated using the transgenic pear fruit. Postharvest 1-MCP and diphenylamine (DPA) treatment inhibited the accumulation of CTols, and thus, mitigated superficial scald development and maintained higher proline content, in association with the up-regulated PbrP5CS activity, PbrP5CS4 &PbrP5CS5 mRNAs as well as the down-regulated PbrProDH activity &PbrProDH4 transcripts. In a further study, we found that exogenous application of proline alleviated superficial scald incidence and index. To that end, the results of our study confirmed that proline was involved in superficial scald development during pear storage.

Emamectin benzoate induced enzymatic and transcriptional alternation in detoxification mechanism of predatory beetle Paederus fuscipes (Coleoptera: Staphylinidae) at the sublethal concentration.

In this study, the detoxification enzyme activity and the transcriptional profile changes in the second instar through RNA-sequencing technology due to emamectin benzoate (EB) were assessed. The cytochrome P450 monooxygenases (P450) enzyme activity was not altered by EB due to the change in concentration and exposure time in all treatments. The glutathione S-transferase (GST) enzyme was not considerably varying in all treatments, while exposure time significantly changed the enzyme activity. Results showed that the esterase (Ests) activity was elevated with the increasing concentrations and exposure time. Two libraries were generated, containing 107,767,542 and 108,142,289 clean reads for the samples treated with LC30 of EB and control. These reads were grouped into 218,070 transcripts and 38,097 unigenes. A total of 2257 differentially expressed genes (DEGs) were identified from these unigenes, of which 599 up-regulated and 1658 were down-regulated. The majority of these DEGs related to pesticides resistance were identified in numerous Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, e.g., steroid hormone biosynthesis, glutathione metabolism, drug metabolism-other enzymes, chemical carcinogenesis, pathways of cancer, metabolism of xenobiotics by cytochrome P450, drug metabolism of cytochrome P450, linoleic acid metabolism, retinol metabolism, and insect hormone biosynthesis. These pathways also shared the same genes as cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), Esterase (Ests), UDP-glucosyltransferases (UGTs), and ATP-binding cassettes (ABCs). A heatmap analysis also showed that regulation of genes in a pathway causes a series of gene expression regulation in subsequent pathways. Our quantitative reverse transcription-PCR (qRT-PCR) results were consistent with the DEG's data of transcriptome analysis. The comprehensive transcriptome sequence resource attained through this study evidence that the EB induces significant modification in enzyme activity and transcriptome profile of Paederus fuscipes, which may enable more significant molecular underpinnings behind the insecticide-resistance mechanism for further investigations.

Lignocellulolytic Potential of the Recently Described Species Aspergillus olivimuriae on Different Solid Wastes

The genus Aspergillus encompasses several species with relevant lignocellulose-degrading capacity, and a novel species, denominated A. olivimuriae, was recently discovered after its isolation from table olive brine. The acquisition of insight into this species and the assessment of its potential relied on a bioinformatics approach, based on the CAZy database, associated with enzymatic activity profiles in solid-state cultures on four different types of waste, including residual thistle biomass (RTB), spent coffee grounds (SCG), digestate solid fraction and barley straw. The CAZy analysis of A. olivimuriae genome showed that the number of predicted genes for each family was close to that of other Aspergillus species, except for cellobiose dehydrogenase, acetyl xylan esterase and polygalacturonases. In A. olivimuriae solid-state cultures, hemicellulose degradation outperformed that of cellulose, and lignin removal did not occur, regardless of the growth substrate. This is in line with its CAZy content and the extent of hemicellulolytic, and ligninolytic activities detected in its solid-state cultures. RTB and barley straw were the substrates enabling the best glycosyl hydrolase production levels. The exception was SCG, the hemicellulose composition of which, mainly made of glucomannans and galactomanans, led to the highest β-mannanase and β-mannosidase production levels (3.72 ± 0.20 and 0.90 ± 0.04 IU g−1 substrate, respectively).

Characterizing the impact of high temperature during grain filling on phytohormone levels, enzyme activity and metabolic profiles of an early indica rice variety.

Global warming results in high temperature stress (HTS), which presents severe challenges worldwide for modern agricultural production and will have significant impacts on the yield and quality of crops. Accumulation of photosynthetic products, activity of enzymes involved in sucrose-starch metabolism, phytohormone levels and metabolic profiling using LC-MS were analysed in the flag leaves and/or developing grains subjected to HTS during the grain-filling stage of an indica rice. HTS induced significant yield loss and reduced the grain quality, with lower amylose content. HTS reduced photosynthetic product accumulation in flag leaves and reduced starch accumulation in developing grains, compared to growth under normal temperatures. The activity of enzymes related to sucrose-starch metabolism were dis-regulated in developing grains grown under high temperature (HT). Moreover, phytohormone homeostasis in flag leaves and developing grains was also dramatically disturbed by HT. Metabolic profiling detected many metabolites with remarkably different relative fold abundances at different time points in the developing grain at HT versus normal temperatures, these metabolites were enriched in several HTS response pathways. The change in phytohormone ratio and auxin level might be associated with the reduction in photosynthetic products and their translocation, and ultimately with reduced starch accumulation in the developing grain. The detected metabolites might have different roles in response to the HTS in developing grain at different development stages. These results provide a theoretical reference and basis for future rice production towards higher quality and yield when grown under HTS.

Cellulolytic and Xylanolytic Microbial Communities Associated With Lignocellulose-Rich Wheat Straw Degradation in Anaerobic Digestion.

The enzymatic hydrolysis of lignocellulosic polymers is generally considered the rate-limiting step to methane production in anaerobic digestion of lignocellulosic biomass. The present study aimed to investigate how the hydrolytic microbial communities of three different types of anaerobic digesters adapted to lignocellulose-rich wheat straw in continuous stirred tank reactors operated for 134 days. Cellulase and xylanase activities were monitored weekly using fluorescently-labeled model substrates and the enzymatic profiles were correlated with changes in microbial community compositions based on 16S rRNA gene amplicon sequencing to identify key species involved in lignocellulose degradation. The enzymatic activity profiles and microbial community changes revealed reactor-specific adaption of phylogenetically different hydrolytic communities. The enzymatic activities correlated significantly with changes in specific taxonomic groups, including representatives of Ruminiclostridium, Caldicoprobacter, Ruminofilibacter, Ruminococcaceae, Treponema, and Clostridia order MBA03, all of which have been linked to cellulolytic and xylanolytic activity in the literature. By identifying microorganisms with similar development as the cellulase and xylanase activities, the proposed correlation method constitutes a promising approach for deciphering essential cellulolytic and xylanolytic microbial groups for anaerobic digestion of lignocellulosic biomass.

How Different Snacks Produce a Distinct Effect in Salivary Protein Composition.

Saliva secretion changes in response to different stimulation. Studies performed in animals and humans suggest that dietary constituents may influence saliva composition, although the dynamics of these changes, and how they are specific for each type of food, are little known. The objective of the present study was to access the short-term effects of different foods in salivation and salivary protein composition. Twelve participants were tested for four snacks (yoghurt, bread, apple and walnuts). Non-stimulated saliva was collected before and at 0′, 5′ and 30′ after each snack intake. Flow rate, total protein, alpha-amylase enzymatic activity and salivary protein profile were analyzed. Yoghurt and apple were the snacks resulting in higher salivary changes, with higher increases in flow rate and alpha-amylase activity immediately after intake. The expression levels of immunoglobulin chains decreased after the intake of all snacks, whereas cystatins and one pink band (proline-rich proteins—PRPs) increased only after yoghurt intake. Walnut’s snack was the one resulting in lower changes, probably due to lower amounts eaten. Even so, it resulted in the increase in one PRPs band. In conclusion, changes in saliva composition varies with foods, with variable changes in proteins related to oral food processing and perception.

Genetic Analysis of Candida glabrata from Candiduric Patients Using Microsatellite Length Polymorphism, Antifungal Susceptibility, and Enzymatic Profiles

Background: Candida glabrata is the second agent of candiduria with increased resistance to antifungals. Microsatellite length polymorphism (MLP) is one of the genotyping techniques used in the epidemiological investigation to improve clinical management. Objectives: We aimed to detect different genotypes of C. glabrata isolates using six microsatellite markers and the MLP technique. Moreover, our genotypes' association with other countries' genotypes was illustrated using a minimum spanning tree. We investigated in vitro antifungal susceptibility and enzymatic activity profiles of the isolates. Methods: Six microsatellite markers were amplified using multiplex-PCR for 22 C. glabrata strains isolated from urine in pediatric patients admitted to the Abuzar Children's Hospital in Ahvaz, Iran. The PCR products were presented for fragment analysis, and the size of the alleles was determined. Antifungal susceptibility tests and extracellular enzyme activities were also performed. Results: Nineteen multilocus genotypes were detected so that 22.7% of the strains had identical genotypes. The isolates were wild-type for amphotericin B (0.0625 - 2 µg/mL), itraconazole (0.125 - 2 µg/mL), and voriconazole (0.0078 - 0.00625 µg/mL). All the isolates were sensitive to fluconazole at the minimum inhibitory concentration (MIC) range (0.0312 - 16 μg/mL), and three of them were resistant to caspofungin (MIC ≥ 0.5 μg/mL). Moreover, 72.7 and 68.2% of the isolates had no phospholipase and esterase activities. The highest potency of enzymatic activity was obtained in hemolysin and proteinase enzymes. A high genetic diversity (19 genotypes of the 22 isolates) existed among the urinary C. glabrata isolates. Based on the minimum spanning tree, two clusters of our genotypes were related to C. glabrata genotypes in a previous study in Iran, and the third cluster was entirely connected with Chinese genotypes. Conclusions: Most of the isolates were the non-wild type for posaconazole but were rarely resistant to other antifungals. Hemolysin and proteinase secreted as the main virulence factors among the urinary C. glabrata isolates.

Safety Evaluation, Biogenic Amine Formation, and Enzymatic Activity Profiles of Autochthonous Enterocin-Producing Greek Cheese Isolates of the Enterococcus faecium/durans Group.

Autochthonous single (Ent+) or multiple (m-Ent+) enterocin-producing strains of dairy enterococci show promise for use as bioprotective adjunct cultures in traditional cheese technologies, provided they possess no pathogenic traits. This study evaluated safety, decarboxylase activity, and enzymatic (API ZYM) activity profiles of nine Ent+ or m-Ent+ Greek cheese isolates previously assigned to four distinct E. faecium (represented by the isolates KE64 (entA), GL31 (entA), KE82 (entA-entB-entP) and KE77 (entA-entB-entP-bac31)) and two E. durans (represented by the isolates KE100 (entP) and KE108 (entP-bac31-cyl)) strain genotypes. No strain was β-hemolytic or harbored vanA and vanB or the virulence genes agg, ace, espA, IS16, hyl, or gelE. All strains were of moderate to high sensitivity to ampicillin, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, penicillin, tetracycline, and vancomycin, except for the E. faecium KE64 and KE82 strains, which were resistant to erythromycin and penicillin. All cheese strains showed moderate to strong esterase-lipase and aminopeptidase activities and formed tyramine, but none formed histamine in vitro. In conclusion, all Ent+ or m-Ent+ strain genotypes of the E. faecium/durans group, except for the cyl-positive E. durans KE108, were safe for use as adjunct cultures in traditional Greek cheeses. Further in situ biotechnological evaluations of the strains in real cheese-making trials are required.