Littérature scientifique sur le sujet « A-glucosidasi acida »

ABSTRACT Aspergillus nidulans possessed an α-glucosidase with strong transglycosylation activity. The enzyme, designated α-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to α-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other α-glucosidases. We propose here that AgdB is a novel α-glucosidase with unusually strong transglycosylation activity.

The α-glucosidase geneaglfromThermus thermophilusHB8 was cloned into expression vector pBV220. The phylogenetic trees of α-glucosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. Evolution analysis indicated the α-glucosidase fromT. thermophileHB8 was distant from the other glycoside hydrolases 4 and 31 α-glucosidases. By weakening the mRNA secondary structure and replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of theaglwas increased 30-fold. The recombinant AGL was purified by the heat treatment, and had a molecular mass of 61 kDa. The optimal activity was at pH 7.8 and 95°C over a 10 min assay. The purified enzyme was stable over a pH range of 5.4-8.6, and had a 1-h half life at 85°C. Kinetic experiments at 90°C withp-nitrophenyl-α-D-glucoside as substrate gave aKm, andVmaxof 0.072 mM and 400 U/mg. Thus, this report provides an industrial means to produce the recombinant α-glucosidase inE. coli.

ABSTRACT Trehalose supports the growth of Thermus thermophilus strain HB27, but the absence of obvious genes for the hydrolysis of this disaccharide in the genome led us to search for enzymes for such a purpose. We expressed a putative α-glucosidase gene (TTC0107), characterized the recombinant enzyme, and found that the preferred substrate was α,α-1,1-trehalose, a new feature among α-glucosidases. The enzyme could also hydrolyze the disaccharides kojibiose and sucrose (α-1,2 linkage), nigerose and turanose (α-1,3), leucrose (α-1,5), isomaltose and palatinose (α-1,6), and maltose (α-1,4) to a lesser extent. Trehalose was not, however, a substrate for the highly homologous α-glucosidase from T. thermophilus strain GK24. The reciprocal replacement of a peptide containing eight amino acids in the α-glucosidases from strains HB27 (LGEHNLPP) and GK24 (EPTAYHTL) reduced the ability of the former to hydrolyze trehalose and provided trehalose-hydrolytic activity to the latter, showing that LGEHNLPP is necessary for trehalose recognition. Furthermore, disruption of the α-glucosidase gene significantly affected the growth of T. thermophilus HB27 in minimal medium supplemented with trehalose, isomaltose, sucrose, or palatinose, to a lesser extent with maltose, but not with cellobiose (not a substrate for the α-glucosidase), indicating that the α-glucosidase is important for the assimilation of those four disaccharides but that it is also implicated in maltose catabolism.

Background β-Glucosidases have attracted considerable attention due to their important roles in various biotechnological processes such as cellulose degradation to make energy and hydrolysis of isoflavone. Microbulbifer thermotolerans (M. thermotolerans) is isolated from deep-sea sediment and has not been researched much yet. As a potential candidate for a variety of biotechnological industries, β-glucosidases from the novel bacterial species should be researched extensively. Methods β-Glucosidase, MtBgl85, from M. thermotolerans DAU221 was purified by His-tag affinity chromatography and confirmed by SDS-PAGE and zymogram. Its biochemical and physiological properties, such as effects of temperature, pH, metal ions, and organic solvents, substrate specificity, and isoflavone hydrolysis, were investigated. Results M. thermotolerans DAU221 showed β-glucosidase activity in a marine broth plate containing 0.1% esculin and 0.25% ammonium iron (III) citrate. The β-glucosidase gene, mtbgl85, was isolated from the whole genome sequence of M. thermotolerans DAU221. The β-glucosidase gene was 2,319 bp and encoded 772 amino acids. The deduced amino acid sequence had a 43% identity with OaBGL84 from Olleya aquimaris and 35% and 32% identity with to CfBgl3A and CfBgl3C from Cellulomonas fimi among bacterial glycosyl hydrolase family 3, respectively. The optimal temperature of MtBgl85 was 50 °C and the optimum pH was 7.0. MtBgl85 activity was strongly reduced in the presence of Hg2+ and Cu2+ ions. As a result of measuring the activity at various concentrations of NaCl, it was confirmed that the activity was maintained up to the concentration of 1 M, but gradually decreased with increasing concentration. MtBgl85 showed higher enzyme stability at non-polar solvents (high Log Pow) than polar solvents (low Log Pow). The hydrolyzed products of isoflavone glycosides and arbutin were analyzed by HPLC.

Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived β-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid substitutions (S28T, Y37H, D44E, R91G, and L115N), showed catalytic efficiency (kcat/Km of 561.72 mM−1 s−1) toward ρ-nitrophenyl-β-d-glucopyranoside (ρNPG) that increased by 23-fold, half-life of inactivation by 10-fold, and further retained transglycosidation activity at 50 °C as compared with the wild-type Bgl1D protein. Site-directed mutagenesis also revealed that Asp44 residue was essential to β-glucosidase activity of Bgl1D. This study improved our understanding of the key amino acids of the novel β-glucosidases and presented a raw material with enhanced catalytic activity and thermostability for the synthesis of butyl glucosides.

In this study, 16 selected edible flowers were evaluated for their content of bioactive compounds (polyphenols, carotenoids, triterpenoids) and for their anti-diabetic (ability to inhibit α-amylase and α-glucosidases) and anti-aging (ability to inhibit acetylcholinesterase and butyrylcholinesterase) activities. Most of the flowers analyzed in this study have not been examined in this respect until now. Contents of the analyzed bioactive compounds differed significantly among the flowers. In particular, the highest contents of carotenoids and triterpenoids were determined in marigold, arnica, lavender, and daisy; in turn, the highest contents of phenolic acids, procyanidin polymers, and total polyphenols were assayed in hawthorn, primrose, and linden blossom. There was a positive correlation between the content of isoprenoids in edible flowers and their anti-aging activity, and between the content of polymeric procyanidins and flowers’ ability to inhibit α-glucosidase. In conclusion, edible flowers may be used to produce functional foods as well as for medical purposes.

Achillea millefolium is a plant used as a component of anti-diabetic preparation but the bioactive compounds responsible for its use are not known. Inhibition of intestinal α-glucosidase is a preferable effect for prevention and treatment of diabetes, and A. millefolium extract showed inhibitory activity against the enzyme. Activity-guided separation of the extract gave four mono- or di-caffeoylquinic acids as the isolate. Quantitation of these four caffeoylquinic acids and the activity of the isolates suggested that these are the major contributor for the α-glucosidase inhibitory activity in the extract. However also, the presence of unidentified, minor, but potent α-glucosidase inhibitor in the isolate was also suggested.

Ceratophyllum demersum L. (CDL) is a traditional Chinese herb to treat many diseases, but research on its anti-diabetic activity is not available. In this research, the α-glucosidase inhibitory ability and phytochemical constituents of CDL extract were firstly studied. Optimal ultrasound-assisted extraction conditions for α-glucosidase inhibitors (AGIs) were optimized by single factor experiment and response surface methodology (RSM), which was confirmed as 70% methanol, liquid-to-solid ratio of 43 (mL/g), extraction time of 54 min, ultrasonic power of 350 W, and extraction temperature of 40 °C. The lowest IC50 value for α-glucosidase inhibition was 0.15 mg dried material/mL (mg DM/mL), which was much lower than that of acarbose (IC50 value of 0.64 mg DM/mL). In total, 80 compounds including 8 organic acids, 11 phenolic acids, 25 flavonoids, 21 fatty acids, and 15 others were identified or tentatively identified from CDL extract by HPLC-QTOF-MS/MS analysis. The results suggested that CDL could be a potential source of α-glucosidase inhibitors. It can also provide useful phytochemical information for research into other bioactivities.

α-glucosidase inhibitors represent an important class of type 2 antidiabetic drugs and they act by lowering postprandial hyperglycemia. Today, only three synthetic inhibitors exist on the market, and there is a need for novel, natural and more efficient molecules exhibiting this activity. In this study, we investigated the ability of Tamarix nilotica ethanolic and aqueous shoot extracts, as well as methanolic fractions prepared from aqueous crude extracts to inhibit α-glucosidase. Both, 50% ethanol and aqueous extracts inhibited α-glucosidase in a concentration-dependent manner, with IC50 values of 12.5 μg/mL and 24.8 μg/mL, respectively. Importantly, α-glucosidase inhibitory activity observed in the T. nilotica crude extracts was considerably higher than pure acarbose (IC50 = 151.1 μg/mL), the most highly prescribed α-glucosidase inhibitor on the market. When T. nilotica crude extracts were fractionated using methanol, enhanced α-glucosidase inhibitory activity was observed in general, with the highest observed α-glucosidase inhibitory activity in the 30% methanol fraction (IC50 = 5.21 μg/mL). Kinetic studies further revealed a competitive reversible mechanism of inhibition by the plant extract. The phytochemical profiles of 50% ethanol extracts, aqueous extracts, and the methanolic fractions were investigated and compared using a metabolomics approach. Statistical analysis revealed significant differences in the contents of the crude extracts and fractions and potentially identified the molecules that were most responsible for these observed variations. Higher α-glucosidase inhibitory activity was associated with an enrichment of terpenoids, fatty acids, and flavonoids. Among the identified molecules, active compounds with known α-glucosidase inhibitory activity were detected, including unsaturated fatty acids, triterpenoids, and flavonoid glycosides. These results put forward T. nilotica as a therapeutic plant for type 2 diabetes and a source of α-glucosidase inhibitors.

A cyanobacterium producing β-glucosidase was isolated from Lake Pamvotis located in Ioannina in Greece. This microorganism, named Pamv7, was identified as Pseudanabaena sp. using phylogenetic characterization. The high-throughput BiologMicroPlate™ method, used for the rapid assessment of heterotrophic potential, indicates that Pseudanabaena sp. metabolizes a wide range of organic substrates such as amino acids, carbohydrates, and carboxylic acids. When the strain grows in a culture medium containing cellobiose as a carbon source, it produces a significant amount of intracellular β-glucosidase. The effect of cellobiose concentration, nitrogen source, and nitrogen concentration of the growth medium, as well as the temperature of the culture, on biomass and β-glucosidase by Pseudanabaena sp., was studied. Biomass and β-glucosidase production by the strain in a lab-scale bioreactor at optimal conditions (10 g/L cellobiose, 1.5 g/L yeast, and 23 ± 1 °C) reached 2.8 g dry weight/L and 44 U/L, respectively. The protein and lipid content of the produced cyanobacterium biomass were 23% and 43 w/w, respectively. This study is the first report of β-glucosidase production by a cyanobacterial strain and concomitant high production of microalgae biomass, making Pseudanabaena sp. a promising microorganism in the field of enzyme biotechnology.

Il glucosio in eccesso viene immagazzinato come glicogeno nel muscolo scheletrico e nel fegato come substrato energetico facilmente disponibile attraverso la via glicolitica. Il malfunzionamento degli enzimi glicolitici provoca disturbi da accumulo di glicogeno come la malattia di Pompe (PD) o glicogenosi di tipo II. PD è una malattia metabolica autosomica recessiva con un'incidenza stimata di 1 su 40000 nati vivi. La malattia di Pompe è causata da difetti a carico dell'enzima lisosomiale acid α-glucosidasi (GAA) o maltasi acida, necessario per la degradazione del glicogeno. Lo spettro di gravità della malattia comprende diversi fenotipi clinici. Essi vanno dalla forma "classica" più grave, caratterizzata da insorgenza durante la prima infanzia, cardiomiopatia grave, decorso progressivo rapido ed esito fatale prima dei due anni, fino ad una forma infantile "intermedia" esprimendo un fenotipo più mite e forme giovanili e adulte caratterizzate dal coinvolgimento prevalente del muscolo scheletrico. La quasi totale carenza dell'enzima GAA causa la forma infantile grave della malattia, mentre la carenza parziale è responsabile delle forme intermedie e lievi. La terapia sostitutiva enzimatica (ERT), in cui l’enzima GAA viene fornito tramite infusione endovenosa è l'unica terapia disponibile dal 2006. Nonostante l'ERT abbia rappresentato una pietra miliare nel trattamento dei pazienti con malattia di Pompe, e abbia dimostrato di essere efficace nella forma infantile grave, non tutti i casi ad esordio tardivo rispondono ugualmente bene a questo trattamento. Pertanto, la correzione del fenotipo muscolare nei casi ad esordio tardivo è ancora ostica, mettendo in luce la necessità di trovare terapie più efficaci. Le difficoltà nel ripristino della funzione muscolare da parte della GAA esogena sono state attribuite ad una concomitante alterazione dell’autofagia. Il processo autofagico è un meccanismo molecolare chiave nel mantenimento dell’omeostasi cellulare, e assicura il corretto turnover delle macromolecole nella cellula. Tuttavia, non è chiaro quali siano le modificazioni dell'autofagia nella malattia di Pompe, poiché non è ancora noto se sia presente un'accelerazione o una riduzione eccessiva di questo processo. In particolare, la recente scoperta di un processo autofagico difettoso nella malattia di Pompe, ha stimolato sia una rivalutazione dei meccanismi patogeni della patologia, sia lo studio di nuovi approcci terapeutici, compresa la ricerca di terapie alternative mirate sia all'accumulo di glicogeno che all'autofagia. Tra le numerose molecole interessanti per il loro effetto di interferire con l'accumulo di glicogeno, abbiamo selezionato l'Acido-3-Bromopiruvico (3-BrPA), un inibitore dell'esochinasi (HK), il quale riveste un ruolo molto importante, essendo un enzima glicolitico. Studi in vitro e in vivo hanno riportato che il 3-BrPA è un efficace farmaco antitumorale, in particolare in quei tipi di tumori in cui le cellule, per crescere e proliferare, dipendono preferibilmente dalla glicolisi per produrre adenosina trifosfato (ATP). La proprietà anticancro di questo particolare composto è dovuta alla sua capacità di inibire la glicolisi. Essa abolisce la produzione di ATP da parte della cellula, impedendo di conseguenza la trasformazione da parte dell’HK del glucosio in glucosio-6-fosfato, e innescando successivamente la modulazione del processo autofagico. Tra le 4 diverse isoforme di esochinasi presenti nei mammiferi (HKI, HKII, HKIII e HKIV), si è visto che l’isoforma HKII è espressa a livello relativamente alto solo nel muscolo scheletrico, nel tessuto adiposo e nel cuore. Lo scopo di questo progetto è quello di testare l’azione del 3-BrPA, valutandone gli effetti sia sul sistema muscolare sia a livello subcellulare, e per fare questo, è stato generato un modello malattia in zebrafish.
Excess glucose is stored as glycogen in skeletal muscle and liver as an energy substrate readily available through the glycolytic pathway. Perturbation of glycolytic enzymes results in glycogen storage disorders such as Pompe disease (PD) or glycogenosis type II. PD is an autosomal recessive metabolic disease with an estimated incidence of 1:40000 live births. PD is due to a defect of the lysosomal enzyme acid α-glucosidase (GAA), or acid maltase, necessary for glycogen degradation. The spectrum of disease severity encompasses a broad continuum of clinical phenotypes ranging from the most severe “classic” form, characterized by early childhood onset, severe cardiomyopathy, rapidly progressive course and fatal outcome before two years of age, to an “intermediate” infantile form expressing a milder phenotype, and to juvenile and adult forms characterized by prevalent involvement of skeletal muscle. The almost total deficiency of the GAA enzyme results in the severe infantile form, while partial deficiency is responsible for the intermediate and mild forms. Enzyme replacement therapy (ERT), where GAA is provided via intravenous infusion is the only therapy available since 2006. While ERT represented a major milestone in the treatment of patients with Pompe disease and it has been shown to be efficacious in infantile severe PD, not all late onset cases respond equally well to this treatment. Therefore, the correction of the skeletal muscle phenotype in late onset cases is still challenging, revealing a need for more effective therapies. GAA difficulties in restoring muscle function have been ascribed to a concomitant altered autophagy, a key molecular mechanism that maintains cellular homeostasis and ensures correct macromolecule turnover in the cell. However, it remains unclear how autophagy is disrupted in PD, since it is yet unknown if an excessive acceleration or reduction of this process is present. Notably, this recent defective autophagy finding in PD has stimulated both a reassessment of the pathogenic mechanisms as well as the investigation of new therapeutic approaches, including search for adjunctive and alternative therapies addressing both glycogen accumulation and autophagy. Among the small molecules to be explored for interfering with glycogen accumulation we have selected the Acid-3-Bromopyruvic (3-BrPA), an inhibitor of hexokinase (HK), which is a key glycolytic enzyme. In vitro and in vivo studies have reported this molecule to be an efficacious anti-tumor drug, in those tumor phenotypes in which cancer cells preferentially depend on glycolysis to produce adenosine triphosphate (ATP) for growth and proliferation. The anti-cancer property of this particular compound is due to its ability to inhibit glycolysis, by abolishing cell ATP production and consequently impeding the transformation by hexokinase of glucose into glucose-6- phosphate, and to trigger modulation of the autophagic process. Among the different hexokinase isoforms HKI, HKII, HKIII, and HKIV found in mammals, HKII is expressed at relatively high level only in skeletal muscle, adipose tissue, and heart. The aim of this project was to use this molecule, as an inhibitor of the key glycolytic enzyme hexokinase-II, to modulate glycogen incorporation into cells. We used zebrafish as in vivo model, in order to evaluate the effect of this specific molecule on the muscular system at subcellular detail. The demonstration of its role as HKII inhibitor and as an autophagy modulator, has created the basis for developing a new strategy to improve muscle function in PD patients.

博士
國立交通大學
應用化學系
90
This study was focused on understanding the catalytic mechanism and identifying the nucleophile and general acid/base catalyst of the family 3 β-glucosidase from Flavobacterium meningosepticum. Recombinant enzyme, fbgl, and mutants were purified from the crude extract of E. coli bearing correspondent genes. With the application of a SP cation-exchanged chromatography, enzymes can be obtained in good quality (> 90% homogeneity). Molecular weight (for all mutants) was analyzed by SDS-PAGE and shown to be ~80 kDa, which was consistent with that derived from DNA sequence. The wild type enzyme possessed highly specific activity on the glycone moiety, while it was relatively broad specificity towards the aglycone portion of the substrate. Its optimal activity was in pH 4.5~5.0. δ-gluconolactone was a competitively strong inhibitor (Ki = 1.1 μM) of the wild type enzyme. Glucose, however, exhibited a moderate product inhibition with Ki = 4.6 mM. The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations including substrate reactivity, secondary kinetic isotope effect. The stereochemistry of the enzymatic hydrolysis was identified as occurring with the retention of anomeric configuration indicating a double-displacement reaction. Based on the kcat values of a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed. This biphasic behavior was consistent with the two-step mechanism involving the formation and the breakdown of a glucosyl-enzyme intermediate. The large Bronsted constant (β1g = -0.85) for the leaving group-dependent portion (pKa of leaving phenols > 7.5) indicates a substantial bond cleavage at the transition state. Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl, o-nitrophenyl, and p-cyanophenyl-β-D-glucopyanoside as substrates were 1.17 ±0.02, 1.19 ±0.02, and 1.04 ±0.02, respectively. These results supported an SN1-like mechanism for the deglucosylation step and an SN2-like mechanism for the glucosylation step. Based on multi-alignment of amino acid sequences of fifteen enzymes from family 3, 11 highly conserved amino acids, including D71, R129, E132, E136, D137, E177, K168, H169, D247, D458 and E473, were studied by means of site-directed mutagenesis and kinetic investigations of the correspondent mutants. Results showed that, Km values of mutants were comparable to that of wild type (0.36 mM for DNPG), with the exception of E473G, Km =0.0004 mM. The kcat values were reduced some 10~3000-fold than that of wild type. The catalytic power of point mutation on D247 or E473 was crippled even more dramatically, e.g. kcat/Km of D247G and D247N were 3x104 and 2x105 times weaker than that of wild type, respectively. Yet, D247E mutant retained at least 20% activity of the wild type (WT) enzyme. Circular dichroism (CD) investigation revealed no significant differences among all mutants. Conduritol-B-epoxide, a potential active site-directed inhibitor, inactivated WT fbgl with a rate of 0.014 s-1, whereas a very slow rate was observed in the case of D247E mutant. These results strongly supported Asp-247 residue functions as the nucleophile of the catalytic reaction. A direct evidence was obtained from another active-site affinity labeling on WT by 2’,4’-dinitrophenyl-2-deoxy -2-fluoro-b-D-glucopyranoside (2F-DNPG) and following by tandem mass spectrometry analysis. The aspartate residue (D247) in the peptide of IVTDYTGINE was identified to be labeled. On the basis of catalytic power analysis of all mutants, E473 residue was the best candidate of the general acid/base catalyst. Further detailed kinetic study confirmed this prediction shown as follows: (1) The kcat and Km value of E473G toward 2’4’-dinitrophenyl-β-D-glucopyranoside (2,4-DNPG) are reduced 3300-fold and 900-fold, respectively, in comparison with that of WT, (2) Unlike the bell-shaped pH profile of WT, the kcat values were virtually invariant with pH over the range of 5.0~9.0, indicating the general acid/base catalyst is absent on E473G mutant, (3) The activity of E473G towards 2,4-DNPG was largely enhanced by the addition of anion such as azide. b-Glucosyl azide was produced, (4) The catalytic activity of E473G towards 2-carboxyphenylβ-glucoside is comparable to that of WT and the correspondent kcat value (E473G) was 60 and 100-fold greater than those of 3-carboxyphenyl and 4-carboxyphenylβ-glucoside catalyzed by E473G, respectively. All of these results highly suggested E473 is the general acid/base catalyst, which was further confirmed by active-site affinity labeling of WT fbgl with N-bromoacetyl b-glucosylamine following by tandem mass spectrometry analysis. The glutamate (E473) in the peptide of SGESSSRANI was found to be labeled.

Gaucher disease (GD) is caused by an inherited deficiency of the human lysosomal enzyme acid β-glucosidase (GBA, EC 3.2.1.45). Absence of functional enzyme results in lysosomal glycolipid accumulation. This disorder primarily affects organs of the reticuloendothelial system and disease severity ranges from mild hepatosplenomegaly to extreme neurological degeneration. Disease symptoms have been shown to be greatly ameliorated by enzyme replacement therapy (ERT). Limitations to therapy include the high cost of current ERT and its inability to treat neurological symptoms. In the present study I sought to produce a GBA-fusion enzyme in an economical manner that can be used to treat neurological GD. I explored the use of Pichia pastoris as an economical recombinant protein expression system for production of human GBA. In addition, I synthesized a protein transduction domain (PTD)-GBA fusion protein for its potential to be used as a neurotherapeutic. The results show that GBA-PTD4 can be expressed and purified from P. pastoris. Hydrophobic interaction chromatography and gel filtration chromatography were successful in purifying GBA-PTD4. Further optimization of expression and purification techniques is required for effective large scale production of recombinant enzyme.
Graduate

Pompe disease, also named acid maltase deficiency and glycogen storage disease type II (GSDII), is a rare autosomal recessive disorder caused by the deficiency of the glycogen-degrading lysosomal enzyme acid α‎-glucosidase. The clinical spectrum of this disease is broad, varying from a lethal infantile-onset generalized myopathy including cardiomyopathy, to late-onset slowly progressive muscle weakness mimicking limb-girdle muscular dystrophy. Respiratory insufficiency is a frequent complication and the main cause of death. The prognosis of Pompe disease has changed considerably with the use of enzyme replacement therapy using recombinant acid α‎-glucosidase (alglucosidase alfa), which has been widely available since 2006. Improvements in survival and major motor achievements can be observed in patients with infantile forms, and recent studies demonstrate improvement of walking distance and stabilization of pulmonary function in late-onset forms. A longer-term study of the safety and efficacy of ERT, based on data gathering across the complete spectrum of Pompe disease via national or international patient registries, is needed in order to formulate more precise guidelines for treatment.

Gaucher disease (GD) is an inherited metabolic disorder caused by the deficiency of enzyme acid β-Glucosidase resulting in the deposition of harmful quantities of lipids/fats. To date, enzyme replacement therapy (ERT) and substrate reduction therapy (SRT) are the only modes of treatment approved by the FDA for Gaucher disease. In this study, we evaluated the ability of microbial bioactive compounds as a drug candidate. The treatment based on molecular docking against selected protein targets plays a crucial role in the future treatment of this disease. Microbial compounds contain bioactive compounds in the form of alkaloids and others of natural origin. Through molecular docking the deep binding affinity of 10 selected compounds present in algae, bacteria, and fungi against the enzyme acid β-Glucosidase of GD using Maestro Schrodinger software, in addition, the ADMET properties are also predicted. Out of these compounds, Lipoxazolidinone C, Cinnamic acid, and Marinopyrrole A, have a sturdy interaction with the Gaucher disease target enzyme, and it can be considered as an effective drug target for Gaucher disease. Our findings reveal a novel discovery towards biology mainly pointing to microbes as a drug formulation. Further, these compounds could be analyzed for their stability through molecular dynamics techniques.

Wildfires are a widespread phenomenon in forests across the Mediterranean Basin but have increased in severity and extent in recent decades. Post-fire treatments are measures that help recover burned vegetation and their functionality but to what extent they also help recover soil functionality is currently unknown. The main objective of this study was to assess the effect of post-fire treatments on ecosystem multifunctionality after a large wildfire in the Cabrera mountain range in 2017 (NW Spain) where close to 10000 Ha of forest were burnt. At the end of 2017 and during 2018, the administration applied different post-fire treatments in high fire severity affected areas: i) straw mulching, ii) woody debris and iii) subsoiling and iv) mechanical hole afforestation. In each treatment, we established ten 2 x 2 m plots and ten adjacent untreated burned plots and collected a composite soil sample from each plot four years after the fire (2021). We calculated regulating services as the standardized mean of total soil organic C (climate regulation), soil water repellence (water regulation) and soil aggregation (soil protection). Supporting services were measured as the standardized mean of mineral N-NH4+ and N-NO3- and available P (soil fertility), β-glucosidase, urease and acid phosphatase (nutrient cycling) and microbial biomass (soil quality). Ecosystem multifunctionality was measured as the standardized mean of all functions measured. Application of straw mulch and woody debris increased regulating ecosystem services in relation to burned control plots. Afforestation with holes had not impact but subsoiling decreased regulating ecosystem services in relation to burned control plots. Post-fire treatments did not have any effect on supporting services. Straw mulch, Woody debris and afforestation with holes improved ecosystem multifunctionality when compared with subsoiling methods. These results show that post-fire stabilisation treatments, in particular straw mulching have a significant positive impact on regulating services and are effective measures in restoring the ecosystem multifunctionality, helping develop effective management based-decisions for the recovery of ecosystem services and functioning after large wildfires.

Justification: Canola meal (CM) is a protein rich co-product of canola oil extraction process, and its use is restricted to animal diet due to the presence of high fibers and antinutritional factors (ANFs) such as glucosinolates and phytic acids. As attempts to provide canola meal with low ANFs and fibers, traditional sprouting process of canola seed followed by sprout defatting and mild washing water pretreatment of hexane extracted CM were applied. The obtained canola sprout meal (CSM) and washed hexane extracted canola meal (WHECM) along with raw hexane extracted canola meal (HECM) underwent submerged and solid-state fermentation. It was noticed that used fungi yielded different outcomes in terms of fiber and ANFs reduction. The objective of this this study was to evaluate the activities of enzymes (cellulase, endoglucanase, and β-glucosidase) produced by three fungal strains (Aureobasidium pullulans, Neurospora crassa and Trichoderma reesei) cultivated on three differently processed canola substrates (CSM, WHECM and HECM) during solid- and submerged- state fermentations using mono and coculture inoculation. Our results showed that cellulase, β-glucosidase and endoglucanase activity significantly varied based on substrate used, mode of fermentation (solid/submerged) and inoculation type (mono/co) even under same strain, highlighting the effect of pre-processed meals, and fermentation conditions on the overall fungal enzymatic activities and their impact on the nutritional composition of the substrate/products.

Introduction: Pompe’s disease is a neuromuscular condition caused by a metabolic disorder of autosomal recessive inheritance. The deficit of acid alpha-glucosidase causes accumulation of glycogen in the lysosomes of the striated and cardiac muscle. It presents in childhood: hypotonia and cardiorespiratory impairment; but at late-onset: axial and waist muscle weakness. Case report: Patient, female, 20 years old, non-consanguineous parents, with good intra-uterus fetal mobility, was born by cesarean delivery weighing 3.7 kilograms and 51 centimeters. She first walked without support and spoke her first words at 13 months of age. By the age of 12, she started progressive thoracolumbar scoliosis and underwent posterior spinal arthrodesis two years later. During the follow-up, muscle weakness was found. Furthermore, she presented macrocephaly, high myopia, fusion of cervical vertebrae, progressive scoliosis, dolichostenomelia and joint hypermobility. Extensive investigation was carried out with laboratory tests that showed CPK elevation, imaging tests and mutation research for facio-scapular-humeral muscular dystrophy and type 2A waistband muscular dystrophy. The incisional biopsy found mixed muscle changes with deposit of amorphous material. Pompe’s disease was confirmed by a significant reduction in alpha-glucosidase activity. The patient evolved with weakness in the legs and fatigue on moderate efforts, but also weakness in the lower limbs, detachable on the right and hyporeflexia, on physical examination. Conclusions: Progressive thoracolumbar scoliosis, refractory to postural and surgical corrections, should be an alert for differential diagnoses. Changes in axial musculature can be suggested and Pompe’s disease, a potentially treatable condition, must become relevant.

Introduction: Pompe disease (PD) affects lysosomal digestion due to absence or low action of the enzyme acid α-glucosidase (GAA), with accumulation of glycogen, causing overflow of enzymes and autophagy, which affects striated muscle. PD is divided into infantile, juvenile, and adult clinical forms, with severity determined by amount of residual GAA activity. Case: P1) 45-year-old man admitted with acute respiratory failure (RF), starts mechanical ventilation. History of weakness, dyspnea, dysphagia. He had decreased proximal muscle strength at lower limbs (LL). Sequencing of GAA gene: autosomal recessive deficiency of two variants. Apnea-hypopnea-index (AHI):10.5. GAA enzyme replacement therapy (ERT) was requested. Judicially denied by disease progression. P2) 40-year-old man presented with loss of muscle strength at LL for 15 years, associated with snoring, daytime somnolence. Brother with similar complaints. He had proximal muscle weakness at LL. Positive genetic panel for PD. AHI:23.5. Judicially released ERT treatment and reported improvement. Discussion: Adult form of PD manifests itself with mild phenotype, with presence of residual GAA activity, which causes different clinical expressions. Main manifestations are symmetric proximal muscle weakness in LL and Gowers’ sign. Frequent death cause in late form is RF, which occurs early, unlike other neuromuscular diseases. In Brazil, PD is underdiagnosed, with approximately 2500 cases. Treatment is performed with Myozyme®, an ERT, not available in SUS, which makes treatment difficult. Conclusion: PD is a serious condition, with high underdiagnosis because of its similarity to other myopathies, which allows disease progression. Furthermore, the variability of GAA mutations allows for distinct phenotypes