Starch degradation in the bean fruit pericarp is characterized by an increase in maltose metabolism.

The bean fruit pericarp accumulates a significant amount of starch, which starts to be degraded 20 days after anthesis (DAA) when seed growth becomes exponential. This period is also characterized by the progressive senescence of the fruit pericarp. However, the chloroplasts maintained their integrity, indicating that starch degradation is a compartmentalized process. The process coincided with a transient increase in maltose and sucrose levels, suggesting that ß-amylase is responsible for starch degradation. Starch degradation in the bean fruit pericarp is also characterized by a large increase in starch phosphorylation, as well as in the activities of cytosolic disproportionating enzyme 2 (DPE2, EC 2.4.1.25) and glucan phosphorylase (PHO2, EC 2.4.1.1). This suggests that the rate of starch degradation in the bean fruit pericarp 20 DAA is dependent on the transformation of starch to a better substrate for ß-amylase and the increase in the rate of cytosolic metabolism of maltose.

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.

High polymorphism in Est-SSR loci for cellulose synthase and ß-amylase of sugarcane varieties (Saccharum spp.) used by the industrial sector for ethanol production.

High and low polymorphisms in simple sequence repeats of expressed sequence tag (EST-SSR) for specific proteins and enzymes, such as ß-amylase, cellulose synthase, xyloglucan endotransglucosylase, fructose 1,6-bisphosphate aldolase, and fructose 1,6-bisphosphatase, were used to illustrate the genetic divergence within and between varieties of sugarcane (Saccharum spp.) and to guide the technological paths to optimize ethanol production from lignocellulose biomass. The varieties RB72454, RB867515, RB92579, and SP813250 on the second stage of cutting, all grown in the state of Paraná (PR), and the varieties RB92579 and SP813250 cultured in the PR state and in Northeastern Brazil, state of Pernambuco (PE), were analyzed using five EST-SSR primers for EstC66, EstC67, EstC68, EstC69, and EstC91 loci. Genetic divergence was evident in the EstC67 and EstC69 loci for ß-amylase and cellulose synthase, respectively, among the four sugarcane varieties. An extremely high level of genetic differentiation was also detected in the EstC67 locus from the RB82579 and SP813250 varieties cultured in the PR and PE states. High polymorphism in SSR of the cellulose synthase locus may explain the high variability of substrates used in pretreatment and enzymatic hydrolysis processes, which has been an obstacle to effective industrial adaptations.

Plantain and banana starches: granule structural characteristics explain the differences in their starch degradation patterns.

Different banana cultivars were used to investigate the influences of starch granule structure and hydrolases on degradation. The highest degrees of starch degradation were observed in dessert bananas during ripening. Scanning electron microscopy images revealed smooth granule surface in the green stage in all cultivars, except for Mysore. The small and round granules were preferentially degraded in all of the cultivars. Terra demonstrated a higher degree of crystallinity and a short amylopectin chain length distribution, resulting in high starch content in the ripe stage. Amylose content and the crystallinity index were more strongly correlated than the distribution of amylopectin branch chain lengths in banana starches. α- and ß-amylase activities were found in both forms, soluble in the pulp and associated with the starch granule. Starch-phosphorylase was not found in Mysore. On the basis of the profile of α-amylase in vitro digestion and the structural characteristics, it could be concluded that the starch of plantains has an arrangement of granules more resistant to enzymes than the starch of dessert bananas.

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...

Mango starch degradation. II. The binding of alpha-amylase and beta-amylase to the starch granule.

During mango ripening, soluble sugars that account for mango sweetening are accumulated through carbon supplied by both photosynthesis and starch degradation. The cultivar Keitt has a characteristic dependence on sugar accumulation during starch degradation, which takes place during ripening, only a few days after detachment from the tree. Most knowledge about starch degradation is based on seeds and leaves currently used as models. However, information about the mango fruit is scarce. This work presents the evaluation of alpha- and beta-amylases in the starch granule surface during fruit development and ripening. Extractable proteins were assayed for amylase activity and detected by immunofluorescence microscopy and correlated to gene expression. The results suggest that both amylases are involved in starch degradation during mango ripening, probably under the dependence of another signal triggered by the detachment from the mother-plant.

Mango starch degradation. I. A microscopic view of the granule during ripening.

The starch content of unripe mango Keitt is around 7% (FW), and it is converted to soluble sugars during the ripening of the detached fruit. Despite the importance of starch-to-soluble sugar metabolism for mango quality, little literature is found on this subject and none concerning the physical aspects of starch degradation. This manuscript presents some changes in the physical aspects of the starch granule during ripening, as analyzed by light microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM). According to the analysis, unripe Keitt-mango-starch being spherical in shape and measuring around 15 microm, has A-type X-ray diffraction pattern with a degree of crystallinity around 21% with slight changes after 8 days of ripening. AFM images of the surface of the granules showed ultra microstructures, which are in agreement with a blocklet-based organization of the granules. The AFM-contrast image of growing layers covering the granule showed fibril-like structures, having 20 nm in diameter, transversally connecting the layer to the granule. The appearance of the partially degraded granules and the pattern of degradation were similar to those observed as a result of amylase activity, suggesting a hydrolytic pathway for the degradation of starch from mango cultivar Keitt. These results provide clues to a better understanding of starch degradation in fruits.

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...

Relationship between grain yield, osmotic adjustment and benzoxazinone content in Triticum aestivum L. cultivars.

Fifteen wheat genotypes were grown under water deficit to ascertain the role of osmotic adjustment (OA) and the concentration of benzoxazinones in sustaining grain yield. A positive correlation between osmotic adjustment capacity and yield was observed in wheat genotypes cultivated under field conditions. The weight gain of plants exposed to drought was in agreement with the OA values (R(2) = 0.93). However, when wheat plants were infested by cereal aphids, this correlation was not found. The benzoxazinones 2,4-dihydroxy-1,4-benzoxa-zin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4 benzoxazin-3-one (DIMBOA) are defensive secondary metabolites present in wheat and others cereals. The content of these compounds varied in wheat genotypes and increased with drought and aphid infestation. A positive correlation between weight gain of irrigated-infested plants and drought-infested plants and the contents of benzoxazinones was observed. These results suggest that plants with better OA capacity and high benzoxazinone content should have better field yields.

Phenylboronate-chitosan resins for adsorption of beta-amylase from soybean extracts.

Isolation and purification of bioproducts from crude extracts can be obtained by affinity methods based on reversible binding of a specific molecule to ligand immobilized in a porous matrix. In the present work, nicrospheres based on chitosan matrix, which incorporated aminophenylboronic acid as a derivative, were prepared and characterized, aimed at developing a beta-amylase adsorption process. Kinetic curves and adsorption isotheriru of the crude extracts as well as the breakthrough curves for a frontal chromatographic separation method of a commercial sample of beta-amylase from soybean are presented. These results were compared to similar data obtained with a comercial microspheres gel based-on agarose.

Amylolytic activity in fruits: comparison of different substrates and methods using banana as model.

Several methodologies have been developed for cereal amylolytic activity estimation, but there is lack of information about the application of these methods for fruits. Mature green banana fruit can achieve 20% of starch content which is degraded during the ripening period in a complex process involving alpha- and beta-amylases and alpha-1,4 and alpha-1,6-glucosidases, besides phosphorylases that can compete for the same substrates. Methods used to determine total hydrolytic activity and individual activity of enzymes involved in starch breakdown were compared for banana extracts in several ripening stages. Total hydrolytic activity was measured by DNS and iodometric methods. Endoamylolytic activity on amylose-azure substrate was also evaluated. BPNPG7 and PNPG5 chromogenic substrates were used for alpha- and beta-amylase activities, respectively. The results showed that methods that depend on the use of thermal treatment or on inhibitors to inactivate one of the enzymes were not adequate. The use of p-nitrophenol derivatives seemed to be the most specific, reproducible, and easiest method employed for single alpha- and beta-amylases activities determination in complex tissues. The DNS and iodometric methods can be used only for initial screenings of total hydrolytic activity, because the nonspecific substrate used in these procedures allows the action of more than one enzyme simultaneously.

Inhibition of beta-amylase activity, starch degradation and sucrose formation by indole-3-acetic acid during banana ripening.

In order to observe the effect of indole-3-acetic acid (IAA) on carbohydrate metabolism, unripe banana (Musa acuminata AAA, cv. Nanicão) slices were infiltrated with the hormone and left to ripen under controlled conditions. The climacteric respiration burst was reduced by the action of IAA, and starch degradation and sucrose formation were delayed. Sucrose synthase (SuSy; EC 2.4.1.13) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) activities and transcript levels were not affected, indicating that prevention of sucrose accumulation was not related to sucrose-metabolizing enzymes. Impairment of sucrose synthesis could be a consequence of lack of substrate, since starch degradation was inhibited. The increase in activity and transcript level of beta-amylase was delayed, indicating that this enzyme could be important in starch-to-sucrose metabolism in bananas and that it might be, at least partially, controlled at the transcriptional level. This is the first report showing that IAA can delay starch degradation, possibly affecting the activity of hydrolytic enzymes such as beta-amylase (EC 3.2.1.2).

Advances in microbial amylases.

This review makes a comprehensive survey of microbial amylases, i.e. alpha-amylase, beta-amylase and glucoamylase. Amylases are among the most important enzymes and are of great significance in present-day biotechnology. Although they can be derived from several sources, such as plants, animals and micro-organisms, the enzymes from microbial sources generally meet industrial demands. Microbial amylases could be potentially useful in the pharmaceutical and fine-chemical industries if enzymes with suitable properties could be prepared. With the advent of new frontiers in biotechnology, the spectrum of amylase application has widened in many other fields, such as clinical, medicinal and analytical chemistries, as well as their widespread application in starch saccharification and in the textile, food, brewing and distilling industries. In this review, after a brief description of the sources of amylases, we discuss the molecular biology of amylases, describing structures, cloning, sequences, and protoplast fusion and mutagenesis. This is followed by sections on their production and finally the properties of various amylases.

Glycogen brain branching enzyme.

Rat brain glycogen branching enzyme was partially purified in order to elucidate its mechanism of action. The alpha1,4-alpha1,6-glucan polysaccharide was synthesized using rat brain branching enzyme under two different elongation conditions: Glc-1-P and phosphorylase or UDP-Glc and glycogen synthase. The products obtained demonstrated that the cpolysaccharides synthesized (pattern of the spectra obtained in the presence of Krisman's reagent, lambda max, parameter A and R, % beta-amylolysis and degree of branching) under different incubation times are nearly constant. These results imply that the degree of branching of a polysaccharide depends only on the enzyme specificity.