Investigation of anti-Alzheimer's activity of aqueous extract of areca nuts (Areca catechu L): in vitro and in vivo studies / Investigación de la actividad anti-Alzheimer del extracto acuoso de nueces de areca (Areca catechu L): estudios in vitro e in vivo

Alzheimer's disease (AD) is an age-related neurodegenerative disorder. Sever cognitive and memory impairments, huge increase in the prevalence of the disease, and lacking definite cure have absorbed worldwide efforts to develop therapeutic approaches. Since many drugs have failed in the clinical trials due to multifactorial nature of AD, symptomatic treatments are still in the center attention and now, nootropic medicinal plants have been found as versatile ameliorators to reverse memory disorders. In this work, anti-Alzheimer's activity of aqueous extract of areca nuts (Areca catechu L.) was investigated via in vitro and in vivo studies. It depicted good amyloid ß (Aß) aggregation inhibitory activity, 82% at 100 µg/mL. In addition, it inhibited beta-secretase 1 (BACE1) with IC50 value of 19.03 µg/mL. Evaluation of neuroprotectivity of the aqueous extract of the plant against H2O2-induced cell death in PC12 neurons revealed 84.5% protection at 1 µg/mL. It should be noted that according to our results obtained from Morris Water Maze (MWM) test, the extract reversed scopolamine-induced memory deficit in rats at concentrations of 1.5 and 3 mg/kg.

La enfermedad de Alzheimer (EA) es un trastorno neurodegenerativo relacionado con la edad. Los severos deterioros cognitivos y de la memoria, el enorme aumento de la prevalencia de la enfermedad y la falta de una cura definitiva han absorbido los esfuerzos mundiales para desarrollar enfoques terapéuticos. Dado que muchos fármacos han fallado en los ensayos clínicos debido a la naturaleza multifactorial de la EA, los tratamientos sintomáticos siguen siendo el centro de atención y ahora, las plantas medicinales nootrópicas se han encontrado como mejoradores versátiles para revertir los trastornos de la memoria. En este trabajo, se investigó la actividad anti-Alzheimer del extracto acuoso de nueces de areca (Areca catechu L.) mediante estudios in vitro e in vivo. Representaba una buena actividad inhibidora de la agregación de amiloide ß (Aß), 82% a 100 µg/mL. Además, inhibió la beta-secretasa 1 (BACE1) con un valor de CI50 de 19,03 µg/mL. La evaluación de la neuroprotección del extracto acuoso de la planta contra la muerte celular inducida por H2O2 en neuronas PC12 reveló una protección del 84,5% a 1 µg/mL. Cabe señalar que, de acuerdo con nuestros resultados obtenidos de la prueba Morris Water Maze (MWM), el extracto revirtió el déficit de memoria inducido por escopolamina en ratas a concentraciones de 1,5 y 3 mg/kg.

One step production of isomalto-oligosaccharides by engineered Yarrowia lipolytica yeast co-displayed β-amylase and α-transglucosidase / 生物工程学报

Isomalto-oligosaccharides (IMO) have good physiochemical properties and excellent physiological functions to make it widely used in food, medicine, feed, cosmetics and other industries. However, the procedures for industrial production of IMO are complicated. Therefore, it is necessary to develop an economical and easy-to-operate method. The genes encoding for β-amylase and α-transglucosidase were fused and co-displayed on the yeast cell surface of Yarrowia lipolytica which can convert liquefied starch to IMO in one step. The highest IMO purity of 75.3% was obtained using the displayed fusion-enzyme at 50 °C. This method showed potential application in IMO production.

Highly efficient enzymatic preparation of isomalto-oligosaccharides from starch using an enzyme cocktail

Background: Current commercial production of isomalto-oligosaccharides (IMOs) commonly involves a lengthy multistage process with low yields. Results: To improve the process efficiency for production of IMOs, we developed a simple and efficient method by using enzyme cocktails composed of the recombinant Bacillus naganoensis pullulanase produced by Bacillus licheniformis, α-amylase from Bacillus amyloliquefaciens, barley bran ß-amylase, and α-transglucosidase from Aspergillus niger to perform simultaneous saccharification and transglycosylation to process the liquefied starch. After 13 h of reacting time, 49.09% IMOs (calculated from the total amount of isomaltose, isomaltotriose, and panose) were produced. Conclusions: Our method of using an enzyme cocktail for the efficient production of IMOs offers an attractive alternative to the process presently in use.

A degradação do amido da banana depende da ação combinada de α-amilase e ß-amilase em regiões de diferentes graus de cristalinidade do grânulo / The banana starch degradation depends on the combined action of α-amylase and ß-amylase in regions of different degrees of crystallinity of the granules

A banana é considerada um bom modelo de estudo para a transformação amido-sacarose, já que acumula um teor alto de amido durante o desenvolvimento, que é degradado durante o amadurecimento. Já foram detectadas em polpa de banana atividade e proteína relativa a várias enzimas supostamente envolvidas no processo de degradação do amido. Entre elas, a α-amilase, a ß-amilase, a amido fosforilase e as glucano-água-diquinases (GWD). Estas enzimas estão envolvidas em dois processos distintos de degradação de amido em plantas: o dependente da ação inicial da α-amilase e o dependente da fosforilação do grânulo pela GWD e PWD e posterior ação da ß-amilase. A dificuldade do estabelecimento da participação efetiva de cada enzima no processo de degradação do amido está associada a muitos fatores, entre eles a não-correlação entre atividade e real envolvimento em um processo, e a acessibilidade da enzima ao seu substrato. Aliado ao estudo da morfologia do grânulo de amido e suas modificações sofridas durante o processo de degradação que ocorre durante o amadurecimento do fruto, estudos in vitro que simulem a ação da enzima sobre o seu substrato poderiam ser mais efetivos no estabelecimento da real ação de dada enzima sobre o suposto substrato. Tentativas no sentido de obter as proteínas relativa à degradação não foram bem sucedidas. Assim, os ensaios de grânulos de amido isolados versus enzimas foram feitos com α-amilase e ß-amilase comerciais. O grau de fosforilação da amilopectina nas posições Glic-6 e Glic-3 foi determinado, condição necessária para o início da degradação do grânulo pela ß-amilase. Os resultados mostraram que os grânulos de amido isolados de bananas recém colhidas, ou verdes, já estão fosforilados e as enzimas responsáveis por esta fosforilação estão associadas aos grânulos. Após 72 h de incubação dos grânulos de amido com as enzimas hidrolítica, os grânulos foram separados do tampão contendo as enzimas e os produtos de hidrólise. Os sobrenadantes foram analisados por cromatografia líquida acoplada a detector amperométrico e os grânulos por Microscopia Eletrônica de Varredura (MEV) e microscopia de força atômica (MFA). Os resultados mostraram que a α-amilase hidrolisa preferencialmente regiões amorfas dos grânulos, com predominância de amilose, expondo as regiões mais cristalinas dos anéis de crescimento, enquanto que a ß-amilase parece atuar preferencialmente nas regiões cristalinas dos grânulos, degradando os bloquetes, que são formados por amilopectina. Pode-se concluir que ambas as enzimas parecem ser importantes no processo de degradação do amido da banana, com diferentes especificidades

Banana is considered a good model to study the starch-sucrose metabolism, since it accumulates a high starch content during development, which is degraded during fruit ripening. It have been detected in banana pulp some proteins and activities of several enzymes supposedly involved in starch degradation process. Among them, α-amylase, ß-amylase, starch phosphorylase and glucan-water-diquinases (GWD). These enzymes are involved in two separate processes of starch degradation in plants: the initial action of α-amylase dependent, and the starch granule phosphorylation by GWD and PWD enzymes and subsequent action of ß-amylase. The difficulty of establishing the effective participation of each enzyme in the starch degradation process is associated with many factors, including the lack of correlation between real activity and involvement in the process, and accessibility of the enzyme to its substrate. Allied to study the morphology of the starch granule and its modifications suffered during the process of degradation, which occurs during the fruit ripening, in vitro studies that simulate the action of the enzyme on its substrate could be more effective in establishing the real action of a given enzyme on the argued substrate. However, attempts to obtain the proteins related to the degradation process were unsuccessful. Thus, assays of isolated starch granules versus enzymes were made with commercial α-amylase and ß-amylase enzymes. The degree of phosphorylation of amylopectin in the Gluc-6 and Gluc-3 positions was determined, a necessary condition for the start of degradation by ß-amylase enzyme. The results showed that the starch granules isolated from freshly harvested bananas, or green, are already phosphorylated and the enzymes responsible for this phosphorylation is associated with the starch granules surface. After 72 h incubation of the starch granules with the hydrolytic enzymes, the granules were separated from the buffer containing the enzymes and the hydrolysis products. The supernatants were analyzed by liquid chromatography coupled with amperometric detector and the granules were visualized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The results showed that the α-amylase preferentially hydrolyzes amorphous regions of the granule, especially amylose, exposing more crystalline regions of the growth rings, whereas ß-amylase appears to act preferentially on crystalline regions of the granule, degrading blocklets that consist of amylopectin. It can be concluded that both enzymes appear to be important in the banana starch degradation process, with different specificities

Partial characterization of cold active amylases and proteases of Streptomyces sp. from Antarctica

The aim of this study was to isolate novel enzyme-producing bacteria from vegetation samples from East Antarctica and also to characterize them genetically and biochemically in order to establish their phylogeny. The ability to grow at low temperature and to produce amylases and proteases cold-active was also tested. The results of the 16S rRNA gene sequence analysis showed that the 4 Alga rRNA was 100 percent identical to the sequences of Streptomyces sp. rRNA from Norway and from the Solomon Islands. The Streptomyces grew well in submerged system at 20ºC, cells multiplication up to stationary phase being drastically increased after 120 h of submerged cultivation. The beta-amylase production reached a maximum peak after seven days, while alpha-amylase and proteases were performing biosynthesis after nine days of submerged cultivation at 20ºC. Newly Streptomyces were able to produce amylase and proteases in a cold environment. The ability to adapt to low temperature of these enzymes could make them valuable ingredients for detergents, the food industry and bioremediation processes which require low temperatures.

Caracterização funcional do gene maBMY que codifica para uma `BETA´-amilase endereçada a plastídeos e expressa durante o amadurecimento da banana / Functional characterization of maBMY gene that encodes a `BETA´-amylase targeted to plastids and expressed during banana ripening

O amadurecimento dos frutos é um processo caracterizado pela ocorrência de diversas alterações bioquímicas que ocorrem em um curto intervalo de tempo e que são importantes para a qualidade desses alimentos. Na banana uma das características mais importantes é o adoçamento do fruto, que ocorre como resultado da degradação do amido e acúmulo de sacarose. Resultados do nosso grupo apontam a ´BETA` amilase como uma enzima importante no processo de mobilização do amido, o que também é visto em estudos recentes utilizando Arabidopsis thaliana como modelo, os quais mostram que a principal via de degradação do amido transitório presente nas folhas ocorre pela ação da ´BETA`-amilase. Entretanto, em bananas, faltam evidências quanto à funcionalidade de um gene de ´BETA`amilase, parcialmente isolado da polpa do fruto, e que é expresso durante o amadurecimento e que parece ser modulado por hormônios vegetais. Em vista disso, esse trabalho objetivou realizar a caracterização funcional desse gene, a qual permitiu constatar que esse gene codifica, de fato, para uma proteína capaz de ser endereçada aos cloroplastos. Também foi observado que o promotor desse gene contém motivos regulatórios para os mesmos hormônios previamente relacionados com a modulação da expressão desse gene em bananas. Essas novas evidências reforçam a idéia de que o produto desse gene de ´BETA`amilase tem um importante papel no processo de degradação do amido durante o amadurecimento da banana...

Cloning, expression and the characterization of beta-amylase from a Bacillus megaterium WS06 / 生物工程学报

A beta-amylase gene (amyG) was cloned from a Bacillus megaterium WS06 and expressed in the Escherichia coli. Nucleotide sequence anlysis showed the amyG gene is composed of 1638 bp (545 amino acid residues with a Mr of 60.194 kD). The AmyG shows 94.5% sequence homologies with beta-amylase from Bacillus megaterium DSM319 and presents a normal beta-amylase primary structure, constituted by three parts: the N-terminal signal sequence, the catalytic domain and the C-terminal starch binding domains. The deduced amino acid sequence revealed that several highly conserved regions of the glycosylhydrolase family 14. The amyG gene was overexpressed using the pET21a vector and Escherichia coli BL21(DE3). The recombinant enzyme was purified 7.4 fold to electrophoretic homogeneity and had a Mr of 57 kD (by SDS-PAGE). The enzyme was optimally active at pH 7.0 and 60 degrees C and showed stability at the temperature below 60 degrees C. This enzyme efficiently hydrolyzed starch to yield maltose from non-reducing chain ends by exo-cleavage mode.

Change of hydrolase activity in germinating seeds of trxS transgenic barley / 生物工程学报

Genetic modification of barley variety can be an efficient way to improve beer quality. The objective of this study was to understand the effect of trxS gene on hydrolases activities in transgenic and non-transgenic barley seeds. The results showed that alpha-amylase, free beta-amylase and limit dextrinase activity were increased in transgenic seeds in comparison with non-transgenic seeds. Sulfhydryl content of protein in transgenic seeds was also higher than that in non-transgenic seeds, suggesting that trxS gene could express in barley seeds, which opens a new way for breeding new barley varieties to improve beer quality.

Estrutura do grânulo de amido de banana e sua relação com as enzimas que atuam no metabolismo amido-sacarose / Structure of the banana starch granule and its relation with the enzymes that attack in the metabolism starch-sucrose

A banana é considerada um bom modelo para o estudo da transformação amido-sacarose, já que acumula um alto teor de amido durante o desenvolvimento que é rapidamente degradado durante o amadurecimento. Várias enzimas e provavelmente mais de uma via metabólica estão envolvidas neste processo. Com isso, o objetivo deste trabalho foi estudar as características estruturais dos grânulos, bem como, a atuação das enzimas envolvidas em sua degradação. Os grânulos de amidos foram isolados de bananas controle (não tratadas) e submetidas a diferentes tratamentos: etileno, 1-MCP, frutos mantidos a 13'graus'C e frutos tratados com etileno e mantidos a 13'graus'C. Os resultados obtidos mostraram alta atividade de enzimas 'alfa' e 'beta'-amilases ligadas ao grânulo tanto por ensaios in vitro como por géis de eletroforese contendo amilopectina como substrato...

Effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism in seedlings of wheat cultivars.

The effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism (alpha and beta amylases, sucrose phosphate synthase, sucrose synthase, acid and alkaline invertases) in wheat (Triticum aestivum L.) was investigated in the seedlings of drought-sensitive (PBW 343) and drought-tolerant (C 306) cultivars. The water deficit was induced by adding 6% mannitol (water potential -0.815 Mpa) in the growth medium. The water deficit reduced starch content in the shoots of tolerant seedlings as compared to the sensitive ones, but increased sucrose content in the shoots and roots of tolerant seedlings, indicating their protective role during stress conditions. It also decreased the alpha-amylase activity in the endosperm of seedlings of both the cultivars, but increased alpha and beta amylase activities in the shoots of tolerant ones. Sucrose phosphate synthase (SPS) activity showed a significant increase at 6 days of seedling growth (DSG) in the shoots of stressed seedlings of tolerant cultivar. However, SPS activity in the roots of stressed seedlings of sensitive cultivar was very low at 4 DSG and appeared significantly only at day 6. Sucrose synthase (SS) activity was lower in the shoots and roots of stressed seedlings of tolerant cultivar than sensitive ones at early stage of seedling growth. Higher acid invertase activity in the shoots of seedlings of tolerant cultivar appeared to be a unique characteristic of this cultivar for stress tolerance. Alkaline invertase activity, although affected under water deficit conditions, but was too low as compared to acid invertase activity to cause any significant affect on sucrose hydrolysis. In conclusion, higher sucrose content with high SPS and low acid invertase and SS activities in the roots under water deficit conditions could be responsible for drought tolerance of C 306.

Relationship between malt qualities and beta-amylase activity and protein content as affected by timing of nitrogen fertilizer application / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The effects of different timing of N fertilizer application at the same rate on grain beta-amylase activity, protein concentration, weight and malt quality of barley were studied. Grain beta-amylase activity and protein concentration were significantly higher in treatments where all top-dressed N fertilizer was applied at booting stage only or equally applied at two-leaf stage and booting stage than in the treatment where all top-dressed N fertilizer was applied at two-leaf age stage only. On the other hand, grain weight and malt extract decreased with increased N application at booting stage. There were obvious differences between barley varieties and experimental years in the grain and malt quality response to the timing of N fertilizer application. It was found that grain protein concentration was significantly and positively correlated with beta-amylase activity, but significantly and negatively correlated with malt extract and Kolbach index. The effect of grain protein concentration on malt quality was predominant over the effect of grain beta-amylase activity.

Chromium in tannery industry effluent and its effect on plant metabolism and growth.

Different dilution levels of tannery treated effluent and their corresponding concentration of chromium (Cr6+) were studied in a petridish culture experiment on seed germination and seedling growth in radish (Raphanus sativus L). The different concentrations of Cr6+ (2, 5 and 10 ppm) and treated tannery effluent (10, 25 and 50%) showed reduction in seedling growth and related enzymatic activities with increase in concentration of Cr6+ in treatments and effluent both. The low concentration of chromium (2 ppm) and effluent dilution (10%) showed significant growth reduction separately. At this concentration of chromium and effluent dilution chlorophyll content, amylase, catalase and protein contents remained unchanged while with increase in Cr6+ concentration (>2ppm) and effluent dilution (> 10%) in treatments showed growth inhibitory effects.

Bioregulation of carbohydrate metabolism in relation to source-sink operation during grain-filling phase of growth in wheat.

Mobilization of free sugars from vegetative tissues to grain and their transformation to starch in relation to activities of some relevant enzymes during growth and development were investigated in wheat (Triticum aestivum L.). Vegetative tissues, viz. flag-leaf, flag-leaf sheath, nodes and internodes contained high concentration of free sugars from 70 DAS to 18 DPA and that was in the order of accumulation--flag-leaf sheath> flag-leaf and internodes > nodes. In these tissues, major portion of 14C appeared in endogenous sucrose, irrespective of the nature of (U-14C]-sugars supplied. In photosynthetic structures above flag-leaf node, namely peduncle, rachis and bracts, the free sugar make-up was maximum at anthesis (90 DAS). Activity of soluble acid invertase (EC 3.2.1.26) was high in these tissues during early stages of grain growth but reverse was true for soluble neutral invertase (EC 3.2.1.27) activity. In apical and basal portions of grain, free sugars were more or less similarly distributed in concentration. Linear and rapid accumulation of starch in endosperm paralleled with a decline in accumulation of this polymer in pericarp-aleurone. In the latter tissue, the activities of starch hydrolyzing enzymes, i.e alpha- and beta-amylases (3.2.1.1 and 3.2.1.2) were high during initial stages of grain growth. During active grain-filling, alkaline inorganic pyrophosphatase (EC 3.6.1.1) seemed to play a vital role during starch accumulation in endosperm, whereas the involvement of 3-PGA phosphatase (EC 3.1.3.38) was almost confined to pericarp-aleurone. Impairement of ear head photosynthesis by shading depressed starch synthesis (approximately 50%) indicating, thereby, the significant role of current photosynthates during grain-filling. The results suggested that grain growth in wheat was influenced by an efficient operation of source as well as regulatory factors, including enzymes, constituting intrinsic potential of grain sink.