Leucine Aminopeptidase Research Articles

Plant Diversity Changes and Its Driving Factors of Abandoned Land at Different Restoration Stages in the Middle of the Qinling Mountains

The process of vegetation restoration is often accompanied by significant changes in aboveground plant diversity. To explore the driving mechanism of litter nutrient-soil nutrient-enzyme activity stoichiometry on aboveground vegetation change is of great importance for maintaining regional biodiversity conservation and ecological stability. Taking typical abandoned farmland of different restoration years （1， 8， 16， 31， and 50 a） in the Qinling Mountains as the research object， the variation characteristics of plant community diversity during vegetation restoration were analyzed through field investigation. Litter nutrients， soil nutrients， and the activities of five extracellular enzymes， including β-1，4-glucosidase （BG）， cellobiohydrolase （CBH）， β-1，4-N-acetylglucosaminidase （NAG）， leucine aminopeptidase （LAP）， and acid phosphatase （AP）， were determined. The characteristics of litter nutrients， soil nutrients， and enzyme stoichiometric ratios during vegetation restoration and the driving mechanism of plant diversity changes were discussed. The results showed that the plant community diversity index firstly decreased and then increased with the increase in vegetation restoration years， and the minimum was reached at 16 years after restoration. The results of principal component analysis showed that there were significant differences between total plant community diversity index and litter-soil-enzyme stoichiometric characteristics in different years of vegetation restoration. The plant community diversity index had a strong positive correlation with litter C∶P ratio and litter N∶P ratio but had a negative correlation with soil enzyme C∶P ratio （EEA C∶P）. The results of redundancy analysis showed that soil EEA C∶P had the highest explanation rate of plant diversity changes during vegetation restoration （25.93%）， followed by soil TP （5.94%）， which was the key factor regulating plant diversity changes. In conclusion， plant species and quantity increased significantly in abandoned farmland in the middle part of the Qinling Mountains at the late stage of vegetation restoration. Changes in the soil environment affected microbial metabolic activities and thus changed enzyme activities. Litter-soil-soil extracellular enzymes affected the community environment and plant diversity through feedback and regulation. EEA C∶P and TP were the main driving factors of aboveground plant diversity change during vegetation restoration.

Land Use Change from Natural Tropical Forests to Managed Ecosystems Reduces Gross Nitrogen Production Rates and Increases the Soil Microbial Nitrogen Limitation.

Understanding the underlying mechanisms of soil microbial nitrogen (N) utilization under land use change is critical to evaluating soil N availability or limitation and its environmental consequences. A combination of soil gross N production and ecoenzymatic stoichiometry provides a promising avenue for nutrient limitation assessment in soil microbial metabolism. Gross N production via 15N tracing and ecoenzymatic stoichiometry through the vector and threshold element ratio (Vector-TER) model were quantified to evaluate the soil microbial N limitation in response to land use changes. We used tropical soil samples from a natural forest ecosystem and three managed ecosystems (paddy, rubber, and eucalyptus sites). Soil extracellular enzyme activities were significantly lower in managed ecosystems than in a natural forest. The Vector-TER model results indicated microbial carbon (C) and N limitations in the natural forest soil, and land use change from the natural forest to managed ecosystems increased the soil microbial N limitation. The soil microbial N limitation was positively related to gross N mineralization (GNM) and nitrification (GN) rates. The decrease in microbial biomass C and N as well as hydrolyzable ammonium N in managed ecosystems led to the decrease in N-acquiring enzymes, inhibiting GNM and GN rates and ultimately increasing the microbial N limitation. Soil GNM was also positively correlated with leucine aminopeptidase and β-N-acetylglucosaminidase. The results highlight that converting tropical natural forests to managed ecosystems can increase the soil microbial N limitation through reducing the soil microbial biomass and gross N production.

Microalgae biomass as a conditioner and regulator of soil quality and fertility.

Characteristics of an acid soil cultivated with Urochloa brizantha cv. Marandu were evaluated in relation to two types of fertilization: a conventional one, chemical based on nitrogen and potassium, and a biofertilizer, based on microalgae biomass. The results were compared among three treatments, control, conventional, and biological fertilization, with seven replications each. The study evaluated microalgae community, total carbon and nitrogen contents, mineral nitrogen, and enzymatic activity. Chlorella vulgaris showed the highest organism density, which can be explained by its rapid growth and high resistance. The highest species diversity was detected in the control 1,380,938 org cm-3 and biological 1,841,250 org cm-3 treatments, with the latter showing a higher density of cyanobacteria, especially Pseudanabaena limnetica with 394,554 org cm-3. The soil treated with chemical fertilization showed higher nitrate (9.14 mg NKg-1 NO3--N) and potassium (52.32 mg dm-3) contents. The highest levels of sulfur (21.73 mg dm-3) and iron (96.46 mgdm-3) were detected in the biological treatment. The chemical treatment showed higher activity of the enzymes acid phosphatase, acetylglucosaminidase, and sulfatase, while α-glucosidase and leucine aminopeptidase stood out in the biological treatment. Soil properties were not significantly affected by the treatments. The use of microalgae biomass derived from wastewater treatment from milking parlors was evaluated and presented as a promising biofertilizer for agriculture, following the line of recovering nutrient-rich wastes. In this sense, although many challenges need to be overcome, the results suggest that microalgal-based fertilizers could lead to low-impact agriculture.

Tryptophan Reduces Intracohort Cannibalism Behavior in Tropical Gar (Atractosteus tropicus) Larvae

The intracohort cannibalism present in tropical gar larvae (A. tropicus) generates great problems in its culture, as in other fish species around the world. The addition of tryptophan (Trp) (10, 20, and 30 g/kg) and a control diet (CD) without Trp were evaluated in A. tropicus larvae regarding growth, survival, cannibalism, behavior, digestive enzymatic activity, and genes related to aggressiveness and/or cannibalism in two stages: 0–13 days after hatching (DAH); and only cannibals (14–24 DAH). In the first stage, no differences were observed in growth parameters; cannibalism was lower with the use of Trp, with the lowest percentage being the 10 g/kg Trp treatment (56.75 ± 2.47%) compared to CD (64.75 ± 1.76%). In the second stage, survival was greater in 10 g/kg Trp (75.00 ± 7.07%) than in CD (23.33 ± 5.77%). Thus, cannibalism was lower with 10 g/kg Trp (20.0 ± 10.0%) compared to CD (76.66 ± 5.77%). Cannibal larvae fed with 10 g/kg Trp had a greater enzymatic activity in acid and alkaline proteases and leucine aminopeptidase, as well as the overexpression of avpi1, crh, and htr1a and the subexpression of tph1, th, sstr1, and hdc (p < 0.05). No aggressive behaviors were recorded in the larvae fed with the 10 g/kg Trp treatment, unlike those fed with CD. The use of 10 g/kg Trp improves survival and reduces cannibalism in A. tropicus larvae.

Accumulation of polyethylene microplastics in river biofilms and effect on the uptake, biotransformation and toxicity of the antimicrobial triclosan

The interaction of multiple stressors in freshwater ecosystems may lead to adverse effects on aquatic communities and their ecological functions. Microplastics (MPs) are a class of contaminants of emerging concern that can exert both direct and indirect ecotoxicological effects. A growing number of studies have investigated MPs-attached microbial communities, but the interaction between MPs and substrate-associated biofilm (i.e., on natural river substrates, such as stones and sediments) remains poorly studied. In this work, the combined effects of polyethylene MPs (PE-MPs) with a particle size of 10–45 μm (2 mg/L) and the antimicrobial triclosan (TCS) (20 μg/L) were investigated on river biofilms through a short-term exposure experiment (72 h). To the best of authors’ knowledge, this is the first time that the combined effects of MPs and chemical contaminants in substrate-associated river biofilms were assessed. Different response parameters were evaluated, including (i) exposure assessment and ii) contaminants effects at different levels: bacterial community composition, antibiotic resistance, extracellular polymeric substances (EPS), photosynthetic efficiency (Yeff), and leucine aminopeptidase activity (LAPA). Triclosan was accumulated in river biofilms (1189–1513 ng/g dw) alongside its biotransformation product methyl-triclosan (20−29 ng/g dw). Also, PE-MPs were detected on biofilms (168−292 MP/cm2), but they had no significant influence on the bioaccumulation and biotransformation of TCS. A moderate shift in bacterial community composition was driven by TCS, regardless of PE-MPs co-exposure (e.g., increased relative abundance of Sphingomonadaceae family). Additionally, Yeff and EPS content were significantly disrupted in TCS-exposed biofilms. Therefore, the most remarkable effects on river biofilms were related to the antimicrobial TCS, whereas single PE-MPs exposure did not alter any of the evaluated parameters. These results demonstrate that biofilms might act as environmental sink of MPs. Although no interaction between PE-MPs and TCS was observed, the possible indirect impact of other MPs-adsorbed contaminants on biofilms should be further assessed.

Methionine sulfoxide reductase 2 regulates Cvt autophagic pathway by altering the stability of Atg19 and Ape1 in Saccharomyces cerevisiae

The reversible oxidation of methionine plays a crucial role in redox regulation of proteins. Methionine oxidation in proteins causes major structural modifications that can destabilize and abrogate their function. The highly conserved methionine sulfoxide reductases protect proteins from oxidative damage by reducing their oxidized methionines, thus restoring their stability and function. Deletion or mutation in conserved methionine sulfoxide reductases leads to aging and several human neurological disorders and also reduces yeast growth on nonfermentable carbon sources. Despite their importance in human health, limited information about their physiological substrates in humans and yeast is available. For the first time, we show that Mxr2 interacts invivo with two core proteins of the cytoplasm to vacuole targeting (Cvt) autophagy pathway, Atg19, and Ape1 in Saccharomyces cerevisiae. Deletion of MXR2 induces instability and early turnover of immature Ape1 and Atg19 proteins and reduces the leucine aminopeptidase activity of Ape1 without affecting the maturation process of Ape1. Additonally, Mxr2 interacts with the immature Ape1, dependent on Met17 present within the propeptide of Ape1 as a single substitution mutation of Met17 to Leu abolishes this interaction. Importantly, Ape1 M17L mutant protein resists oxidative stress-induced degradation in WT and mxr2Δ cells. By identifying Atg19 and Ape1 as cytosolic substrates of Mxr2, our study maps the hitherto unexplored connection between Mxr2 and the Cvt autophagy pathway and sheds light on Mxr2-dependent oxidative regulation of the Cvt pathway.

Effects of Organic Fertilizer of Kitchen Waste on Soil Microbial Activity and Function

Changes in soil microbial activity and ecological function can be used to assess the level of soil fertility and the stability of ecosystems. To assess the fertility and safety of organic fertilizer of kitchen waste (OFK), soils containing 0% (CK), 1%, 3%, and 5% OFK were cultured, and the physical, chemical, and microbial properties of the soils were measured dynamically with routine agrochemical analysis measures and amplicon sequencing. The results showed that compared with those in CK, the contents of organic matter, available phosphorus, available potassium, NH4+-N, and NO3--N in soils with OFK increased by 23.80%-35.13%, 13.29%-29.72%, 16.91%-39.37%, 164.7%-340.2%, and 28.56%-32.71%, respectively. The activities of hydrolases related to the cycle of carbon, nitrogen, and phosphorus (α-glucosidase, leucine aminopeptidase, acid phosphatase, etc.) were also significantly higher than those of the CK treatment. OFK stimulated the growth of soil microorganisms and increased the carbon content of the microbial biomass. The amplicon sequencing analysis found that the microbial community structures of different treatments were significantly different at both the class and genus levels. In addition, it was found that the abundance of beneficial microbes in the soils with OFK increased, whereas pathogenic microbes decreased. RDA results confirmed that soil properties (including soil pH, organic matter, available nutrients, and microbial biomass) had a significant impact on microbial community structure. The results of investing bacterial community based on PICRUSt and FAPROTAX revealed that the function of the soil bacterial community was similar in the four treatments, but OFK supply significantly improved the microbial carbon utilization and metabolic ability. Moreover, by using the FUNGuild software, we found that the application of OFK increased the proportion of saprotroph-symbiotroph and symbiotroph and stimulated the growth of ectomycorrhizal fungi-undefined saprophytic fungi but inhibited plant and animal pathogenic fungi in soil. These results implied that OFK could promote the establishment of symbiotic relationships and inhibit the growth of pathogenic fungi. In summary, OFK could improve soil fertility and hydrolase activity, stimulate the growth of beneficial microorganisms, and defend against pathogens, indicating a promising use as safe and efficient organic fertilizer.

Effects of Different Forest Types on Stoichiometric Characteristics of Carbon, Nitrogen, and Phosphorus in Tropical Soils, China

The stoichiometric characteristics of carbon (C), nitrogen (N), and phosphorus (P) in soil can directly reflect the level of soil fertility. In this study, a secondary forest, two kinds of plantation forests (Dacrycarpus imbricatus and Pinus caribaea Morelet), and a primary forest in tropical areas were selected for soil sample collection and analysis. We found that the soil [leucine aminopeptidase (LAP) + β-N-acetylglucosidase (NAG)]/acid phosphatase (APho) value was less than 1, indicating that there was P limitation. In addition, we also found that compared with primary forest, the β-glucosidase (BG) activity and (LAP + NAG) activity of the secondary forest were lower, and the microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) contents were higher. The contents of total nitrogen (TN) and MBN in the Dacrycarpus imbricatus forest soil were significantly higher than those in the primary forest, but the activity of (LAP + NAG) was lower. The structural equation model showed that the factors that significantly affected the total nutrients were the available nutrients (0.396**) and the enzyme activities (0.600**). For the long-term requirements of sustainable development, ecological adjustment and soil nutrient optimization of plantations should be carried out scientifically.

Soil extracellular enzyme stoichiometry reveals the nutrient limitations in soil microbial metabolism under different carbon input manipulations

Changes in the quality and quantity of litter and root inputs due to climate change and human activities can influence below-ground biogeochemical processes in forest ecosystems. However, it is unclear whether and how much aboveground litter and root inputs affect soil microbial metabolism and nutrient limitation mechanisms. In this study, according to a 4-years field manipulation experiment, litter and root manipulations (control (CK), double litter input (DL), no litter (NL), no root (NR), and no inputs (NI)) were set up to analyze the extracellular enzyme activities and stoichiometric ratios characteristics of 0–10 cm and 10–20 cm soils, explore the metabolic limitations of microorganisms, and clarify the main driving factors restricting nutrient limitation. The results showed that the enzyme activities associated with the C cycling (β-1,4-glucosidase (BG), cellulose disaccharide hydrolase (CBH)) and N cycling (β-1,4-N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP)) in DL treatment were significantly higher than those in NR treatment. Moreover, enzyme activities related to P cycling are significantly higher in comparison to other treatments. The acid phosphatase (AP), which is related to the P cycle, showed the highest activity under NR treatment. In addition, there was no significant difference in soil microbial metabolic limitation by the different carbon inputs, which did not change the original nutrient limitation pattern. The main drivers of microbial nutrient metabolic limitation included soil physicochemical properties, soil total nutrients, and available nutrients, among which soil SWC and pH presented the greatest influence on microbial C limitation and soil total nutrients showed the greatest influence on microbial N limitation. Changes in soil carbon input altered soil extracellular enzyme activities and their stoichiometric ratios by affecting soil physicochemical properties, total nutrients. This study provides data for the understanding of material cycling in forest ecosystems under environmental change.

Influencing mechanism of stand age to the accumulation of microbial residue carbon in the Pinus masso-niana plantations

Clarifying the accumulation pattern of soil microbial residue carbon and its contribution to soil organic carbon (SOC) across stand age is helpful to understand the mechanism underlying soil carbon cycling. In this study, we analyzed the differences of amino sugar content, physicochemical properties and microbial composition in surface soil (0-10 cm) in young (6 a), middle-aged (13 a), near-mature (29 a), mature (38 a) and over-mature (57 a) Pinus massoniana plantations of subtropical China, quantified the microbial residue carbon content and its contribution to SOC, and discussed the mechanism. The results showed that SOC, total nitrogen, amorphous iron oxide and leucine aminopeptidase contents in the middle-aged plantation were significantly lower than those in the mature plantation. Soil pH and fungal/bacteria in young plantation were significantly higher than those in other age groups. Across the stand age gradient, the ranges of microbial, fungal and bacterial residue carbon were 7.52-14.63, 4.03-8.00 and 3.48-6.63 g·kg-1, respectively. The contents of all the residue carbon were significantly higher in the mature plantation than that of the middle-aged plantation, which were positively affected by soil total nitrogen content. The contribution of microbial, fungal, and bacterial residue carbon to SOC was 59.7%-72.3%, 33.4%-45.6%, and 24.3%-30.8%, respectively. The contribution of fungal residue carbon to SOC in young plantation was significantly higher than that in other age groups, and the contribution of bacterial residue carbon to SOC in middle-aged plantation was significantly higher than that in young and near-mature plantations, both of which were affected by soil inorganic nitrogen. Fungal residue carbon content was 1.2-1.7 times as that of bacterial residue carbon content, and dominated for the accumulation of microbial residue carbon. Results of the partial least squares model showed that stand age, soil environmental factors (such as leucine aminopeptidase, amorphous iron oxide, pH, and total nitrogen), bacterial residue carbon, fungal residue carbon and the contribution of bacterial residue carbon to SOC had total effects on the contribution of fungal residue carbon to SOC (-0.37, -1.16, 0.90, 1.09, and 0.83, respectively). In conclusion, stand age promoted the accumulation of microbial residue carbon but did not increase its contribution to SOC.

Identification of atrial fibrillation-related genes through transcriptome data analysis and Mendelian randomization.

Atrial fibrillation (AF) is a common persistent arrhythmia characterized by rapid and chaotic atrial electrical activity, potentially leading to severe complications such as thromboembolism, heart failure, and stroke, significantly affecting patient quality of life and safety. As the global population ages, the prevalence of AF is on the rise, placing considerable strains on individuals and healthcare systems. This study utilizes bioinformatics and Mendelian Randomization (MR) to analyze transcriptome data and genome-wide association study (GWAS) summary statistics, aiming to identify biomarkers causally associated with AF and explore their potential pathogenic pathways. We obtained AF microarray datasets GSE41177 and GSE79768 from the Gene Expression Omnibus (GEO) database, merged them, and corrected for batch effects to pinpoint differentially expressed genes (DEGs). We gathered exposure data from expression quantitative trait loci (eQTL) and outcome data from AF GWAS through the IEU Open GWAS database. We employed inverse variance weighting (IVW), MR-Egger, weighted median, and weighted model approaches for MR analysis to assess exposure-outcome causality. IVW was the primary method, supplemented by other techniques. The robustness of our results was evaluated using Cochran's Q test, MR-Egger intercept, MR-PRESSO, and leave-one-out sensitivity analysis. A "Veen" diagram visualized the overlap of DEGs with significant eQTL genes from MR analysis, referred to as common genes (CGs). Additional analyses, including Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and immune cell infiltration studies, were conducted on these intersecting genes to reveal their roles in AF pathogenesis. The combined dataset revealed 355 differentially expressed genes (DEGs), with 228 showing significant upregulation and 127 downregulated. Mendelian randomization (MR) analysis identified that the autocrine motility factor receptor (AMFR) [IVW: OR = 0.977; 95% CI, 0.956-0.998; P = 0.030], leucine aminopeptidase 3 (LAP3) [IVW: OR = 0.967; 95% CI, 0.934-0.997; P = 0.048], Rab acceptor 1 (RABAC1) [IVW: OR = 0.928; 95% CI, 0.875-0.985; P = 0.015], and tryptase beta 2 (TPSB2) [IVW: OR = 0.971; 95% CI, 0.943-0.999; P = 0.049] are associated with a reduced risk of atrial fibrillation (AF). Conversely, GTPase-activating SH3 domain-binding protein 2 (G3BP2) [IVW: OR = 1.030; 95% CI, 1.004-1.056; P = 0.024], integrin subunit beta 2 (ITGB2) [IVW: OR = 1.050; 95% CI, 1.017-1.084; P = 0.003], glutaminyl-peptide cyclotransferase (QPCT) [IVW: OR = 1.080; 95% CI, 1.010-0.997; P = 1.154], and tripartite motif containing 22 (TRIM22) [IVW: OR = 1.048; 95% CI, 1.003-1.095; P = 0.035] are positively associated with AF risk. Sensitivity analyses indicated a lack of heterogeneity or horizontal pleiotropy (P > 0.05), and leave-one-out analysis did not reveal any single nucleotide polymorphisms (SNPs) impacting the MR results significantly. GO and KEGG analyses showed that CG is involved in processes such as protein polyubiquitination, neutrophil degranulation, specific and tertiary granule formation, protein-macromolecule adaptor activity, molecular adaptor activity, and the SREBP signaling pathway, all significantly enriched. The analysis of immune cell infiltration demonstrated associations of CG with various immune cells, including plasma cells, CD8T cells, resting memory CD4T cells, regulatory T cells (Tregs), gamma delta T cells, activated NK cells, activated mast cells, and neutrophils. By integrating bioinformatics and MR approaches, genes such as AMFR, G3BP2, ITGB2, LAP3, QPCT, RABAC1, TPSB2, and TRIM22 are identified as causally linked to AF, enhancing our understanding of its molecular foundations. This strategy may facilitate the development of more precise biomarkers and therapeutic targets for AF diagnosis and treatment.

Indirect influence of soil enzymes and their stoichiometry on soil organic carbon response to warming and nitrogen deposition in the Tibetan Plateau alpine meadow.

Despite extensive research on the impact of warming and nitrogen deposition on soil organic carbon components, the response mechanisms of microbial community composition and enzyme activity to soil organic carbon remain poorly understood. This study investigated the effects of warming and nitrogen deposition on soil organic carbon components in the Tibetan Plateau alpine meadow and elucidated the regulatory mechanisms of microbial characteristics, including soil microbial community, enzyme activity, and stoichiometry, on organic carbon components. Results indicated that both warming and nitrogen deposition significantly increased soil organic carbon, readily oxidizable carbon, dissolved organic carbon, and microbial biomass carbon. The interaction between warming and nitrogen deposition influenced soil carbon components, with soil organic carbon, readily oxidizable carbon, and dissolved organic carbon reaching maximum values in the W0N32 treatment, while microbial biomass carbon peaked in the W3N32 treatment. Warming and nitrogen deposition also significantly increased soil Cellobiohydrolase, β-1,4-N-acetylglucosaminidase, leucine aminopeptidase, and alkaline phosphatase. Warming decreased the soil enzyme C: N ratio and C:P ratio but increased the soil enzyme N:P ratio, while nitrogen deposition had the opposite effect. The bacterial Chao1 index and Shannon index increased significantly under warming conditions, particularly in the N32 treatment, whereas there were no significant changes in the fungal Chao1 index and Shannon index with warming and nitrogen addition. Structural equation modeling revealed that soil organic carbon components were directly influenced by the negative impact of warming and the positive impact of nitrogen deposition. Furthermore, warming and nitrogen deposition altered soil bacterial community composition, specifically Gemmatimonadota and Nitrospirota, resulting in a positive impact on soil enzyme activity, particularly soil alkaline phosphatase and β-xylosidase, and enzyme stoichiometry, including N:P and C:P ratios. In summary, changes in soil organic carbon components under warming and nitrogen deposition in the alpine meadows of the Tibetan Plateau primarily depend on the composition of soil bacterial communities, soil enzyme activity, and stoichiometric characteristics.

Safety evaluation of the food enzyme leucyl aminopeptidase from the non-genetically modified Aspergillus sp. strain AE-MB.

The food enzyme leucyl aminopeptidase (EC 3.4.11.1) is produced with the non-genetically modified Aspergillus sp. strain AE-MB by Amano Enzyme Inc. The food enzyme is considered free from viable cells of the production organism. It is intended to be used in five food manufacturing processes: processing of dairy products for the production of (1) flavouring preparations; processing of plant- and fungal-derived products for the production of (2) protein hydrolysates; processing of meat and fish products for the production of (3) protein hydrolysates, (4) modified meat and fish products and processing of (5) yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 2.273 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 183 mg TOS/kg bw per day. The calculated margin of exposure for each age group was 135 (infants), 81 (toddlers), 83 (children), 109 (adolescents), 160 (adults) and 184 (the elderly). A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. The safety of the food enzyme could not be established given the derived margins of exposure. Therefore, the Panel concluded that this food enzyme could not be considered safe under the intended conditions of use.

Microbial carbon and nitrogen limitation in Larix gmelinii forests along an altitudinal gradient: Evidence from ecoenzymatic stoichiometry and vector analysis

Mountain ecosystems have obvious variations in environmental factors at small spatial scales and thus provide a natural platform for investigating climate change. To investigate the characteristics of extracellular enzymes and microbial metabolic limitations in the hostile environmental conditions of cold temperate mountain ecosystems, soil physicochemical properties and enzyme activities were measured in the 0–10 cm and 10–20 cm soil layers of Larix gmelinii forests at elevations of 830 m, 950 m, 1100 m, and 1300 m. Our results showed that altitude and soil depth had a significant effect on 1,4-β-glucosidase, 1,4-β-N-acetyl-glucosaminidase, leucine aminopeptidase, and acid phosphatase activities. Extracellular enzyme activities decreased with increasing altitude and soil depth. The stoichiometric ratio of soil C-, N-, and P-acquiring enzymes after logarithmic transformation was 1:1.27:0.72 across all altitudes and soil depths. The metabolism of the microorganisms was mainly limited by C and N, and a high C limitation was observed at higher-altitude sites. With increased soil depth, the N limitation gradually weakened. Moreover, soil bulk density and pH were the main factors affecting extracellular enzyme activities and microbial nutrient constraints. Our study revealed that the carbon and nitrogen limitation of soil microbes was induced by the variation in soil factors in a cold temperate mountain forest ecosystem. This finding provides important insights into the mechanism of microbial metabolic limitation in the face of climate change.

Enzymatic Activity and Amino Acids Production of Predominant Fungi from Traditional Meju during Soybean Fermentation.

To investigate the effect of the predominant fungal species from Korean traditional meju and doenjang on soybean fermentation, the enzymatic activity and amino acid production of twenty-two fungal strains were assessed through solid- and liquid-state soybean fermentation. Enzymatic activity analyses of solid-state fermented soybeans revealed different enzyme activities involving protease, leucine aminopeptidase (LAP), carboxypeptidase (CaP), glutaminase, γ-glutamyl transferase (GGT), and amylase, depending on the fungal species. These enzymatic activities significantly affected the amino acid profile throughout liquid-state fermentation. Strains belonging to Mucoromycota, including Lichtheimia, Mucor, Rhizomucor, and Rhizopus, produced smaller amounts of total amino acids and umami-producing amino acids, such as glutamic acid and aspartic acid, than strains belonging to Aspergillus subgenus circumdati. The genera Penicillium and Scopulariopsis produced large amounts of total amino acids and glutamic acid, suggesting that these genera play an essential role in producing umami and kokumi tastes in fermented soybean products. Strains belonging to Aspergillus subgenus circumdati, including A. oryzae, showed the highest amino acid content, including glutamic acid, suggesting the potential benefits of A. oryzae as a starter for soybean fermentation. This study showed the potential of traditional meju strains as starters for soybean fermentation. However, further analysis of processes such as the production of G-peptide for kokumi taste and volatile compounds for flavor and safety is needed.

Precisely Constructing Renal-Clearable and LAP-Activatable Ratiometric Molecular Probes for Early Diagnosis of Acute and Chronic Kidney Injury Via Optimizing Asymmetric DPP Dyes.

Fluorescence analysis is an increasingly important contributor to the early diagnosis of kidney diseases. To achieve precise visualization of the kidneys and early diagnosis of related diseases, an asymmetric pyrrolopyrrolidone (DPP) dye platform with C-aromatic substituents and N-lipophilic/hydrophilic modification was constructed. Based on these, we developed the renal-clearable, water-soluble, and kidney injury biomarker leucine aminopeptidase (LAP) activated ratiometric fluorescent probe DPP-S-L. In the mouse model of cisplatin-induced acute kidney injury and during the development of type 2 diabetes to diabetic kidney disease, we visualized for the first time the upregulation of LAP in the kidney and urine by dual-channel ratiometric fluorescence signal and diagnosed the kidney injury earlier and more sensitively than blood/urine enzyme detection and tissue analysis. This study showcases an excellent asymmetric DPP dye platform and renal-clearable ratiometric fluorescent probe design strategy that is extended to determination and visualization of other biomarkers for early disease diagnosis.

Short-term continuous monocropping reduces peanut yield mainly via altering soil enzyme activity and fungal community

Continuous monocropping can lead to soil sickness and increase of soil-borne disease, which finally reduces crop yield. Microorganisms benefit plants by increasing nutrient availability, participating in auxin synthesis, and defending against pathogens. However, little is known about the influence of short-term successive peanuts cropping on soil properties, enzyme activities, its yield, plant-associated microbes, and their potential correlations in peanut production. Here, we examined the community structure, composition, network structure and function of microbes in the rhizosphere and bulk soils under different monocropping years. Moreover, we assessed the impact of changes in the soil micro-environment and associated soil microbes on peanut yield. Our results showed that increase of monocropping year significantly decreased most soil properties, enzyme activities and peanut yield (p < 0.05). Principal co-ordinates analysis (PCoA) and analysis of similarities (ANOSIM) indicated that monocropping year significantly influenced the fungal community structure in the rhizosphere and bulk soils (p < 0.01), while had no effect on the bacterial community. With the increase of continuous monocropping year, peanut selectively decreased (e.g., Candidatus_Entotheonella, Bacillus and Bryobacter) or increased (e.g., Nitrospira, Nocardioides, Ensifer, Gaiella, and Novosphingobium) the abundance of some beneficial bacterial genera in the rhizosphere. Continuous monocropping significantly increased the abundance of plant pathogens (e.g., Plectosphaerella, Colletotrichum, Lectera, Gibberella, Metarhizium, and Microdochium) in the rhizosphere and negatively affected the balance of fungal community. Besides, these species were correlated negatively with L-leucine aminopeptidase (LAP) activity. Network co-occurrence analysis showed that continuous monocropping simplified the interaction network of bacteria and fungi. Random forest and partial least squares path modeling (PLS-PM) analysis further showed that fungal community, pathogen abundance, soil pH, and LAP activity negatively affected peanut yield. In conclusion, short-term continuous monocropping decreased LAP activity and increased potential fungal pathogens abundance, leading to reduction of peanut yield.

Altered intra-annual precipitation patterns affect the N-limitation status of soil microorganisms in a semiarid alpine grassland

The ongoing intensification of the hydrological cycle due to global climate change alters intra-annual precipitation variability. Changes in precipitation patterns lead to disparities in soil moisture; however, the responses of soil extracellular enzyme activities (EEAs) and microbial metabolism limitations to them is unclear. This study conducted an in situ field experiment in the alpine grasslands of the Kunlun Mountains to simulate the same amount of precipitation but change the distribution time of precipitation within the plant growing season. We examined the effects of variability in intra-annual precipitation patterns on the stoichiometry of topsoil (0–5 and 5–20 cm) properties, microbial biomass, and EEAs. Results showed that altered precipitation patterns significantly increased soil moisture’s disparity index (D) by 57 %–89 %. The activity of nitrogen-acquiring enzymes (L-leucine aminopeptidase) was increased by 20.82–32.08 % (P < 0.05) with increased variability of precipitation, while phosphorus-acquiring enzymes (Acid phosphatase) and carbon-acquiring enzymes (β-glucosidase) both decrease by 9.25–23.35 % and by 17.86–33.04 %, respectively (P < 0.05). We determined that soil microorganisms in the study region were metabolism-limited by nitrogen (vector angles < 45◦), and increased precipitation variability exacerbated nitrogen limitation (decrescent vector angles) and alleviated carbon limitation (shortened vector length). In addition, soil EEAs can be both interactively influenced by biotic and abiotic factors, with abiotic factors (i.e., D, soil organic carbon content, and soil total nitrogen content) having a stronger effect. Our study provides an effective strategy for regulating soil EEAs, microbial biomass, and microbial metabolism limitation and contributes to developing nutrient management and restoration strategies in semiarid alpine grassland ecosystems.

Role of sodium octanoate in regulating survival, growth, intestinal development, digestive and absorptive capacities, antioxidant capacity and innate immunity of large yellow croaker (Larimichthys crocea) larvae

The present study was conducted to investigate the effects of dietary sodium octanoate (So) on the survival, growth, intestinal development, digestive and absorptive capacities, antioxidant capacity and innate immunity of Larimichthys crocea larvae. Larvae (initial body weight: 7.44 ± 0.53 mg) were fed with either a control diet or diets with different levels (0.1%, 0.2%, 0.4%, and 0.8%) of So for 30 days. The results showed that larvae fed diets with 0.1%–0.4% So had significantly increased survival compared to the control. Compared to the control, larvae fed the diet with 0.4% So showed dramatically improved growth performance, although there were no significant differences between other diets. Notably, larvae fed diets with 0.2%–0.4% So displayed significantly higher fold height of intestine compared to the control group. Increased levels of selected intestinal cell proliferation and differentiation gene markers (pcna and odc) and increased activity of intestinal brush border enzymes (alkaline phosphatase and leucine aminopeptidase) further affirmed the promotional effect of So on intestinal development. Moreover, for genes related to the intestinal apical junction complex, larvae fed diets with 0.1%–0.8% So showed significantly higher mRNA expression levels of occludin and cateninα1 than the control group. Meanwhile, compared to the control, the expression of claudin1, afadin and cadherin2 significantly increased in larvae fed diets with 0.1%–0.4% So. Also, larvae fed diets with 0.1%–0.8% So showed significantly higher activity of lipase than the control group, whereas the activity of trypsin was significantly higher only in larvae fed the diet with 0.4% So. Furthermore, absorption capacity, as reflected by the activity of brush border enzymes (Na+-K+-Adenosinetriphosphatase and creatine kinase), significantly increased in larvae fed diets with 0.2%–0.4% So compared to the control. Furthermore, larvae fed the diet with 0.4% So exhibited improved antioxidant enzyme activity (catalase and total antioxidant capacity), upregulated antioxidant related gene expression (sod2, sod3, cat, and nrf2), increased antioxidant substance levels (reduced glutathione), and decreased malondialdehyde content compared to the control group. Moreover, larvae fed the diet with 0.4% So showed the highest activity of innate immune-related enzymes (lysozyme, acid phosphatase, inducible nitric oxide synthase and total nitric oxide synthase) and the highest mRNA expression of anti-inflammatory cytokines (il4/13 and arg1). In summary, the diet supplemented with 0.4% So may optimally improve larval survival and growth probably by enhancing intestinal development, digestion and absorption, antioxidant capacity, and innate immunity.

Engineering a highly selective leucine aminopeptidase near-infrared fluorescence probe for early diagnosis of diabetic nephropathy

Diabetic nephropathy (DN) is one of the most serious and general complications of diabetes, while schedules for prevention and treatment are unsatisfactory. Leucine aminopeptidase (LAP) as one of the biomarkers of DN is associated with glomerular and/or tubular injury. Imaging of LAP activity in DN disease model in vivo is thus beneficial for early diagnosis and prevention of DN, but such a strategy is still lacking. Herein, an enzyme-activated probe HD-LAP with a NIR fluorescence emission for specific detection of LAP activity in the DN model is designed and synthesized. HD-LAP has a significant fluorescence enhancement after reacted with LAP and shows a NIR fluorescence emission at 704 nm based on intramolecular charge transfer mechanism. Moreover, HD-LAP can be employed to image LAP activity in HK-2 and HepG2 cells. More importantly, HD-LAP is the first example to real-time image LAP in DN mice and clinical serum samples. These results demonstrated that HD-LAP is promising as a powerful tool for the research on LAP associated diabetic diseases in future.