Hydrolase Enzyme Research Articles

A Optimization of culture conditions to express AA9 Polysaccharide monooxygenases AN3860 in Escherichia coli

Lignocellulose biomass is a copious source for second generation biomaterial production. The participant of Polysaccharide monooxygenases enzyme (PMO) in the reactions which convert lignocellulose biomass into monosaccharides enhances the activity and improve the efficiency of hydrolysis of hydrolase enzymes on lignocellulose substrate. Enzyme AN3860, obtained from Aspergillus nidulans strain belonging to AA9 PMO, is expected to catalyze flexibly at C1 and C4 carbon positions of β-glycosidic linkage. As an enzyme with high potential of improving cellulose crystals hydrolysis capacity, AN3860 was successfully cloned into the expression system of E. coli BL21 (DE3) strain. In this study, the culture process of recombinant strain with AN3680 gene is optimized to increase the target proteins yield, thus ensure the outcome of purification process, and save production cost. The results demonstrate that the E. coli recombinant strains grow sufficiently in TB (Terrific Broth) culture media and the highest yield of AN3680 protein achieved when the concentration of Isopropyl β-D-1-thiogalactopyranoside (IPTG) is 0.05 mM and the temperature of the reaction is 30 0C at 150 rpm. After 6 hours of induction, the biomass reaches 500 mg/L and the yield of AN3860 account for (7-10) % total protein generated. The recombinant AN3860 protein is later harvested on larger scale and purified by Ni-NTA column chromatography method for analysis of bioactivities on lignocellulose substrates in the future.
 ® 2022 Journal of Science and Technology - NTTU

Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity

Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5–9.0 and temperatures from 30–70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.

Wastewater bacteria remediating the pharmaceutical metformin: Genomes, plasmids and products.

Metformin is used globally to treat type II diabetes, has demonstrated anti-ageing and COVID mitigation effects and is a major anthropogenic pollutant to be bioremediated by wastewater treatment plants (WWTPs). Metformin is not adsorbed well by activated carbon and toxic N-chloro derivatives can form in chlorinated water. Most earlier studies on metformin biodegradation have used wastewater consortia and details of the genomes, relevant genes, metabolic products, and potential for horizontal gene transfer are lacking. Here, two metformin-biodegrading bacteria from a WWTP were isolated and their biodegradation characterized. Aminobacter sp. MET metabolized metformin stoichiometrically to guanylurea, an intermediate known to accumulate in some environments including WWTPs. Pseudomonas mendocina MET completely metabolized metformin and utilized all the nitrogen atoms for growth. Pseudomonas mendocina MET also metabolized metformin breakdown products sometimes observed in WWTPs: 1-N-methylbiguanide, biguanide, guanylurea, and guanidine. The genome of each bacterium was obtained. Genes involved in the transport of guanylurea in Aminobacter sp. MET were expressed heterologously and shown to serve as an antiporter to expel the toxic guanidinium compound. A novel guanylurea hydrolase enzyme was identified in Pseudomonas mendocina MET, purified, and characterized. The Aminobacter and Pseudomonas each contained one plasmid of 160kb and 90kb, respectively. In total, these studies are significant for the bioremediation of a major pollutant in WWTPs today.

Soil Organic Carbon Distribution, Enzyme Activities, and the Temperature Sensitivity of a Tropical Rainforest in Wuzhishan, Hainan Island

Soil carbon (C) distribution, which is deeply correlated with soil extracellular enzyme activity and its temperature sensitivity (Q10), are important for predicting the soil organic carbon (SOC) pools under climate warming. However, the high spatial heterogeneity in tropical forest soils makes the predicted results vary significantly. Thus, a total of 87 soil samples of three forest types, eight slope orientations, and four vegetation types were selected from Wuzhishan in Hainan Island, China. SOC distribution, the activities of six soil enzymes, and Q10 at 10, 20, 30, and 40 °C were investigated to supplement the tropical data. The results showed that: (1) SOC ranged from 24.82 to 87.72 g/kg. SOC in the primary forest was significantly higher than that of the secondary forest, and SOC of the cloud forests and hilltop scrub at higher elevations was significantly higher than that of the lowland rainforests and montane rainforests at lower elevations. However, the amount of recalcitrant carbon in the primary forest was the lowest. (2) Under lab temperature conditions, the acid phosphatase and β-1,4-glucosidase in the plantation forest were significantly higher than that of the secondary forest, and the polyphenol oxidase and catalase in the plantation forest were significantly higher than that of the primary forest. Enzyme stoichiometry analysis indicated that microbials were limited by nitrogen in the study area. The six soil enzyme activities were strongly correlated with SOC and total nitrogen. (3) The Q10 of soil enzymes ranged from 0.61 to 1.92 under three temperature gradients. Most hydrolases enzymes (Q10 > 1) showed a positive response with temperatures from 10 to 30 °C, and showed a negative response (Q10 < 1) with temperatures from 30 to 40 °C. We concluded that the negative response of enzyme Q10 with global warming would slow down the SOC decomposition. Primary tropical forests could still sequester SOC; however, their ability to do so may be vulnerable to climate change, as the amount of soil C quality index was low.

Identification of fungal lignocellulose-degrading biocatalysts secreted by Phanerochaete chrysosporium via activity-based protein profiling

Activity-based protein profiling (ABPP) has emerged as a versatile biochemical method for studying enzyme activity under various physiological conditions, with applications so far mainly in biomedicine. Here, we show the potential of ABPP in the discovery of biocatalysts from the thermophilic and lignocellulose-degrading white rot fungus Phanerochaete chrysosporium. By employing a comparative ABPP-based functional screen, including a direct profiling of wood substrate-bound enzymes, we identify those lignocellulose-degrading carbohydrate esterase (CE1 and CE15) and glycoside hydrolase (GH3, GH5, GH16, GH17, GH18, GH25, GH30, GH74 and GH79) enzymes specifically active in presence of the substrate. As expression of fungal enzymes remains challenging, our ABPP-mediated approach represents a preselection procedure for focusing experimental efforts on the most promising biocatalysts. Furthermore, this approach may also allow the functional annotation of domains-of-unknown functions (DUFs). The ABPP-based biocatalyst screening described here may thus allow the identification of active enzymes in a process of interest and the elucidation of novel biocatalysts that share no sequence similarity to known counterparts.

Temporal and spatial evolution of enzymatic degradation of amorphous PET plastics

Biocatalytic degradation is an emerging strategy aiming for energy-efficient recycling of poly(ethylene terephthalate) (PET), the most commonly used thermoplastic polyester. Besides material composition and physico-chemical parameters, the degradation kinetics is co-determined by the evolving nanotopography. In this study, the 3-dimensional development of the surface characteristics of an amorphous PET film, reacted with a highly effective hydrolase enzyme for up to 24 h, was explored by vertical scanning interferometry and confocal microscopy. The spatio-temporal analysis unveiled that the degradation process is not uniform with respect to reaction time and spatial reactivity distribution. An early phase of an unspecific roughness evolution is followed by an advanced phase characterized by a circular degradation pattern, consisting of shallow pits that are steadily renewed over time. The data suggest a hindrance of degradation during the initial roughening process, demonstrating the potential role of targeted surface modification in the large-scale treatment of plastic waste.

Mediterranean grassland succession as an indicator of changes in ecosystem biodiversity and functionality

The abandonment of agricultural lands triggers a secondary succession of plant species which implies important changes in soil quality. Annual Mediterranean grasslands are known to be persistent on abandoned agriculture lands in the western Mediterranean. We used plant taxonomic and functional approaches to determine the role of Mediterranean grasslands as an indicator of changes in ecosystem biodiversity and functionality. We tested the hypothesis that Mediterranean grasslands are a suitable model for monitoring biodiversity and soil fertility in a secondary succession. Soil and vegetation features on 21 permanent plots were monitored in 2016 and 2020. Numerical classifications based on floristic composition showed two different plant communities independently of the sampling year: early-stage grasslands in the first post-abandonment decade and late-stage grasslands after the first post-abandonment decade. Generalized linear model and redundancy analysis also revealed differences in growth forms, functional traits and soil functionality between communities. Late-stage grasslands was characterized by enriched bryophyte coverage and an impoverishment in hemicryptophytes and plant latex segregators growing on soils with a higher hydrolase enzyme activity and TOC content compared to early-stage grassland. Our results suggest that annual Mediterranean grasslands growing on siliceous soils denoting a mature-stage succession, and floristically characterized by the symbiont plant with Ascomycota, Tuberaria guttata, and a high bryophyte cover, are worthy of recognition for conservation.

Arginine ADP-Ribosylation: Chemical Synthesis of Post-Translationally Modified Ubiquitin Proteins.

We describe the development and optimization of a methodology to prepare peptides and proteins modified on the arginine residue with an adenosine-di-phosphate-ribosyl (ADPr) group. Our method comprises reacting an ornithine containing polypeptide on-resin with an α-linked anomeric isothiourea N-riboside, ensuing installment of a phosphomonoester at the 5'-hydroxyl of the ribosyl moiety followed by the conversion into the adenosine diphosphate. We use this method to obtain four regioisomers of ADP-ribosylated ubiquitin (UbADPr), each modified with an ADP-ribosyl residue on a different arginine position within the ubiquitin (Ub) protein (Arg42, Arg54, Arg72, and Arg74) as the first reported examples of fully synthetic arginine-linked ADPr-modified proteins. We show the chemically prepared Arg-linked UbADPr to be accepted and processed by Legionella enzymes and compare the entire suite of four Arg-linked UbADPr regioisomers in a variety of biochemical experiments, allowing us to profile the activity and selectivity of Legionella pneumophila ligase and hydrolase enzymes.

Cell wall composition and enzyme-related activities in eggplant as affected by hot water, 1-MCP and calcium chloride treatments.

The effects of postharvest applications of hot water (HWT) (45, 50, and 55 °C), 1-MCP (1, 5, and 10 μL/L), and CaCl2 (1, 2, and 3%) on polygalacturonase (PG), pectin methylesterase (PME), α-galactosidase (α-Gal), β-galactosidase (β-Gal) and β-1,4-glucanase (β-1,4-Glu) activities, and the fruit firmness and cell wall composition of eggplant fruit were investigated. The results showed that the decrease in the eggplants firmness was delayed by HWT, 1-MCP, and CaCl2 treatments during storage compared with the control. However, HWTs were less effective than the 1-MCP and CaCl2 treatments. The results show that 1-MCP and CaCl2 treatments inhibited the depolymerization of water (WSP), CDTA (CSP), and sodium carbonate (SSP) soluble polyuronides. The results suggest that 1-MCP (5 and 10 μL/L) and CaCl2 (1, 2, and 3%) could prevent eggplant softening by inhibiting hydrolase enzymes and reducing the disintegration of the polysaccharides. In addition, 1-MCP and CaCl2 were more effective than hot water treatment in extending postharvest storage life. There is a significantly high correlation between firmness, polyuronide content and cell wall enzyme activity.

Persistence, leaching and associated toxicity risks of insecticide pyriproxyfen in soil ecosystem.

Pyriproxyfen is a pyridine-based insecticide used for pest control in fruits and vegetables. It is a potent endocrine disruptor and hormone imitator. Considering its potential hazards to non-target organisms and the associated environment, a lab study was conducted for assessing persistence, mobility in sandy loam soil and associated risk to various non-target organisms and soil enzymes. Pyriproxyfen formulation was applied at 0.05 and 0.10 µg g-1 soil which was equivalent to recommended and double dose of 100 and 200 g a.i. ha-1, respectively. Three methods namely QuEChERS, liquid-solid extraction (LSE) and matrix solid phase dispersion (MSPD) were compared for achieving efficient sample preparation. MSPD was applied for final analysis as it gave better recoveries (94.2 to 104.3%) over other methods with limits of detection and quantification (LOD and LOQ) as 0.0001 and 0.0005 µg g-1, respectively. Dissipation followed first order kinetics with half-lives of 7.6 and 8.2 days in both doses but residues retained over 45 days in soil. Leaching studies conducted at 50 and 100 µg of pyriproxyfen showed extremely poor leaching potential. Retention of over 90% residues in top 5 cm soil surface indicated minimal threat of ground and surface water contamination. Toxicological study demonstrated very different behaviour toward different enzymatic activities. Pyriproxyfen was relatively toxic for alkaline phosphatase and fluorescein diacetate hydrolase enzymes. β-glucosidase activity was triggered whereas arylsulfatase activity remained unaffected. Unacceptable risk to soil invertebrates at double dose application clearly indicated that its longer persistence in soil could be toxic to other non-target organisms and needs further investigations.

The Supplementation of FloraMax-B11 Did Not Affect the Bile Acid Neosynthesis and the Enterohepatic Circulation in Broiler Chickens.

Most probiotics possess bile salt hydrolase enzymes and may increase bile acid excretion and negatively affect fat digestion and absorption. Therefore, the study objective was to determine the time course effects of a commercial probiotic (P) FloraMax-B11 (FM) supplementation on bile acid neosynthesis and enterohepatic circulation in broiler chickens. Fertile Ross 708 eggs were incubated under standard commercial conditions. At hatch, chicks (n = 550) were randomly assigned to 5 treatment groups (n = 5 replicates per treatment group) with 22 birds per pen. The 5 treatment groups consisted of: control group (C, normal water from hatch to 35 days of age without supplements); P3, water supplemented with FM for the first 3 days post-hatch followed by normal water until day 35; P10, water supplemented with FM for the first 10 days post-hatch followed by normal water until day 35; P35, water supplemented with FM from hatch to day 35; and AGP, water supplemented with antibiotic growth promoter (AGP) from hatch until day 35. Ileum, liver, and plasma were collected at hatch, days 3, 10, 21, and 35 post-hatch. The relative mRNA expression of genes involved in bile acid synthesis (CYP7A1, CYP8B1, FXR, FGFR4, and FGF19) and transport (ASBT, I-BABP, OSTα, OSTβ, and BSEP) as well as ileal deoxycholic acid and plasma cholic acid were determined. There was no FM and AGP interaction for any of the response criteria. No FM or AGP effects were observed (p > 0.05) for any genes, except FGF19, which expression was increased (p < 0.0001) in AGP compared to P35. No FM or AGP effects were observed (p > 0.05) for levels of deoxycholic and cholic acids. However, all the genes, deoxycholic acid, and plasma cholic acid were affected by age (p < 0.0001). In general, the data indicate that FM did not negatively impact bile acid metabolism and enterohepatic circulation, which appeared to be age dependent. However, more research should be conducted to confirm these results and investigate the effects of FM on bile acid metabolism, fat digestion, and intestinal microbiota in broiler chickens.

Synthesis, Docking Study and Biological Evaluation of Amide-Based Soluble Epoxide Hydrolase Inhibitors with Novel Secondary Pharmacophore of Pyrimidin-2-ol.

Soluble epoxide hydrolase enzyme (sEH) is one of the most promising and emerging targets to develop drugs for multiple disease indications, including hypertension, diabetes, stroke, dyslipidemia, pain, etc. Most inhibitor scaffolds have a urea or amide moiety to mimic the active-site transition state. In this regard, we developed a series of amide sEH inhibitors with a pyrimidin-2-ol ring as a new secondary pharmacophore, which was subjected to in vitro evaluation. Compound 4w (4-chloro-N-{4-[6-(4-chlorophenyl)-2-hydroxypyrimidin-4-yl]phenyl}benzamide), which contains 4-chloro substituent in both terminal phenyl rings, exhibited the most inhibitory activity against sEH with an IC50 value of 1.2 nM. Molecular docking analysis of the synthesized compounds revealed that the greater number of hydrogen bonding interactions of the amide group as the primary pharmacophore with Asp-353, Tyr-383, and Tyr-466 as the key catalytic residue triad of the enzyme played a critical role and led to a more favorable binding affinity. Pharmacokinetic properties of the synthesized compounds were calculated in silico, and all ADMET indices fell within acceptable ranges. Altogether, the results of this work could provide useful information on 4,6-diphenylpyrimidin-2-olas sEH inhibitors which can be utilized in further development in this area.

Species Composition and Hydrolase Enzyme (EC.3) Activity of Fungi Isolated from Thasala Mangroves, Nakhon Si Thammarat Province, Southern Thailand

In the present study, we aimed to investigate diversity of manglicolous and endobiotic fungi associated with mangrove plants from Thasala mangroves, Nakhon Si Thammarat province, southern Thailand. Extracellular hydrolase enzymes (EC.3) including amylase, cellulase, lipase, protease, and pectinase from isolated fungi were evaluated. A total of 31 obligate marine fungal species was recorded from various Thasala mangroves sites. These included 24 ascomycetes (77.4 %), 1 mucoromycete (3.2 %), 3 basidiomycetes (9.7 %), 4 asexual morphs (12.9 %), and tentatively identified fungi (9.7 %). The average percentage occurrence of the fungi recorded from each site ranged from 0.4 to 16%. At all sites, the common filamentous marine fungi were Cumulospora sp., Halorosellinia oceanica, Kallichroma tethys, Leptosphaeria sp., Rimora mangrovei, Lulworthia sp., Phoma sp., Halenospora varia, Halocyphina villosa, Nia vibrissa, and Sclerococcum haliotrephum. Also, the most frequent marine fungi include Eutypella naqsii, Sammeyersia grandispora, Marinosphaera mangrovei, Nemania maritima, and Verruculina enalia. Seven mangrove plants were selected for endophytic fungal isolation including Acanthus ebracteatus, Acrostichum aureum, Avicennia alba, Bruguiera cylindrica, Rhizophora apiculata, R. mucronata, and Sonneratia alba. The isolation rate (IR) and colonization rate (CR) of fungal endobiota varied for all plant studied. The isolation rate ranged from 24 - 53.3 %, while colonization rate varied from 29.3 - 61.3 %. The fungal isolates were screened for the production of hydrolase enzymes (EC.3), of which 37.2 % exhibited enzyme activities. The 16 out of 28 obligate marine fungi (57.1 %) and 13 out of 50 fungal endophytes (26 %) had enzyme production efficiency toward specific substrates or an enzymatic index (EI) higher than 1.4. None of the fungal isolates depicted lipase activity. Additionally, the environmental stressors (temperature, pH, salinity) affecting enzyme production were evaluated in order to discover potential candidates for industrial application.
 HIGHLIGHTS
 
 Diversity of obligate marine fungal species was estimated from Thasala mangroves
 The obligate marine fungi and fungal endophytes had hydrolase enzyme (EC.3) production efficiency toward specific substrates or an enzymatic index (EI) higher than 1.4
 The temperature, pH, and salinity affecting enzyme production were evaluated in order to discover potential candidates for industrial application
 
 GRAPHICAL ABSTRACT

S3069 Lifesaving but Tiresome Treatment for Tyrosinemia Type 1

Introduction: Tyrosinemia type I (HT1) is a rare autosomal recessive condition caused by a genetic mutation disrupting proper functioning of the fumarylacetoacetate hydrolase (FAH) enzyme, the terminal enzyme in the tyrosine catabolism pathway. The below case exemplifies the difficulty for adherence as children transition to adulthood of the standard therapy that prolongs life and decreases need for orthotopic liver transplantation (OLT). Case Description/Methods: A 23-year-old male with known tyrosinemia type I presents with progressively worsening back, lower extremity, and abdominal pain over the last six months. He had been lost to follow-up from Genetics clinic for the last ten years. He adhered to nitisinone therapy and diet modifications until around five years ago. The patient was admitted to the hospital for neurological crises due to non-adherence to HT1 therapy. On admission, vital signs were stable and his physical exam significant for mild tenderness of abdomen at RUQ and tenderness in the lumbar and thoracic region of back. Pertinent lab findings were albumin of 3.2, alkaline phosphatase of 356. AST, ALT, Bili, CBC, BMP WNL. Amino acid panel found tyrosine level of 62. AFP level of 188. MRI Abdomen with and without contrast found cirrhotic liver morphology, developing portal hypertension (borderline enlarged spleen, collateral vasculature, but no ascites), and a lesion of hepatic segment 8 measuring 2.3cm x 1.4cm in diameter. Patient was planned to be restarted on nitisinone with pending further evaluation of liver lesion. (Figure) Discussion: The above case demonstrates a patient with the rare genetic disorder Tyrosinemia type 1 with non-adherence to nitisinone and diet modification therapies. The presentation of neurological crises and abdominal pain with development of hepatomas/hepatocellular carcinoma (HCC) is typical for untreated or non-adherent patients with HT1. Nitisinone has been shown to be highly effective for this condition with 4-year survival rates of 94% compared to 29% of diet modification alone. Prior to nitisinone therapy, most patients required OLT. Despite the clear benefits, recent evidence is finding increasing non-adherence as patients grow older into adolescence and adulthood. This is consistent with adherence problems with early diagnosis chronic diseases with long-term therapies. More research is needed to elucidate major barriers for non-compliance and to refine guidelines regarding frequency of follow-up and monitoring of serum AFP to screen for HCC and encourage compliance.Figure 1.: MRI Abdomen with contrast with findings of a hepatic lesion on segment 8.

Carbohydrate active enzyme system in rumen fungi: a review

Hydrolysis and dehydration reactions of carbohydrates, which are used as energy raw materials by all living things in nature, are controlled by Carbohydrate Active Enzyme (CAZy) systems. These enzymes are also used in different industrial areas today. There are different types of microorganisms that have the CAZy system and are used in the industrial sector. Apart from current organisms, there are also rumen fungi within the group of candidate microorganisms with the CAZy system. It has been reported that xylanase (EC3.2.1.8 and EC3.2.1.37) enzyme, a member of the glycoside hydrolase enzyme family obtained from Trichoderma sp. and used especially in areas such as bread, paper, and feed industry, is more synthesized in rumen fungi such as Orpinomyces sp. and Neocallimastix sp. Therefore, this study reviews Neocallimastixsp., Orpinomyces sp., Caecomyces sp., Piromyces sp., and Anaeromyces sp., registered in the CAZy and Mycocosm database for rumen fungi to have both CAZy enzyme activity and to be an alternative microorganism in the industry. Furthermore the CAZy enzyme activities of the strains are investigated. The review shows thatNeocallimax sp. and Orpinomyces sp. areconsidered as candidate microorganisms.

Regulation of gut microbiota-bile acids axis by probiotics in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by chronic and relapsing inflammation of gastrointestinal tract, with steadily increased incidence and prevalence worldwide. Although the precise pathogenesis remains unclear, gut microbiota, bile acids (BAs), and aberrant immune response play essential roles in the development of IBD. Lately, gut dysbiosis including certain decreased beneficial bacteria and increased pathogens and aberrant BAs metabolism have been reported in IBD. The bacteria inhabited in human gut have critical functions in BA biotransformation. Patients with active IBD have elevated primary and conjugated BAs and decreased secondary BAs, accompanied by the impaired transformation activities (mainly deconjugation and 7α-dehydroxylation) of gut microbiota. Probiotics have exhibited certain positive effects by different mechanisms in the therapy of IBD. This review discussed the effectiveness of probiotics in certain clinical and animal model studies that might involve in gut microbiota-BAs axis. More importantly, the possible mechanisms of probiotics on regulating gut microbiota-BAs axis in IBD were elucidated, which we focused on the elevated gut bacteria containing bile salt hydrolase or BA-inducible enzymes at genus/species level that might participate in the BA biotransformation. Furthermore, beneficial effects exerted by activation of BA-activated receptors on intestinal immunity were also summarized, which might partially explain the protect effects and mechanisms of probiotics on IBD. Therefore, this review will provide new insights into a better understanding of probiotics in the therapy targeting gut microbiota-BAs axis of IBD.

The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines.

Sequence variants or mutations in the GBA gene are numerically the most important risk factor for Parkinson disease (PD). The GBA gene encodes for the lysosomal hydrolase enzyme, glucocerebrosidase (GCase). GBA mutations often reduce GCase activity and lead to the impairment of the autophagy-lysosomal pathway, which is important in the turnover of alpha-synuclein, accumulation of which is a key pathological hallmark of PD. Although the E326K variant is one of the most common GBA variants associated with PD, there is limited understanding of its biochemical effects. We have characterized homozygous and heterozygous E326K variants in human fibroblasts. We found that E326K variants did not cause a significant loss of GCase protein or activity, endoplasmic reticulum (ER) retention or ER stress, in contrast to the L444P GBA mutation. This was confirmed in human dopaminergic SH-SY5Y neuroblastoma cell lines overexpressing GCase with either E326K or L444P protein. Despite no loss of the GCase activity, a significant increase in insoluble alpha-synuclein aggregates in E326K and L444P mutants was observed. Notably, SH-SY5Y overexpressing E326K demonstrated a significant increase in the lipid droplet number under basal conditions, which was exacerbated following treatment with the fatty acid oleic acid. Similarly, a significant increase in lipid droplet formation following lipid loading was observed in heterozygous and homozygous E326K fibroblasts. In conclusion, the work presented here demonstrates that the E326K mutation behaves differently to the common loss of function GBA mutations; however, lipid dyshomeostasis and alpha-synuclein pathology are still evident.

Screening Lipolytic from Soil Bacterial Contaminated Oil

Lipase (triasilglycerol acylhydrolase, E.C, 3.1.1.3) is an enzyme can hydrolyze long chains of triglycerides[1]. Lipase is a water-soluble enzyme and naturally lipase catalyze the hydrolysis of ester bonds in water insoluble lipid substrate and long-chain acylglycerol synthesis, diacylglycerol, monoacylglycerol, and glycerol[2]. Hydrolase enzyme lipase can be used potentialy produced fatty acid which precursors in the chemical industry[3]. The research aims to study the lipolytic activity of bacteria isolated from soil contaminated with oil. Screening of lipolytic bacteria was performed in Rhodamin-B agar plate containing olive oil. The lipolytic activity was determined by spectrofotometry method toward p-nitrofenil palmitat as a substrate. The result this research showed that there are 15 of 150 isolates bacteria from soil contaminated with oil was confirmed by observing the zone of hydrolysis formed around the growth of colony on Rhodamin B agar plate containing olive oil. One of the lipolytic bacteria identified as higher activity was produced lipase with activity of 7,41 U/ml

Structural and functional characterisation of a stable, broad-specificity multimeric sialidase from the oral pathogen Tannerella forsythia.

Sialidases are glycosyl hydrolase enzymes targeting the glycosidic bond between terminal sialic acids and underlying sugars. The NanH sialidase of Tannerella forsythia, one of the bacteria associated with severe periodontal disease plays a role in virulence. Here, we show that this broad-specificity enzyme (but higher affinity for α2,3 over α2,6 linked sialic acids) digests complex glycans but not those containing Neu5,9Ac. Furthermore, we show it to be a highly stable dimeric enzyme and present a thorough structural analysis of the native enzyme in its apo-form and in complex with a sialic acid analogue/ inhibitor (Oseltamivir). We also use non-catalytic (D237A) variant to characterise molecular interactions while in complex with the natural substrates 3- and 6-siallylactose. This dataset also reveals the NanH carbohydrate-binding module (CBM, CAZy CBM 93) has a novel fold made of antiparallel beta-strands. The catalytic domain structure contains novel features that include a non-prolyl cis-peptide and an uncommon arginine sidechain rotamer (R306) proximal to the active site. Via a mutagenesis programme, we identified key active site residues (D237, R212 and Y518) and probed the effects of mutation of residues in proximity to the glycosidic linkage within 2,3 and 2,6-linked substrates. These data revealed that mutagenesis of R306 and residues S235 and V236 adjacent to the acid–base catalyst D237 influence the linkage specificity preference of this bacterial sialidase, opening up possibilities for enzyme engineering for glycotechology applications and providing key structural information that for in silico design of specific inhibitors of this enzyme for the treatment of periodontitis.

Deep insights into the anaerobic co-digestion of waste activated sludge with concentrated leachate under different salinity stresses.

Treatment of high-salinity organic wastewater (e.g., concentrated leachate) is a major challenge. Anaerobic co-digestion can effectively treat high-salinity organic wastewater and recover energy. In this study, the concentrated landfill leachate and waste activated sludge (WAS) were anaerobic co-digested in the lab-scale continuous stirred tank reactors (CSTR) to understand their co-digestion performance under different salinity stresses. As revealed by the results, when the salinity was low (<10 g/L), the removal ratio of organic matter in the digester was kept at a high level (>91.3%), and the concentration of total volatile fatty acids (TVFAs) was low (<100 mg COD/L), indicating that the digester could operate efficiently and stably. However, when the salinity level was elevated from 10 g/L to 30 g/L, the removal ratio of organic matter in the digester decreased from ~91.3% to ~64.5%, the TVFAs continued to accumulate, the yields of biogas and methane also dropped sharply, and the performance of the digester decreased gradually. The results of microbial community and diversity analysis showed that there is limited adaptability of microbial community to high salinity in such process. Salinity could cause significant changes in the microbial community and diversity, thereby affecting the digestive performance. Metagenomic analysis showed that under high salinity conditions, the content of genes encoding hydrolase and methanogenic enzyme decreased, whereas the pathway of acetotrophic methanogenesis was weakened. Mechanism study showed that with the increase of salinity, the activity of microbial cells decreased, the structure of sludge flocs was damaged more significantly, and the extracellular polymeric substances (EPS) secreted by microbe increased continuously, which was used to resist the toxic effects of salinity stresses on microorganisms. The results of this study could provide certain theoretical guidance for anaerobic digestion under salinity stresses.