Hydrolase Activity Research Articles

Nitrogen addition accelerates litter decomposition and arsenic release of Pteris vittata in arsenic-contaminated soil from mine

The arsenic (As) release from litter decomposition of As-hyperaccumulator (Pteris vittata L.) in mine areas poses an ecological risk for metal dispersion into the soil. However, the effect of atmospheric nitrogen (N) deposition on the litter decomposition of As-hyperaccumulator in the tailing mine area remains poorly understood. In this study, we conducted a microcosm experiment to investigate the As release during the decomposition of P. vittata litter under four gradients of N addition (0, 5, 10, and 20 mg N g−1). The N10 treatment (10 mg N g−1) enhanced As release from P. vittata litter by 1.2–2.6 folds compared to control. Furthermore, Streptomyces, Pantoea, and Curtobacterium were found to primarily affect the As release during the litter decomposition process. Additionally, N addition decreased the soil pH, subsequently increased the microbial biomass, as well as hydrolase activities (NAG) which regulated N release. Thereby, N addition increased the As release from P. vittata litter and then transferred to the soil. Moreover, this process caused a transformation of non-labile As fractions into labile forms, resulting in an increase of available As concentration by 13.02–20.16% within the soil after a 90-day incubation period. Our findings provide valuable insights into assessing the ecological risk associated with As release from the decomposition of P. vittata litter towards the soil, particularly under elevated atmospheric N deposition.

The mitogen-activated protein kinase module CcSte11-CcSte7-CcPmk1 regulates pathogenicity via the transcription factor CcSte12 in Cytospora chrysosperma

The pathogen Cytospora chrysosperma is the causal agent of poplar canker disease and causes considerable economic losses in China. Mitogen-activated protein kinase (MAPK) cascades play a crucial role in mediating cellular responses and Pmk1-MAPKs are indispensable for pathogenic related processes in plant pathogenic fungi. In previous studies, we demonstrated that the CcPmk1 acts as a core regulator of fungal pathogenicity by modulating a small number of master downstream targets, such as CcSte12. In this study, we identified and characterized two upstream components of CcPmk1: MAPKKK CcSte11 and MAPKK CcSte7. Deletion of CcSte11 and CcSte7, resulted in slowed growth, loss of sporulation and virulence, similar to the defects observed in the CcPmk1 deletion mutant. In addition, CcSte11, CcSte7 and CcPmk1 interact with each other, and the upstream adaptor protein CcSte50 interact with CcSte11 and CcSte7. Moreover, we explored the global regulation network of CcSte12 by transcriptional analysis between CcSte12 deletion mutants and wild-type during the simulated infection process. Two hydrolase activity GO terms (GO:0004553 and GO:0016798) and starch and sucrose metabolism (mgr00500) KEGG pathway were significantly enriched in the down-regulated genes of CcSte12 deletion mutants. In addition, a subset of glycosyl hydrolase genes and putative effector genes were significantly down-regulated in the CcSte12 deletion mutant, which might be important for fungal pathogenicity. Especially, CcSte12 bound to the CcSp84 promoter region containing the TGAAACA motif. Moreover, comparison of CcSte12-regulated genes with CcPmk1-regulated genes revealed 116 overlapping regulated genes in both CcSte12 and CcPmk1, including some virulence-associated genes. Taken together, the protein complexes CcSte11-CcSte7-CcPmk1 receive signals transmitted by upstream CcSte50 and transmit signals to downstream CcSte12, which regulates hydrolase, effectors and other genes to promote virulence. Overall, these results indicate that the CcPmk1-MAPK signaling pathway of C. chrysosperma plays a key role in the pathogenicity.

The Effect of p.G2019S Mutation in the LRRK2 Gene on the Activity of Lysosomal Hydrolases and the Clinical Features of Parkinson's Disease Associated with p.N370S Mutation in the GBA1 Gene.

Mutations in the glucocerebrosidase (GBA1) and leucine-rich repeat kinase 2 (LRRK2) genes, encoding lysosomal enzyme glucocerebrosidase (GCase) and leucine-rich repeat kinase 2 (LRRK2), respectively, are the most common related to Parkinson's disease (PD). Recent data suggest a possible functional interaction between GCase and LRRK2 and their involvement in sphingolipid metabolism. The aim of the present study was to describe the clinical course and evaluate the lysosomal enzyme activities and sphingolipid concentrations in blood of patients with PD associated with dual mutations p.N370S GBA1 and p.G2019S LRRK2 (p.N370S/GBA-p.G2019S/LRRK2-PD) as well as in blood of asymptomatic mutation carriers (p.N370S/GBA1-p.G2019S/LRRK2-carrier). One patient with p.N370S/GBA1-p.G2019S/LRRK2-PD and one p.N370S/GBA1-p.G2019S/LRRK2-carrier were enrolled. GBA1-associated PD (GBA1-PD), LRRK2-associated PD (LRRK2-PD), sporadic PD (sPD) patients were described earlier by our research group. A neuropsychiatric examination of the p.N370S/GBA1-p.G2019S/LRRK2-PD patient was carried out using scales (Montreal Cognitive Assessment scale (MoCA), Mini-mental State Examination scale (MMSE), Frontal Assessment Batter scale (FAB), Hospital Anxiety, and Depression Scale (HADS), etc). Lysosomal enzyme activity (GCase, alpha-galactosidase [GLA], acid sphingomyelinase [ASMase], galactosylcerebrosidase [GALC]) and sphingolipid concentrations (hexasylsphingosine [HexSph], lysoglobotriaosylsphingosine [LysoGb3], lysosphingomyelin [LysoSM]) were assessed with high-performance liquid chromatography-tandem mass spectrometry in blood. The following comparison with the previously described groups of GBA1-PD and sPD patients were conducted. Clinical features of p.N370S/GBA1-p.G2019S/LRRK2-PD included an early age of onset of the disease (46 years) and mild cognitive and affective disorders (MMSE = 29, MoCA = 23), despite a long (24 years) course of the disease. Interestingly, no differences were found in hydrolase activity and lysosphingolipid concentrations between the p.N370S/GBA1-p.G2019S/LRRK2-PD patient and GBA1-PD patients. However, GCase activity was lower in these groups than in LRRK2-PD, sPD, and controls. Additionally, the p.N370S/GBA1-p.G2019S/LRRK2-PD patient was characterized by a pronounced decreased in ASMase activity and increased LysoSM concentration compared to the p.N370S/GBA1-p.G2019S/LRRK2-carrier (p = 0.023, p = 0.027, respectively). Based on one patient, our results indicate a protective effect of the p.G2019S mutation in the LRRK2 gene on clinical course of p.N370S/GBA1-PD. The identified pronounced alteration of ASMase activity and LysoSM concentration in p.N370S/GBA1-p.G2019S/LRRK2-PD provide the basis for the further research.

Inoculation of cadmium-tolerant bacteria to regulate microbial activity and key bacterial population in cadmium-contaminated soils during bioremediation

The perennial ryegrass Lolium perenne can be used in conjunction with cadmium (Cd)-tolerant bacteria such as Cdq4–2 (Enterococcus spp.) for bioremediation of Cd-contaminated soil. In this study, a theoretical basis was provided to increase the efficiency of L. perenne remediation of Cd-contaminated soil using microorganisms to maintain the stability of the soil microbiome. The experimental design involved three treatment groups: CK (soil without Cd addition) as the control, 20 mg·kg–1 Cd-contaminated soil, and 20 mg·kg–1 Cd-contaminated soil + Cdq4–2, all planted with L. perenne. The soil was collected on day 60 to determine the soil microbial activity and bacterial community structure and to analyze the correlation between soil variables, the bacterial community, available Cd content in the soil, Cd accumulation, and L. perenne growth. The soil microbial activity and bacterial community diversity decreased under Cd stress, and the soil microbial community composition was changed; while inoculation with Cdq4–2 significantly increased soil basal respiration and the activities of urease, invertase, and fluorescein diacetate (FDA) hydrolase by 83.65%, 79.72%, 19.88%, and 96.15% respectively; and the stability of the community structure was also enhanced. The Actinobacteriota biomass, the amount of available Cd, and the above- and belowground Cd content of L. perenne were significantly negatively correlated with the total phosphorus, total potassium, and pH. The activity of urease, invertase, and FDA hydrolase were significantly positively correlated with the biomasses of Acidobacteriota and L. perenne and significantly negatively correlated with the Chloroflexi biomass. Further, the available soil Cd content and the above- and belowground Cd levels of L. perenne were significantly positively correlated with the Actinobacteriota biomass and significantly negatively correlated with the Gemmatimonadetes biomass. Overall, inoculating Cd-tolerant bacteria improved the microbial activity, diversity, and abundance, and changed the microbial community composition, facilitating the remediation of Cd-contaminated soil by L. perenne.

Polysaccharide preferred minority-dominant community assembly and exoenzyme enrichment in transparent exopolymer particles: Implication for global carbon cycle in water

The ubiquitous transparent exopolymer particles (TEPs) are an important organic carbon pool and an ideal microhabitat for bacteria in aquatic environments. They play a crucial role in the global carbon cycle. Organic matter transformation and carbon turnover in TEPs strongly depend on the assembly of their associated bacterial communities and enzyme activity. However, the mechanisms of bacterial community assembly and their potential effects on the organic carbon cycle in TEPs are still unclear. In this study, we comparatively explored the community assembly of TEP-associated bacteria and bacterioplankton from surface freshwater using metagenomics. It was found that the bacterial community assembly in TEPs followed a minority-dominant rule and was governed by homogeneous selection. Pseudomonadota and Actinomycetota, which are responsible for polysaccharide degradation, serve as taxon-specific biomarkers among the abundant and diverse bacteria in TEPs. The network of TEP-associated bacteria displayed stronger robustness than that of bacterioplankton. Bin 76 (majorly Acinetobacter) was the overwhelmingly dominant taxa in TEPs, whereas there was no clearly dominant taxa in TEP-free water. Exoenzyme analysis showed that 64 out of 71 identified polysaccharide hydrolases were markedly linked with the dominant bin 76 in TEPs, while no such linkage was observed for bacterioplankton. Generally, Acinetobacter, which is capable of utilizing polysaccharides, is preferred to be assembled in TEPs together with high polysaccharide hydrolase activity. This may significantly accelerate the turnover of organic carbon in the giant global TEP pool. These findings are important for a deep understanding of the carbon cycle in water.

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.

Effect of Microplastics and Phenanthrene on Soil Chemical Properties, Enzymatic Activities, and Microbial Communities

Microplastic and polycyclic aromatic hydrocarbon (PAHs) pollution have received increasing attention due to their ubiquitous distribution and potential risks in soils. However, the effects of microplastics-PAHs combined pollution on soil ecosystems remain unclear. Polyethylene (PE)/polypropylene (PP) and phenanthrene (PHE) were selected as the representatives of microplastics and PAHs, respectively. A 300-day soil microcosm experiment was conducted to study the single and combined effects of PE/PP and PHE on soil chemical properties, enzymatic activities, and bacterial communities (i.e., quantity, composition, and function), using the soil agricultural chemical analysis method and 16S amplicon sequencing technology. The interactions of soil properties, enzyme activities, and flora in the presence of PE/PP and PHE were analyzed. The results showed that the addition of PE/PP and PHE slightly changed the pH, available phosphorus (AP), and microbial quantity (i.e., bacteria, actinomycetes, and mold) but considerably increased the fluorescein diacetate hydrolase (FDAse) activity. There was a significant enhancement of soil organic matter (SOM) and urease activity in PE, PP, PHE-PE, and PHE-PP amended systems. PHE, PHE-PE, and PHE-PP obviously increased the dehydrogenase/neutral phosphatase activities and available nitrogen (AN) content. PHE had little effect on the microbial community. The PE, PP, PHE-PE, and PHE-PP addition influenced the microbial community to some extent. PE/PP and PHE showed positive effects on the energy production, growth, and reproduction of soil microorganisms and then accelerated the metabolism/degradation of pollutants and membrane transport. The changes in AN and SOM induced by PE/PP and PHE were the key factors affecting soil enzyme activities. Alterations in AN, AP, and pH were mainly responsible for the increase in microbial population. The changes in the microbial community were related to soil chemical properties and enzyme activities, and SOM had a significant effect on the microbial community. The presence of different carbon sources (PE/PP and PHE) in the soil and the microbial interaction also affected the microbiota. In conclusion, the addition of single or combined pollutants of PE/PP and PHE influenced the soil chemical properties, enzymatic activities, bacterial communities, and their interaction processes, thus facilitating the adaptation of the microbial community to pollutant stress.

Bioactivity and genome analysis of Bacillus amyloliquefaciens GL18 isolated from the rhizosphere of Kobresia myosuroides in an alpine meadow.

The unique eco-environment of the Qinghai-Tibet Plateau breeds abundant microbial resources. In this research, Bacillus amyloliquefaciens GL18, isolated from the rhizosphere of Kobresia myosuroides from an alpine meadow, and the antagonistic activity, bacteriostatic hydrolase activity, and low temperature, salt, and drought resistance of it were determined and analysed. The seedlings of Avena sativa were root-irrigated using bacteria suspensions (cell concentration 1 × 107cfu/mL) of GL18, and the growth-promoting effect of GL18 on it was determined under cold, salt and drought stress, respectively. The whole genome of GL18 was sequenced, and its functional genes were analysed. GL18 presented significant antagonistic activity to Fusarium graminearum, Fusarium acuminatum, Fusarium oxysporum and Aspergillus niger (inhibition zone diameter > 17mm). Transparent zones formed on four hydrolase detection media, indicating that GL18 secreted cellulase, protease, pectinase and β-1,3-glucanase. GL18 tolerated conditions of 10°C, 11% NaCl and 15% PEG-6000, presenting cold, salt and drought resistance. GL18 improved the cold, salt and drought tolerance of A. sativa and it showed significant growth effects under different stress. The total length of the GL18 genome was 3,915,550bp, and the number of coding DNA sequence was 3726. Compared with the clusters of orthologous groups of proteins, gene ontology and kyoto encyclopedia of genes and genomes databases, 3088, 2869 and 2357 functional genes were annotated, respectively. GL18 contained gene clusters related to antibacterial substances, functional genes related to the synthesis of plant growth-promoting substances, and encoding genes related to stress resistance. This study identified an excellent Bacillus strain and provided a theoretical basis for improving stress resistance and promoting the growth of herbages under abiotic stress.

Brown sugar as a carbon source can make agricultural organic waste compost enter the secondary thermophilic stage and promote compost decomposition.

To enhance the efficiency of composting agricultural organic waste (AOW), this study aimed to examine the impact of inoculating tomato straw compost with two distinct microbial agents: ZymoZone (ZZ), a composite microbial agent derived from the straw compost and Effective Microorganisms (EM), a commercial microbial agent. Furthermore, in order to reactivate the microorganisms within the compost during the initial high temperature phase, 10% brown sugar was introduced as a carbon source. The objective of this addition was to assess its influence on the composting process. The findings revealed that compared to the control (CK) group, the ZZ and EM treatments extended the first high-temperature phase by 2 and 1day, respectively. Furthermore, with the addition of 10% brown sugar, the ZZ and EM treatments remained in the second high-temperature phase for 8 and 7days, respectively, while the CK treatment had already entered the cooling stage by then. Notably, the inoculation of microbial agents and the addition of brown sugar substantially augmented the activity of lignocellulose-related hydrolases, thereby promoting the degradation of lignocellulose in the ZZ and EM treatment groups. This was confirmed by FTIR analysis, which demonstrated that the addition of microbial agents facilitated the degradation of specific substances, leading to reduced absorbance in the corresponding spectra. XRD analysis further indicated a notable reduction in cellulose crystallinity for both the ZZ (8.00%) and EM (7.73%) treatments. Hence, the incorporation of microbial agents and brown sugar in tomato straw compost effectively enhances the composting process and improves the quality of compost products.

Perception of butenolides by Bacillus subtilis via the α/β hydrolase RsbQ

The regulation of behavioral and developmental decisions by small molecules is common to all domains of life. In plants, strigolactones and karrikins are butenolide growth regulators that influence several aspects of plant growth and development, as well as interactions with symbiotic fungi.1,2,3 DWARF14 (D14) and KARRIKIN INSENSITIVE2 (KAI2) are homologous enzyme-receptors that perceive strigolactones and karrikins, respectively, and that require hydrolase activity to effect signal transduction.4,5,6,7 RsbQ, a homolog of D14 and KAI2 from the gram-positive bacterium Bacillus subtilis, regulates growth responses to nutritional stress via the alternative transcription factor SigmaB (σB).8,9 However, the molecular function of RsbQ is unknown. Here, we show that RsbQ perceives butenolide compounds that are bioactive in plants. RsbQ is thermally destabilized by the synthetic strigolactone GR24 and its desmethyl butenolide equivalent dGR24. We show that, like D14 and KAI2, RsbQ is a functional butenolide hydrolase that undergoes covalent modification of the catalytic histidine residue. Exogenous application of both GR24 and dGR24 inhibited the endogenous signaling function of RsbQ invivo, with dGR24 being 10-fold more potent. Application of dGR24 to B.subtilis phenocopied loss-of-function rsbQ mutations and led to a significant downregulation of σB-regulated transcripts. We also discovered that exogenous butenolides promoted the transition from planktonic to biofilm growth. Our results suggest that butenolides may serve as inter-kingdom signaling compounds between plants and bacteria to help shape rhizosphere communities.

Invitro and Invivo Analysis of Human Milk Lactic Acid Bacteria Isolates for Their Anti-hypercholesterolemia Actions.

The aim of this study was to evaluate the cholesterol lowering ability of Lactic Acid Bacteria (LAB) isolated from human breast milk under in vitro and in vivo conditions. Six LAB isolates namely Lacticaseibacillus casei 1A, Lactobacillus gasseri 5A, Enterococcus faecium 2C, Limosilactobacillus fermentum 3D, Pediococcus acidilactici 1C, and Lactiplantibacillus plantarum 7A, were examined for their bile resistance, bile salt hydrolase activity, cholesterol assimilation and viability in cholesterol rich; DeMan Rogosa and Sharpe broth, simulated gastric, small and upper intestinal conditions. During in vivo experiments, two putative LAB isolates were orally gavage to BALB/c mice, fed with normal basal and cholesterol rich (HCD) diets, daily for a period of 4 weeks. Blood serum analysis including total serum cholesterol, triglycerides, high-density and low-density lipoprotein (LDL) cholesterol levels and total fecal LAB counts of the animals were determined. The isolates in study showed bile resistance and bile salt hydrolysis activity, while significant differences (P < 0.05) were seen in their cholesterol assimilation ability. L. gasseri 5A (195.67%) and L. plantarum 7A (193.78%) displayed highest cholesterol removal percentages, respectively. Animals in HCD, fed with L. gasseri 5A and L. plantarum 7A showed decreased levels of total cholesterol and LDL, compared to the control groups. In HCD group liver weight was increased, while fecal LAB counts were decreased. No changes were observed in behavior or body weight in all experimental groups. In conclusion, L. gasseri 5A and L. plantarum 7A isolated from human breast milk demonstrates significant hypocholesterolaemic actions in vitro and in vivo and might be considered a promising candidates for preventing hypercholesterolemia in man and animals.

PET-degrading microorganisms isolated from residues of the SAV biosphere reserve and identification of carboxylic ester hydrolase activity during their growth in the presence of polymer

The “Sistema Arrecifal Veracruzano” (SAV) is a vital marine ecosystem; its resources are continually perturbed due to contamination by anthropogenic activities, with multiple contaminants such as plastics. In aquatic ecosystems, plastics are almost immediately coated by inorganic and organic matter, which is then colonized by microbes to form a biofilm on plastic surfaces. This work aimed to isolate and identify plastic-degrading microorganisms isolated mainly from plastic residues of the SAV biosphere reserve. Eight bacteria and three fungi were isolated from a biofilm in plastic residues from islands of SAV. All the bacteria and one fungus showed evidence of degrading PET, over 10% for two bacteria and 17% for the fungus. All fungi belong to the genus Aspergillus, and bacteria belong to the genera Aneurinibacillus, Bordetella, Bacillus, and Lysinibacillus. Aneurinibacillus migulanus and Aspergillus flavus showed the highest values for PET degradation. Carboxylic ester hydrolase (CEH) activity was detected in all crude extracts from fungi and bacteria growing with PET triturates as a carbon source; the maximum CEH in bacteria was 255 U mg-1 and 780 U mg-1 for fungi.

Cholesterol lowering in diet-induced hypercholesterolemic mice using Lactobacillus bile salt hydrolases with different substrate specificities.

The cholesterol-lowering effect of lactic acid bacteria with high activity of bile salt hydrolase (BSH) is unclear. We believe that distinguishing BSH substrate specificity is necessary to study the effect of various BSH enzymes. We engineered a BSH mutant enzyme recombinant strain named F67A, which exclusively hydrolyzes taurocholic acid (TCA) using site-directed mutagenesis, and a previously lab-constructed BSH recombinant strain, YB81 that exclusively hydrolyzes glycocholic acid (GCA). We also constructed the recombinant strain named NB5462, which carries the empty pSIP411 plasmid and was used as a blank control strain. The intestinal flora in pseudo-germ-free (PGF) mice in which intestinal flora were eliminated via antibiotics, and F67A successfully reduced serum cholesterol levels in high-cholesterol diet-fed mice, whereas YB81 did not yield the same results. However, YB81 regained its cholesterol-lowering capacity in specific pathogen-free (SPF) mice with intact intestinal flora. The cholesterol-lowering mechanism of F67A involved modifying the bile acid pool through BSH enzyme activity. This adjustment regulated the expression of intestinal farnesoid X receptor and subsequently elevated hepatic cholesterol 7α-hydroxylase (CYP7A1), effectively reducing cholesterol levels. Conversely, GCA, the substrate of YB81, was found in minimal quantities in mice, preventing it from inducing changes in bile acid pools. In the presence of intestinal flora, the YB81 BSH enzyme induced notable alterations in bile acids by regulating changes in the intestinal flora and BSH within the flora, ultimately resulting in cholesterol reduction. This is the first study investigating the substrate specificity of BSH, demonstrating that different substrate-specific BSH enzymes exhibit cholesterol-lowering properties. Additionally, we elaborate on the mechanism of BSH-mediated enterohepatic axis regulation.

Enzyme characterization of lactic acid bacteria isolated from duck excreta.

The production of lignocellulosic biomass waste in the agricultural sector of Indonesia is quite high annually. Utilization of lignocellulosic biomass waste through fermentation technology can be used as feed and biofuel. Fermentation technology requires the involvement of micro-organisms such as bacteria (lactic acid bacteria or LAB). LABs can be isolated from various sources, such as duck excreta. However, there have not been many reports of LAB from duck excreta. The present study aimed to characterize LAB enzymes isolated from duck excreta and obtain LAB enzymes with superior fermentation properties. A total of 11 LAB cultures obtained from duck excreta in Yogyakarta, Indonesia, were tested. Enzyme characterization of each LAB was performed using the API ZYM kit (BioMérieux, Marcy-I'Etoile, France). The bacterial cell suspension was dropped onto the API ZYM™ cupule using a pipette and incubated for 4 h at 37°C. After incubation, ZYM A and ZYM B were dripped onto the API ZYM cupule, and color changes were observed for approximately 10 s under a strong light source. Esterase activity was moderate for all LABs. The activity of α-chymotrypsin, β-glucuronidase, α-fucosidase, and α-mannosidase was not observed in a total of 10 LAB. The phosphohydrolase and amino peptidase enzyme activity of seven LABs was strong. Only six LAB samples showed protease activity. The glycosyl hydrolase (GH) activity was observed in a total of 8 LAB, while the activity of 2 LAB was strong (Lactococcus lactis subsp. lactis K5 and Lactobacillus brevis M4A). A total of 2 LABs have superior properties. L. lactis subsp. lactis K5 and L. brevis M4A have a high potential to be used in fermentation. They have the potential for further research, such as their effectiveness in fermentation, lignocellulose hydrolysis, feed additives, molecular characterization to detect specific enzymes, and their specific activities.

Effects of thinning on accumulation of soil microbial residue carbon of Picea asperata plantations in sub-alpine region of western Sichuan, China

Microbial residues are an important component of soil organic carbon (SOC). It is unclear how long-term thinning affects the accumulation characteristics of microbial residue carbon (C). We analyzed the differences in soil physicochemical properties, microbial communities, extracellular enzyme activities, and microbial residue C in topsoil (0-10 cm) and subsoil (20-30 cm) in Picea asperata plantation of non-thinned (control, 4950 trees·hm-2) and thinned for 14 years (1160 trees·hm-2) stands, aiming to reveal the regulatory mechanism of thinning on microbial residue C accumulation. The results showed that thinning significantly increased SOC content, total nitrogen content, available phosphorus content, the proportion of particulate organic C, soil water content, C-cycle hydrolase, and acid phosphatase activities, but significantly reduced the proportion of mineral-associated organic C. Thinning significantly affected the content of fungal and microbial residue C, and the contribution of microbial residue C to SOC, and these effects were independent of soil layer. The content of fungal and microbial residue C was 25.0% and 24.5% higher under thinning treatments. However, thinning significantly decreased the contribution of microbial residue C to SOC by 12.3%, indicating an increase in the proportion of plant-derived C in SOC. Stepwise regression analysis showed that total nitrogen and soil water content were key factors influencing fungal and micro-bial residue C accumulation. In summary, thinning promoted microbial residue C sequestration by altering soil pro-perties and changed the composition of SOC sources.

Selective fluorescence detection of acetylsalicylic acid, succinic acid and ascorbic acid based on a responsive lanthanide metal fluorescent coordination polymer.

A fluorescent sensor for highly selective and ultrasensitive detection of acetylsalicylic acid (ASA), succinic acid (SA), and ascorbic acid (AA) was reported. The water-soluble fluorescent ligand salicylic acid (Sal) was generated through catalyzing ASA by the hydrolase activity of zeolitic-imidazolate framework-8 (ZIF-8) or natural esterase (Est). The Sal can coordinate with 2-methylimidazole (2-MIm) and Ln(III) to form a fluorescent lanthanide coordination polymer (LCP), which has a fluorescence emission peak with the maximum wavelength at 412 nm (the excitation wavelength at 300 nm). Therefore, the detection of ASA can be achieved through the fluorescence intensity changes of LCPs in the system, which has comparable sensitivity and good selectivity (linear range of 0.031-1.00 mM and LODs of 11.72 and 3.22 μM) as compared to a direct reaction between Est/ZIF-8 and ASA for detecting ASA (linear range of 0.05-1.20 mM and limits of detection (LODs) of 4.43 and 4.58 μM). Furthermore, upon the addition of SA and AA, the fluorescence intensity of the reaction system can be enhanced and weakened through changing the energy resonance transfer pathways and affecting the enzymatic reaction process, respectively, realizing their sensitive and selective fluorescence detection. The established fluorescent sensors can work well in a wide linear range of SA concentrations from 0 to 2.50 mM (Est-based reaction system) and 0 to 1.50 mM (ZIF-8-based reaction system) with the LODs of 0.032 and 0.028 mM, respectively. The linear ranges of AA concentrations are from 0.0078 to 0.25 mM (Est-based reaction system) and 0.0078 to 0.13 mM (ZIF-8-based reaction system) with the LODs of 2.54 and 3.80 μM, respectively. The established sensors were successfully used in the detection of SA in rabbit plasma, with a recovery of 84.0%-98.7%. Additionally, the contents of ASA in Aspirin Enteric-Coated tablets and AA in vitamin C tablets were also determined by the developed methods.

Transcriptome differential expression analysis of defoliation in different lemon varieties under drought treatment.

'Allen Eureka' is a bud variety of Eureka lemon with excellent fruiting traits, but severe winter defoliation affects the following year's yield, and the response mechanism of lemon defoliation is currently unknown. Two lemon cultivars ('Allen Eureka' and 'Yunning No. 1') with different defoliation traits were used as materials to investigate the molecular regulatory mechanisms of different leaf abscission periods in lemons. The petiole abscission zone was collected at three different defoliation stages, namely, the predefoliation stage (k15), the middefoliation stage (k30), and the postdefoliation stage (k45). Transcriptome sequencing was performed to analyze the gene expression differences between these two cultivars. A total of 1141, 2695, and 1433 differentially expressed genes (DEGs) were obtained in k15, k30, and k45, respectively, and the number of DEGs in k30 was the largest. GO analysis revealed that the DEGs between the two cultivars were mainly enriched in processes related to hydrolase activity, chitinase activity, oxidoreductase activity, and transcription regulator activity in the defoliation stages. KEGG analysis showed that the DEGs were concentrated in k30, which involved plant hormone signal transduction, phenylpropanoid biosynthesis, and biosynthesis of amino acids. The expression trends of some DEGs suggested their roles in regulating defoliation in Lemon. Seven genes were obtained by WGCNA, including sorbitol dehydrogenase (CL9G068822012_alt, CL9G068820012_alt, CL9G068818012_alt), abscisic acid 8'-hydroxylase (CL8G064053012_alt, CL8G064054012_alt), and asparagine synthetase (CL8G065162012_alt, CL8G065151012_alt), suggesting that these genes may be involved in the regulation of lemon leaf abscission.

Increasing the productivity of hydrolase producers by induced mutagenesis and their comparative characteristics

In order to study the increase in productivity of natural hydrolase producers B. subtilis (producer of amylolytic and proteolytic enzymes) and T. harzianum (producer of xylolytic and cellulolytic enzymes), the efficiency of various approaches to non-directed induced mutagenesis was studied and their comparative characteristics were carried out. Using chemical and physical mutagenesis, new highly active producers of extracellular hydrolytic enzymes B. subtilis strain MB085-1457, B. subtilis strain MB085-2701, B. subtilis strain MB085-3689, T. harzianum strain MF091-962, T. harzianum strain MF091-2093 and T. harzianum strain MF091-3416 were obtained. Comparison of various methods of induced mutagenesis, namely methods of mutagenesis using nitrosoguanidine and nitrosomethylurea, UV and gamma irradiation showed that the greatest efficiency was achieved after mutagenesis using gamma rays. Various methods of induced mutagenesis can be used not only to create effective schemes for increasing the level of accumulation of target enzymes of producers, but also to obtain new drugs based on highly productive strains of microorganisms with hydrolase activity for the purpose of using them in resource-saving technologies for processing agricultural waste (into useful resources) and reducing the negative impact on the environment.

Altered Sphingolipid Hydrolase Activities and Alpha-Synuclein Level in Late-Onset Schizophrenia.

Recent data described that patients with lysosomal storage disorders (LSDs) may have clinical schizophrenia (SCZ) features. Disruption of lipid metabolism in SCZ pathogenesis was found. Clinical features of schizophrenia (SCZ) have been demonstrated in patients with several lysosomal storage disorders (LSDs). Taking into account the critical role of lysosomal function for neuronal cells' lysosomal dysfunction could be proposed in SCZ pathogenesis. The current study analyzed lysosomal enzyme activities and the alpha-synuclein level in the blood of patients with late-onset SCZ. In total, 52 SCZ patients with late-onset SCZ, 180 sporadic Parkinson's disease (sPD) patients, and 176 controls were recruited. The enzymatic activity of enzymes associated with mucopolysaccharidosis (alpha-L-Iduronidase (IDUA)), glycogenosis (acid alpha-glucosidase (GAA)) and sphingolipidosis (galactosylceramidase (GALC), glucocerebrosidase (GCase), alpha-galactosidase (GLA), acid sphingomyelinase (ASMase)) and concentration of lysosphingolipids (hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), and lysosphingomyelin (LysoSM)) were measured using LC-MS/MS. The alpha-synuclein level was estimated in magnetically separated CD45+ blood cells using the enzyme-linked immunosorbent assay (ELISA). Additionally, NGS analysis of 11 LSDs genes was conducted in 21 early-onset SCZ patients and 23 controls using the gene panel PGRNseq-NDD. Decreased ASMase, increased GLA activities, and increased HexSpn, LysoGb3, and LysoSM concentrations along with an accumulation of the alpha-synuclein level were observed in late-onset SCZ patients in comparison to the controls (p < 0.05). Four rare deleterious variants among LSDs genes causing mucopolysaccharidosis type I (IDUA (rs532731688, rs74385837) and type III (HGSNAT (rs766835582)) and sphingolipidosis (metachromatic leukodystrophy (ARSA (rs201251634)) were identified in five patients from the group of early-onset SCZ patients but not in the controls. Our findings supported the role of sphingolipid metabolism in SCZ pathogenesis. Aberrant enzyme activities and compounds of sphingolipids associated with ceramide metabolism may lead to accumulation of alpha-synuclein and may be critical in SCZ pathogenesis.

ABHD6 drives endocytosis of AMPA receptors to regulate synaptic plasticity and learning flexibility

Trafficking of α‐Amino‐3–hydroxy‐5–methylisoxazole‐4–propionic acid (AMPA) receptors (AMPARs), mediated by AMPAR interacting proteins, enabled neurons to maintain tuning capabilities at rest or active state. α/β-Hydrolase domain-containing 6 (ABHD6), an endocannabinoid hydrolase, was an AMPAR auxiliary subunit found to negatively regulate the surface delivery of AMPARs. While ABHD6 was found to prevent AMPAR tetramerization in endoplasmic reticulum, ABHD6 was also reported to localize at postsynaptic site. Yet, the role of ABHD6 interacting with AMPAR at postsynaptic site, and the physiological significance of ABHD6 regulating AMPAR trafficking remains elusive. Here, we generated the ABHD6 knockout (ABHD6KO) mice and found that deletion of ABHD6 selectively enhanced AMPAR-mediated basal synaptic responses and the surface expression of postsynaptic AMPARs. Furthermore, we found that loss of ABHD6 impaired hippocampal long-term depression (LTD) and synaptic downscaling in hippocampal synapses. AMPAR internalization assays revealed that ABHD6 was essential for neuronal activity-dependent endocytosis of surface AMPARs, which is independent of ABHD6's hydrolase activity. The defects of AMPAR endocytosis and LTD are expressed as deficits in learning flexibility in ABHD6KO mice. Collectively, we demonstrated that ABHD6 is an endocytic accessory protein promoting AMPAR endocytosis, thereby contributes to the formation of LTD, synaptic downscaling and reversal learning.