Solid state fermentation to obtain vegetable products bio-enriched with isoflavone aglycones using lactic cultures / Fermentación en sustrato sólido utilizando cultivos lácticos para la obtención de productos vegetales bioenriquecidos con isoflavonas agliconas

The consumption of soybean isoflavones (IS) is associated with several beneficial properties on human health. Some lactic acid bacteria possess ß-glucosidase enzyme, that allows to obtain the active form of IS (aglycone). The solid state fermentation (SSF) has received great attention in the last years in order to obtain several valuable compounds. SSF, using soybean as substrate and Lactobacillus rhamnosus CRL 981 as starter, was studied in the present work. Sucrose was added into soybean paste to study the effect on the behavior of the selected strain. The development of L. rhamnosus CRL 981 through pH and recount measures, sugar intake, organic acid production, ß-glucosidase activity and IS conversion were analyzed. No significant differences in growth and acidity were observed between soybean pastes with and without sucrose added, but the production of lactic acid was higher in the latter paste. The ß-glucosidase activity was detected in both pastes and the complete hydrolysis of IS at 12 h of fermentation was observed. Also, this strain was able to increase the free amino acids in soybean paste. SSF, using soybean as substrate and L. rhamnosus CRL 981 as starter culture, is an alternative process to obtain a soybean product bio-enriched in active IS with attractive nutritional characteristics.

El consumo de isoflavonas de soja (IS) está asociado a diversos beneficios para la salud humana. Ciertas bacterias lácticas poseen la enzima ß-glucosidasa, que permite obtener la forma bioactiva (agliconas) de las IS. La fermentación en sustrato sólido (FSS) ha recibido gran atención en los últimos anos debido a sus numerosas ventajas, y permite la obtención de productos con valor agregado. En el presente trabajo se estudió la FSS utilizando soja como sustrato y Lactobacillus rhamnosus CRL981 como cultivo iniciador. Con el fin de estudiar el efecto de una fuente de carbono externa sobre el comportamiento de la cepa seleccionada, se adicionó sacarosa a la pasta de soja. Se evaluó el crecimiento de L. rhamnosus CRL 981 a través de medidas de pH y recuento en placa. Además, se analizó el consumo de azúcares, producción de ácidos orgánicos, actividad ß-glucosidasa y conversión de IS. No se observaron diferencias significativas en el crecimiento y acidez entre las pastas de soja sin adición de sacarosa y con ella, sin embargo, la producción de ácido láctico fue mayor en esta última. La actividad de ß-glucosidasa se detectó en ambas pastas y se observó la hidrólisis completa de IS a las 12 h de fermentación. Además, esta cepa fue capaz de aumentar los aminoácidos libres en la pasta de soja. La FSS, utilizando soja como sustrato y L. rhamnosus CRL 981 como cultivo iniciador, es un proceso alternativo para obtener un producto de soja bioenriquecido en IS bioactivas con características nutricionales atractivas.

Cellulolytic ability of a promising Irpex lacteus (Basidiomycota: Polyporales) strain from the subtropical rainforest of Misiones province, Argentina / Capacidad celulolítica de una cepa prometedora de Irpex lacteus (Basidiomycota: Polyporales) de la selva subtropical de la provincia de Misiones, Argentina

Abstract The cellulolytic activity of fungi growing in the subtropical rainforest of Misiones (Argentina) represents a challenge in the technological development of the production of cellulosic bioethanol in the region using native sources. These fungi are promising to obtain sustainable enzyme cocktails using their enzymes. Cellulolytic ability of 22 white-rot fungi isolated from the subtropical rainforest of Misiones-Argentina in agar medium with two types of cellulosic substrates, carboxy-methylcellulose or crystalline cellulose, were comparatively analyzed, and the activity of two cellulolytic enzymes was evaluated in liquid medium. Although all isolates were able to grow and degrade both substrates in agar medium, and to produce total cellulase Filter paper (FPase) and endo-β-1,4-glucanase (EG) activities in broth, the isolate Irpex sp. LBM 034 showed the greatest enzymatic levels (FPase, 65.45 U L-1; EG, 221.21 U L-1). Therefore, the ITS sequence of this fungus was sequenced and analyzed through a phylogenetic analysis. These results indicate that the isolate LBM 034, corresponding to Irpex lacteus, has a promising cellulolytic ability and enzymes such as EG useful in sustainable saccharification of cellulosic materials in the region. Rev. Biol. Trop. 66(3): 1034-1045. Epub 2018 September 01.

Resumen La actividad celulolítica de hongos autóctonos asociados a la selva subtropical de Misiones (Argentina) representa un desafío en el desarrollo tecnológico de la producción de bioetanol celulósico en la región, mediante el uso de recursos nativos. Los sistemas enzimáticos de estos hongos tienen potencial aplicación en la obtención de cocteles enzimáticos rentables. La habilidad celulolítica de 22 hongos causantes de pudrición blanca se analizó comparativamente, que fueron aislados de la selva subtropical de Misiones-Argentina, en cultivos agarizados con dos tipos de sustratos celulósicos, carboxi-metilcelulosa o celulosa cristalina. También se evaluó la actividad de dos enzimas celulolíticas en cultivos líquidos. Aunque todos los aislamientos fueron capaces de crecer y degradar ambos sustratos en medio agarizado y revelar actividad celulolítica total y endo-β-1,4-glucanasa en cultivo líquido, el aislamiento Irpex sp. LBM 034 mostró las mayores actividades en papel de filtro con 65.45 U L-1 y endo-β-1,4-glucanasa con 221.21 U L-1, respectivamente. Por tanto, se secuenció y analizó la secuencia ITS de este hongo a través de un análisis filogenético. Estos resultados indicaron que el aislamiento LBM 034, correspondiente a Irpex lacteus, tiene una habilidad celulolítica prometedora en la producción de enzimas con actividad endo-β-1,4-glucanasa, útil en la sacarificación sustentable de materiales celulósicos de la región.

Production of thermostable ß-glucosidase and CMCase by Penicillium sp: LMI01 isolated from the Amazon region

Background: Cellulolytic enzymes of microbial origin have great industrial importance because of their wide application in various industrial sectors. Fungi are considered the most efficient producers of these enzymes. Bioprospecting survey to identify fungal sources of biomass-hydrolyzing enzymes from a high-diversity environment is an important approach to discover interesting strains for bioprocess uses. In this study, we evaluated the production of endoglucanase (CMCase) and ß-glucosidase, enzymes from the lignocellulolytic complex, produced by a native fungus. Penicillium sp. LMI01 was isolated from decaying plant material in the Amazon region, and its performance was compared with that of the standard isolate Trichoderma reesei QM9414 under submerged fermentation conditions. Results: The effectiveness of LMI01 was similar to that of QM9414 in volumetric enzyme activity (U/mL); however, the specific enzyme activity (U/mg) of the former was higher, corresponding to 24.170 U/mg of CMCase and 1.345 U/mg of ß-glucosidase. The enzymes produced by LMI01 had the following physicochemical properties: CMCase activity was optimal at pH 4.2 and the ß-glucosidase activity was optimal at pH 6.0. Both CMCase and ß-glucosidase had an optimum temperature at 60°C and were thermostable between 50 and 60°C. The electrophoretic profile of the proteins secreted by LMI01 indicated that this isolate produced at least two enzymes with CMCase activity, with approximate molecular masses of 50 and 35 kDa, and ß-glucosidases with molecular masses between 70 and 100 kDa. Conclusions: The effectiveness and characteristics of these enzymes indicate that LMI01 can be an alternative for the hydrolysis of lignocellulosic materials and should be tested in commercial formulations.

In vivo assay to identify bacteria with ß-glucosidase activity

Background: ß-Glucosidase assay is performed with purified or semipurified enzymes extracted from cell lysis. However, in screening studies, to find bacteria with ß-glucosidase activity among many tested bacteria, a fast method without cell lysis is desirable. In that objective, we report an in vivo ß-glucosidase assay as a fast method to find a ß-glucosidase producer strain. Results: The method consists in growing the strains for testing in a medium supplemented with the artificial substrate p-nitrophenyl-ß-glucopyranoside (pNPG). The presence of ß-glucosidases converts the substrate to p-nitrophenol (pNP), a molecule that can be easily measured in the supernatant spectrophotometrically at 405 nm. The assay was evaluated using two Bifidobacterium strains: Bifidobacterium longum B7254 strain that lacks ß-glucosidase activity and Bifidobacterium pseudocatenulatum B7003 strain that shows ß-glucosidase activity. The addition of sodium carbonate during pNP measurement increases the sensitivity of pNP detection and avoids the masking of absorbance by the culture medium. Furthermore, we show that pNP is a stable enzymatic product, not metabolized by bacteria, but with an inhibitory effect on cell growth. The ß-glucosidase activity was measured as units of enzyme per gram per minute per dry cell weight. This method also allowed the identification of Lactobacillus strains with higher ß-glucosidase activity among several lactobacillus species. Conclusion: This in vivo ß-glucosidase assay can be used as an enzymatic test on living cells without cell disruption. The method is simple, quantitative, and recommended, especially in studies screening for bacteria not only with ß-glucosidase activity but also with high ß-glucosidase activity.

Microbial transformation of ginsenosides extracted from Panax ginseng adventitious roots in an airlift bioreactor

Background: Ginsenoside is the most important secondary metabolite in ginseng. Natural sources of wild ginseng have been overexploited. Although root culture can reduce the length of the growth cycle of ginseng, the number of species of ginsenosides is reduced and their contents are lower in the adventitious roots of ginseng than in the roots of ginseng cultivated in the field. Results: In this study, 147 strains of ß-glucosidase-producing microorganisms were isolated from soil. Of these, strain K35 showed excellent activity for converting major ginsenosides into rare ginsenosides, and a NCBI BLAST of its 16S rDNA gene sequence showed that it was most closely related to Penicillium sp. (HQ608083.1). Strain K35 was used to ferment the adventitious root extract, and the fermentation products were analyzed by high-performance liquid chromatography. The results showed that the content of the rare ginsenoside CK was 0.253 mg mL-1 under the optimal converting conditions of 9 d of fermentation at pH 7.0 in LL medium, which was significantly higher than that in the adventitious roots of ginseng. Conclusion: These findings may not only solve the problem of low productivity of metabolite in ginseng root culture but may also result in the development of a new valuable method of manufacturing ginsenoside CK.

Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.

Production of beta-glucosidase on solid-state fermentation by Lichtheimia ramosa in agroindustrial residues: characterization and catalytic properties of the enzymatic extract

Background β-Glucosidases catalyze the hydrolysis of cellobiose and cellodextrins, releasing glucose as the main product. This enzyme is used in the food, pharmaceutical, and biofuel industries. The aim of this work is to improve the β-glucosidase production by the fungus Lichtheimia ramosa by solid-state fermentation (SSF) using various agroindustrial residues and to evaluate the catalytic properties of this enzyme. Results A high production of β-glucosidase, about 274 U/g of dry substrate (or 27.4 U/mL), was obtained by cultivating the fungus on wheat bran with 65% of initial substrate moisture, at 96 h of incubation at 35°C. The enzymatic extract also exhibited carboxymethylcellulase (CMCase), xylanase, and β-xylosidase activities. The optimal activity of β-glucosidase was observed at pH 5.5 and 65°C and was stable over a pH range of 3.5-10.5. The enzyme maintained its activity (about 98% residual activity) after 1 h at 55°C. The enzyme was subject to reversible competitive inhibition with glucose and showed high catalytic activity in solutions containing up to 10% of ethanol. Conclusions β-Glucosidase characteristics associated with its ability to hydrolyze cellobiose, underscore the utility of this enzyme in diverse industrial processes.

Chemical modification of Aspergillus niger β-glucosidase and its catalytic properties

Aspergillus niger β-glucosidase was modified by covalent coupling to periodate activated polysaccharides (glycosylation). The conjugated enzyme to activated starch showed the highest specific activity (128.5 U/mg protein). Compared to the native enzyme, the conjugated form exhibited: a higher optimal reaction temperature, a lower Ea (activation energy), a higher Km (Michaelis constant) and Vmax (maximal reaction rate), and improved thermal stability. The calculated t1/2 (half-life) values of heat in-activation at 60 °C and 70 °C were 245.7 and 54.5 min respectively, whereas at these temperatures the native enzyme was less stable (t1/2 of 200.0 and 49.5 min respectively). The conjugated enzyme retained 32.3 and 29.7%, respectively from its initial activity in presence of 5 mM Sodium Dodecyl Sulphate (SDS) and p-Chloro Mercuri Benzoate (p-CMB), while the native enzyme showed a remarkable loss of activity (retained activity 1.61 and 13.7%, respectively). The present work has established the potential of glycosylation to enhance the catalytic properties of β-glucosidase enzyme, making this enzyme potentially feasible for biotechnological applications.

Regulation of gene expression: Cryptic β-glucoside (bgl) operon of Escherichia coli as a paradigm

Bacteria have evolved various mechanisms to extract utilizable substrates from available resources and consequently acquire fitness advantage over competitors. One of the strategies is the exploitation of cryptic cellular functions encoded by genetic systems that are silent under laboratory conditions, such as the bgl (β-glucoside) operon of E. coli. The bgl operon of Escherichia coli, involved in the uptake and utilization of aromatic β-glucosides salicin and arbutin, is maintained in a silent state in the wild type organism by the presence of structural elements in the regulatory region. This operon can be activated by mutations that disrupt these negative elements. The fact that the silent bgl operon is retained without accumulating deleterious mutations seems paradoxical from an evolutionary view point. Although this operon appears to be silent, specific physiological conditions might be able to regulate its expression and/or the operon might be carrying out function(s) apart from the utilization of aromatic β-glucosides. This is consistent with the observations that the activated operon confers a Growth Advantage in Stationary Phase (GASP) phenotype to Bgl+ cells and exerts its regulation on at least twelve downstream target genes.

Study of enzyme replacement therapy for Gaucher Disease: comparative analysis of clinical and laboratory parameters at diagnosis and after two, five and ten years of treatment

Objective: To evaluate the impact of enzyme replacement therapy for Gaucher Disease on clinical and laboratory parameters after two, five and ten years of treatment. Methods: Data were collected from patient records and analyzed using BioEstat software (version 5.0). Student's t-test, Analysis of Variance (ANOVA), Wilcoxon test and Kruskal–Wallis test were used for statistical analysis. Hepatomegaly and splenomegaly were analyzed using the Kappa test. Results: There was a significant increase in hemoglobin levels (p-value <0.01) and platelet counts (p-value = 0.01) within two years of therapy. At the same time, the frequencies of splenomegaly (p-value <0.01) and hepatomegaly (p-value <0.05) reduced. These results were similar at five and ten years of enzyme replacement therapy. Conclusions: There are substantial and quick (within two years) laboratory and clinical responses to enzyme replacement therapy. These improvements continue as long as enzyme replacement therapy is administered every two weeks, as recommended by the literature...

Optimal immobilization of Beta-glucosidase into chitosan beads using response surface methodology

Background: β-Glucosidase is known as an effective catalyst for the hydrolysis of various glycosides and immobilization is one of the most efficient strategies to improve its activity recovery and properties. Results: Crosslinking-adsorption-crosslinking method was employed to immobilize β-glucosidase into chitosan beads and response surface methodology (RSM) was used to optimize the immobilized conditions of the maximum activity recovery. Enzyme concentration and adsorption time were found to be significant influence factors, and the maximum activity recovery (50.75%) obtained from response surface methodology was in excellent agreement with experimental value (50.81%). Furthermore, various characteristics of immobilized β-glucosidase were evaluated. Compared to the free β-glucosidase, the immobilized enzyme exhibited broader pH and temperature ranges, enhanced thermal stability, better storage stability and reusability and higher accessibility of the substrate to the immobilized β-glucosidase. Conclusion: Response surface methodology (RSM) was proved to be much economical for optimum immobilization of β-glucosidase into chitosan beads.

Informe de evaluación rápida de tecnología sobre seguridad y efectividad de la imiglucerasa para enfermedad de Gaucher tipo I / Rapid evaluation report of technology on the safety and effectiveness of imiglucerase for Gaucher disease type I

La Enfermedad de Gaucher es la enfermedad más común de depósito lisosomal, pertenece al grupo de enfermedades catalogadas como errores innatos del metabolismo y se trasmite por herencia autosómica recesiva. (1) Se caracteriza por la presencia de mutaciones patológicas en los genes que codifican la enzima ß- glucosidasa-ácida lisosomal produciendo su deficiencia parcial o total, lo que origina depósitos anormales de material glicolípido (glucocerebrósidos) en los lisosomas de los macrófagos mononucleares de los órganos ricos en células de la línea monocitosmacrófagos como el hígado, bazo, médula ósea y parénquima de los nódulos linfáticos. Se reporta después de la intervención aumento sostenido de la hemogloblina, disminución de la hepatomegalia, disminución del volumen esplénico en pacientes con esplenomeglia moderada y severa, resolución del dolor óseo, los niños que reciben la TRE pueden llegar a alcanzar la talla normal en alrededor de 3 años de tratamiento, respuesta a hipertensión pulmonar. Los pacientes con TRE tuvieron mejores puntajes para el funcionamiento físico, el rol físico, el dolor corporal, la salud general y vitalidad. Se recomienda cubrir con generación de evidencia.(AU)

Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1

The production of xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 ºC and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources; in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, β-xylosidase and ferulic acid esterase to a peak concentrations of 44,880; 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest β-glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L), which was not detected in the T. reesei culture.

Enhancement of escherichia coli cellulolytic activity by co-production of beta-glucosidase and endoglucanase enzymes

Cellulase is a group of enzymes (endoglucanase, exoglucanase and beta-glucosidase) required for cellulosic feedstock hydrolysis during bioethanol production. The use of recombinant cellulase is a strategy to reduce the enzyme cost. In this context, the present work describes the construction of a cellulase expression vector (pEglABglA), which allowed constitutive co-expression of endoglucanase A (EglA) from an endophytic Bacillus pumilus and the hyperthermophilic beta-glucosidase A (BglA) from Fervidobacterium sp. in Escherichia coli. When compared to the non-modified strain DH5 alpha, the recombinant Escherichia coli DH5 alpha (pEglABglA) reduced fivefold the viscosity of the carboxymethylcellulose medium (CMC-M). Also, it presented almost 30-fold increase in reducing sugar released from CMC-M, enabling the recombinant strain to grow using CMC as the sole carbon and energy source. When cultivated in rich media, specific growth rates of recombinant E. coli strains BL21, JM101 and Top10 were higher than those of DH5 alpha and DH10B strains. The constructed plasmid (pEglABglA) can be used as backbone for further cellulase gene addition, which may enhance even more E. coli cellulolytic capacity and growth rate.

Simultaneous saccharification and fermentation process of different cellulosic substrates using a recombinant Saccharomyces cerevisiae harbouring the beta-glucosidase gene

In Brazil, the production of ethanol from sugarcane produces large amounts of lignocellulosic residues (bagasse and straw), which have been driving research and development for the production of second generation ethanol. In the present work, a recombinant Saccharomyces cerevisiae strain expressing the beta-glucosidase gene from Humicola grisea was used for ethanol production from three different cellulosic sources by simultaneous saccharification and fermentation. Initially, a enzymatic pre-hydrolysis step was done with a solid:liquid ratio of 1:4, and an enzymatic load of 25 filter paper activity (FPU).g-1 of cellulosic substrate. Using sugarcane bagasse pretreated cellulignin, crystalline cellulose and carboxymethyl cellulose, 51.7 g L-1, 41.7 g L-1 and 13.8 g L-1 of ethanol was obtained, respectively, at the end of 55 hrs of fermentation. The highest ethanol productivity (0.94 g L-1 hrs-1) was achieved using sugarcane bagasse pretreated cellulignin. The use of a recombinant S. cerevisiae led to extremely low glucose concentrations when compared to other works reported in literature.

Aspectos clínicos, bioquímicos, moleculares y tratamiento de 2 pacientes con enfermedad de Gaucher / Clinical, biochemical and molecular features and the treatment of two patients presenting with Gaucher's disease

La enfermedad de Gaucher es una entidad hereditaria del metabolismo de los esfingolípidos con un patrón de herencia autosómico recesivo determinada por una deficiencia de la actividad de la enzima b-glucosidasa ácida. En este trabajo se presentan 2 pacientes en edad pediátrica, uno del sexo femenino y otro del masculino, ambos con anemia y hepatoesplenomegalia confirmadas por ultrasonido. El aspirado de médula ósea mostró infiltración por células de almacenamiento, niveles bajos de la actividad enzimática de b-glucocerebrosidasa y el diagnóstico molecular de las posibles mutaciones conocidas confirmaron la enfermedad en ambos pacientes que se encuentran en tratamiento con terapia enzimática sustitutiva (imiglucerasa), con evolución favorable en los aspectos clínicos y humorales.

Gaucher's disease is a hereditary entity related to sphingolipids metabolism with an autosomal recessive hereditary pattern determined by a failure of the acid b-glucosidase enzyme. In present paper authors present the case of two pediatric patients (1 female and 1 male) both presenting with anemia and hepatosplenomegaly by ultrasound (US). Bone marrow aspirate showed infiltration by storage cells, low levels of enzymatic activity of b-glucocerebroside and a molecular diagnosis of potential known mutations confirmed the disease in both patients, who are under treatment with substitutive enzymatic therapy (imiglucerase) with a favorable course in clinical and humoral features.

Characterization of the interdependency between residues that bind the substrate in a â-glycosidase

The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the â-glycosidase from Spodoptera frugiperda (Sfâgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfâgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl â-galactoside and p-nitrophenyl â-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfâgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on â-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

Purification and characterization of beta-glucosidase from Melanocarpus sp. MTCC 3922

This study reports the purification and characterization of beta-glucosidase from a newly isolated thermophilic fungus, Melanocarpus sp. Microbial Type Culture Collection (MTCC) 3922. The molecular weight of beta-glucosidase was determined to be ~ 92 and 102 kDa with SDS PAGE and gel filtration, respectively, and pI of ~ 4.1. It was optimally active at 60 C and pH 6.0, though was stable at 50 C and pH 5.0 - 6.0. The presence of DTT, mercaptoethanol and metal ions such as Na+, K+, Ca2+, Mg2+and Zn2+ positively influenced the activity of beta-glucosidase but the activity was inhibited in the presence of CuSO4. beta-Glucosidase recognized pNP- beta-glucopyranoside (pNPG) as the preferred substrate, and showed very low affinity for pNP- beta-D-cellobioside. Km and Vmax for the hydrolysis of pNPG by beta-glucosidase was calculated as 3.3 mM and 43.68 ‘molmin-1mg protein-1, respectively and k cat was quantified as 4 x 10³ min-1. beta-Glucosidase activity was enhanced appreciably in the presence of alcohols (methanol and ethanol) moreover, purified beta-glucosidase showed putative transglycosylation activity that was positively catalyzed in presence of methanol as an acceptor molecule

Bioassay screening of Amazonian plants

OBJECTIVE: The purpose of this study was to evaluate several biological activities of thirty plant extracts collected in the North West Amazon (Ecuador). Some of these plants are being used for their reputed medicinal properties by the natives of this region. METHODS: Five in vitro bioassays were used to screen the plant material. 1. The brine shrimp lethality examination (BSLT) in microplate is a general test that seems capable of detecting a broad spectrum of bioactivity present in crude plant extracts. 2. Free radical scavenging properties were studied in a colorimetric assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH). 3. The beta-glucosidase inhibition test is thought to be a method for the evaluation of anti-AIDS, anti-diabetic or anti-obesity compounds. 4. The xanthine oxidase inhibition assay is used to identify potential anti-gout agents. 5. The antibacterial activity that is being used to isolate and identify antibiotic drugs. RESULTS: In the BSLT, we found that Piscidia carthagenensis demonstrated very good activity with a LC50: 21.81 micrograms/mL. It is considered that plant extracts with low LC50 values may contain metabolites with cytotoxic, antifungal, insecticidal or pesticide activities. In the antioxidant activity bioassay, several plant extracts were confirmed to have excellent free radical scavenging properties. Rhus juglandifolia and Clusia venusta leaves exhibited an ED50: 3.12 micrograms/mL and 3.61 micrograms/mL, respectively. Piper reticulatum (84 per cent), Inga heteroptera (77 per cent), Clusia venusta (70.9 per cent), and Rhus juglandifolia (70.5 per cent) showed fairly good inhibition activity for beta-glucosidase. On the other hand, none of the plant extracts was capable of inhibiting xanthine oxidase. Finally, the Gram-positive microorganisms Staphylococcus aureus and Corynebacterium diphteriae were found to be sensitive to the majority of the plant extracts, whereas the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, and Salmonella typhi were proved to be resistant toward the plant extracts. CONCLUSIONS: It is important to continue investigating our plant kingdom, especially the world tropical reserves as an alternative for finding new or better drugs. It should be essential to follow-up this type of investigation to isolate and elucidate the active principles of the bio-positive plants.

Serum hexosaminidase and á-glucuronidase activities in infants: effects of age and sex

We investigated the effect of age and sex on the serum activity of hexosaminidase (HEX) and á-glucuronidase (BGLU) in 275 normal term infants aged 12 h to 12 months. Up to six weeks of life, HEX was significantly higher in boys (P<=0.023). During the age period of 1-26 weeks, BGLU was also higher in boys, but differences were significant only at 2-6 and 7-15 weeks (P<=0.016). The developmental pattern of HEX and BGLU was sex dependent. HEX activity increased in both sexes from 4-7 days of life, reaching a maximum of 1.4-fold the birth value at 2-6 weeks of age in boys (P<0.001) and a maximum of 1.6-fold at 7-15 weeks in girls (P<0.001). HEX activity gradually decreased thereafter, reaching significantly lower levels at 27-53 weeks than during the first three days of life in boys (P = 0.002) and the same level of this age interval in girls. BGLU increased in both sexes from 4-7 days of age, showing a maximum increase at 7-15 weeks (3.3-fold in boys and 2.9-fold in girls, both P<0.001). Then BGLU decreased in boys to a value similar to that observed at 4-7 days of age. In girls, BGLU remained elevated until the end of the first year of life. These results indicate a variation of HEX and BGLU activities during the first year of life and a sex influence on their developmental pattern. This observation should be considered in the diagnosis of GM2 gangliosidosis and mucopolysaccharidosis type VII