A simplified and miniaturized glucometer-based assay for the detection of β-glucosidase activity / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

Screening of thermotolerant and thermophilic fungi aiming β-xylosidase and arabinanase production

Plant cell wall is mainly composed by cellulose, hemicellulose and lignin. The heterogeneous structure and composition of the hemicellulose are key impediments to its depolymerization and subsequent use in fermentation processes. Thus, this study aimed to perform a screening of thermophilic and thermotolerant filamentous fungi collected from different regions of the São Paulo state, and analyze the production of β-xylosidase and arabinanase at different temperatures. These enzymes are important to cell wall degradation and synthesis of end products as xylose and arabinose, respectively, which are significant sugars to fermentation and ethanol production. A total of 12 fungal species were analyzed and 9 of them grew at 45 ºC, suggesting a thermophilic or thermotolerant character. Additionally Aspergillus thermomutatus anamorph of Neosartorya and A. parasiticus grew at 50 ºC. Aspergillus niger and Aspergillus thermomutatus were the filamentous fungi with the most expressive production of β-xylosidase and arabinanase, respectively. In general for most of the tested microorganisms, β-xylosidase and arabinanase activities from mycelial extract (intracellular form) were higher in cultures grown at high temperatures (35-40 ºC), while the correspondent extracellular activities were favorably secreted from cultures at 30 ºC. This study contributes to catalogue isolated fungi of the state of São Paulo, and these findings could be promising sources for thermophilic and thermotolerant microorganisms, which are industrially important due to their enzymes.

Atividade das glicosidases na presença de chá verde e de chá preto / Activity of glycosidases in the presence of green tea and black tea

Várias plantas têm sido consideradas produtos terapêuticos, dentre elas destacam-se os chás verde e preto, popularmente utilizados para controle da hiperglicemia e obesidade. Objetivou-se neste trabalho avaliar o potencial inibitório sobre as enzimas α-amilase, α e β-glicosidases e o teor de compostos fenólicos do chá verde e do chá preto. O teor de compostos fenólicos encontrados foram de 80,8 ± 0,43 mg g-1 no chá preto e 32,0 ± 0,12 mg g-1 no chá verde. O chá verde e o chá preto, em condições de consumo, inibiram as enzimas em estudo, porém, após a simulação do fluido gástrico o inibidor presente no chá verde perdeu a ação. O chá preto deixou de inibir a α-amilase e apresentou inibição inalterada para α e β-glicosidases. Tais resultados sugerem que o chá preto pode ser auxiliar em dietas de restrição de carboidratos.

Several plants have been considered therapeutic products, including green and black tea, popularly used to control hyperglycemia and obesity. This study aimed to evaluate the inhibitory potential of the enzymes α-amylase, α and β-glycosidases, as well as the content of phenolic compounds in green tea and black tea. The concentrations of phenolic compounds found were 80.8 ± 0.43 mg g-1 in black tea and 32.0 ± 0.12 mg g-1 in green tea. Under the tested conditions of use, green and black tea inhibited the enzyme under study. However, after simulation of the gastric fluid, the inhibitor present in green tea lost its action. Black tea no longer inhibited a-amylase and showed unaltered inhibition for α and β-glycosidases. These results suggest that black tea can be helpful in diets restricting carbohydrates.

Cloning and expression of antiviral/ribosome-inactivating protein from Bougainvillea xbuttiana.

A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP)from the leaves of Bougainvillea x buttiana was isolated.The cDNA consisted of 1364 nucleotides with an open reading frame (ORF)of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids.The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species.The deduced protein has been designated BBAP1 (Bougainvillea x buttiana antiviral protein1).The ORF was cloned into an expression vector and expressed in E.coli as a fusion protein of approximately 78 kDa.The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity,and imparted a high level of resistance against the tobacco mosaic virus (TMV).

Production of thermostable glucoamylase by newly isolated Aspergillus flavus A 1.1 and Thermomyces lanuginosus A 13.37

Entre 13 linhagens de fungos filamentosos isolados a partir de amostras de solo agrícola, tubérculos e de material em compostagem, duas foram selecionadas em função da capacidade de crescer em meio líquido contendo amido como única fonte de carbono, a 45ºC, e produzir consideráveis quantidades de glucoamilase. Essas linhagens, identificadas como Aspergillus flavus A1.1 e Thermomyces lanuginosus A13.37, foram utilizadas para desenvolvimento de experimentos para avaliar os efeitos do tipo de amido (milho e mandioca), do pH inicial do meio de cultura (4,0; 5,0 e 6,0) e da temperatura de incubação (40 e 45ºC), em um modelo fatorial (2x3x2), sobre a produção da glucoamilase. O tipo de amido usado como fonte de carbono para o cultivo dos fungos influenciou significativamente a produção de glucoamilase por A. flavus, sendo obtida uma maior quantidade da enzima em meio contendo amido de mandioca do que em meio com amido de milho. A produção da enzima por T. lanuginosus também foi maior em meio contendo amido de mandioca, porém, a diferença não foi estatisticamente significativa. As atividades enzimáticas sobre amido (0,3 per center), maltose (0,3 per center) ou sobre mistura de 0,3 per center de amido com 0,1 per center de maltose, indicaram que as enzimas de Aspergillus hidrolisaram, preferencialmente, o amido, embora tenham mostrado atividade considerável sobre a maltose. A maior liberação de glicose a partir da mistura de substratos sugeriu que o fungo em questão possa secretar dois tipos diferentes de enzimas. Enzimas produzidas por T. lanuginosus hidrolisaram o amido e a maltose e não liberaram maiores teores de glicose quando o substrato constou de mistura de amido e maltose. As enzimas de Aspergillus e Thermomyces apresentaram elevada temperatura ótima de atividade (65 e 70ºC, respectivamente) com boa termoestabilidade na ausência de substrato (manutenção de 50 per ceter da atividade por 5 e 8h respectivamente), além de estabilidade em ampla faixa de pH. Os resultados apresentados indicam uma importante fonte alternativa de glucoamilase para uso no processamento industrial de amido.

Activity of glycosidases from freshwater heterotrophic microorganisms on the degradation of extracellular polysaccharide produced by Anabaena spiroides (Cyanobacteria)

A atividade de glicosidases durante a degradação do polissacarídeo extracelular (EPS) produzido por Anabaena spiroides foi detectada e quantificada utilizando-se MUF-substratos (MUF-monossacarídeos). O consumo total do polissacarídeo efetuou-se em duas fases, uma primeira de alta atividade enzimática que rapidamente consumiu 41 per center do polissacarídeo e uma segunda, mais lenta, que consumiu o polissacarídeo restante (59 per center). A mudança de fase coincidiu com a sucessão de uma população de bactérias cocóides por outra de bacilos. A biomassa bacteriana, quantificada por contagens de células, aumentou com a degradação do EPS. As atividades registradas através dos substratos 4-MUF-a-D- e 4-MUF-b-D- glicosídeo foram mais altas quando comparadas aos demais substratos testados que foram: MUF-a-L-ramnopiranosídeo, MUF-b-D-galactosídeo, MUF-a-D-manopiranosídeo, MUF-b-D-fucosídeo, MUF-b-D-manopiranosídeo, MUF-a-L-arabinopiranosídeo, e MUF-b-L-fucosídeo. A fluorescência emitida a partir de cada um dos diferentes MUF-substratos foi, de modo geral, proporcional à concentração dos monossacarídeos correspondentes constituintes do polissacarídeo, um indício da susceptibilidade ao ataque enzimático microbiano do EPS produzido por A. spiroides.

Alteraciones bioquímicas y citoquímicas de hidrolasas ácidas lisosomales en leucemias / Biochemical and citochemical alterations of lisosomal acid hydrolases in leukemias

La actividad enzimática total de ß-galactosidasa (ß-gal), hexosaminidasa (hex) y fosfatasa ácida (Fac) fue determinada bioquímicamente, tanto en suero como en sobrenadantes de homogenatos celulares de sujetos normales y pacientes portadores de leucemias, empleándose sustratos paranitrofenilados específicos. La actividad de ß-gal en leucemias mieloides, tanto agudas (LMA-M1) como crónicas (LMC), sólo mostró un incremento significativo en sobrenadantes de homogenato celulares, respecto a los valores de neutrófilos normales. En leucemias linfoidales agudas (LLA), como crónicas (LLC), su comportamiento no ofreció variaciones. Tanto los sueros como los sobrenadantes de homogenatos celulares de LMA-M1 y LMC mostraron un franco incremento en la actividad de hex, mientras en LLA y LLC esta actividad no mostró variaciones. La actividad sérica de fosfatasa ácida estuvo incrementada en el 86% de las LMC. En los sobrenadantes de homogenatos celulares de LLA y LLC, esta actividad enzimática se mostró significativamente disminuida respecto de los valores de linfocitos normales. En los tres casos de LMA-M4 analizados, fue observado en el contenido celular niveles elevados de ß-gal, hex y Fac (lo que estaría correlacionado con la presencia de monocitos y/o monoblastos normales, con alto contenido de hidrolasas ácidas). Citoquímicamente fue demostrado en médula ósea y sangre periférica de pacientes con LMA-M1 una ligera o nula actividad de hex, en tanto que en LLA la reacción fue localizada asimétricamente en un polo celular o en gránulos citoplasmáticos. Los resultados encontrados demuestran una gran heterogeneidad en el contenido lisosomal de los diferentes tipos de leucemias