Avaliação da qualidade do mel e atividade da enzima invertase em Apis mellifera L. africanizadas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Método de obtenção de suporte a ser utiilizado na imobilizaçao de enzima invertase e a imobilização de enzima invertase

Onde o método de obtenção de suporte a ser utilizado na imobilização de enzima invertase compreende as etapas de: extração das substâncias húmicas existentes no solo.

Characterization of the co-purified invertase and beta-glucosidase of a multifunctional extract from Aspergillus terreus

The filamentous fungus Aspergillus terreus secretes both invertase and beta-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a beta-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0-6.0 and 55-65 A degrees C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0-10.0 and maintained full activity for up to 1 h at 55 A degrees C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn2+ (161 %), Co2+ (68 %) and Mg2+ (61 %) and was inhibited by Al3+, Ag+, Fe2+ and Fe3+. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K-M = 22 mM). However, in the presence of Mn2+, the apparent affinity and V-max for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V-max was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and beta-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.

Uso de invertase imobilizada em pó de sabugo de milho para produção de açúcar invertido

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Invertase from a Candida stellata strain isolated from grape: production and physico-chemical characterization

Invertases are enzymes which hydrolyze the sucrose and are widely employed in food and pharmaceutical industries. In this work, the screening of autochthonous grape yeasts from Brazil was carried out in order to investigate their invertase production potential. Yeasts belonging to Saccharomyces, Hanseniaspora, Sporidiobolus, Issatchenkia, Candida, Cryptococcus and Pichia genera were analyzed by submerged fermentation (SbmF) using sucrose as substrate. Among them, Candida stellata strain (N5 strain) was selected as the best producer (10.6 U/ml after 48 hours of SbmF). This invertase showed optimal activity at pH 3.0 and 55°C, demonstrating appropriate characters for application in several industrial processes, which includes high temperatures and acid pHs. In addition, this invertase extract presented tolerance to low concentrations of ethanol, suggesting that it could also be suitable for application at the beginning of alcoholic fermentation. These data provide promising prospects of the use of this new invertase in food and ethanol industry.

A Nicotiana attenuata cell wall invertase inhibitor (NaCWII) reduces growth and increases secondary metabolite biosynthesis in herbivore-attacked plants

Plant invertases are sucrolytic enzymes that are essential for the regulation of carbohydrate metabolism and source–sink relationships. While their activity has been well documented during abiotic and biotic stresses, the role of proteinaceous invertase inhibitors in regulating these changes is unknown. Here, we identify a putative Nicotiana attenuata cell wall invertase inhibitor (NaCWII) which is strongly up-regulated in a jasmonate (JA)-dependent manner following simulated attack by the specialist herbivore Manduca sexta. To understand the role of NaCWII in planta, we silenced its expression by RNA interference and measured changes in primary and secondary metabolism and plant growth following simulated herbivory. NaCWII-silenced plants displayed a stronger depletion of carbohydrates and a reduced capacity to increase secondary metabolite pools relative to their empty vector control counterparts. This coincided with the attenuation of herbivore-induced CWI inhibition and growth suppression characteristic of wild-type plants. Together our findings suggest that NaCWII may act as a regulatory switch located downstream of JA accumulation which fine-tunes the plant's balance between growth and defense metabolism under herbivore attack. Although carbohydrates are not typically viewed as key factors in plant growth and defense, our study shows that interfering with their catabolism strongly influences plant responses to herbivory.

Cloning of a Tobacco Apoplasmic Invertase Inhibitor1 : Proof of Function of the Recombinant Protein and Expression Analysis during Plant Development

Higher plants express several isoforms of vacuolar and cell wall invertases (CWI), some of which are inactivated by inhibitory proteins at certain stages of plant development. We have purified an apoplasmic inhibitor (INH) of tobacco (Nicotiana tabacum) CWI to homogeneity. Based on sequences from tryptic fragments, we have isolated a full-length INH-encoding cDNA clone (Nt-inh1) via a reverse transcriptase-polymerase chain reaction. Southern-blot analysis revealed that INH is encoded by a single- or low-copy gene. Comparison with expressed sequence tag clones from Arabidopsis thaliana and Citrus unshiu indicated the presence of Nt-inh1-related proteins in other plants. The recombinant Nt-inh1-encoded protein inhibits CWI from tobacco and Chenopodium rubrum suspension-cultured cells and vacuolar invertase from tomato (Lycopersicon esculentum) fruit, whereas yeast invertase is not affected. However, only in the homologous system is the inhibition modulated by the concentration of Suc as previously shown for INH isolated from tobacco cells. Highly specific binding of INH to CWI could be shown by affinity chromatography of a total cell wall protein fraction on immobilized recombinant Nt-inh1 protein. RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate.

Apoplastic Sugars, Fructans, Fructan Exohydrolase, and Invertase in Winter Oat: Responses to Second-Phase Cold Hardening

Changes in apoplastic carbohydrate concentrations and activities of carbohydrate-degrading enzymes were determined in crown tissues of oat (Avena sativa L., cv Wintok) during cold hardening. During second-phase hardening (−3°C for 3 d) levels of fructan, sucrose, glucose, and fructose in the apoplast increased significantly above that in nonhardened and first-phase-hardened plants. The extent of the increase in apoplastic fructan during second-phase hardening varied with the degree of fructan polymerization (DP) (e.g. DP3 and DP4 increased to a greater extent than DP7 and DP > 7). Activities of invertase and fructan exohydrolase in the crown apoplast increased approximately 4-fold over nonhardened and first-phase-hardened plants. Apoplastic fluid extracted from nonhardened, first-phase-hardened, and second-phase-hardened crown tissues had low levels, of symplastic contamination, as determined by malate dehydrogenase activity. The significance of these results in relation to increases in freezing tolerance from second-phase hardening is discussed.

Split invertase polypeptides form functional complexes in the yeast periplasm in vivo.

The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.

Production of beta-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of beta-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of beta-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular beta-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 degrees C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 degrees C. beta-Fructofuranosidase activity was slightly activated by Cu(2+), Mn(2+), Mg(2+), and Na(+) at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K(d) values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively. (C) 2008 Elsevier Ltd. All rights reserved.

Biochemical properties of an extracellular beta-D-fructofuranosidase II produced by Aspergillus phoenicis under Solid-Sate Fermentation using soy bran as substrate

The filamentous fungus A. phoenicis produced high levels of beta-D-fructofuranosidase (FFase) when grown for 72 hrs under Solid-State Fermentation (SSF), using soy bran moistened with tap water (1:0.5 w/v) as substrate/carbon source. Two isoforms (I and II) were obtained, and FFase II was purified 18-fold to apparent homogeneity with 14% recovery. The native molecular mass of the glycoprotein (12% of carbohydrate content) was 158.5 kDa with two subunits of 85 kDa estimated by SDS-PAGE. Optima of temperature and pH were 55 degrees C and 4.5. The enzyme was stable for more than 1 hr at 50 degrees C and was also stable in a pH range from 7.0 to 8.0. FFase II retained 80% of activity after storage at 4 degrees C by 200 hrs. Dichroism analysis showed the presence of random and beta-sheet structure. A. phoenicis FFase II was activated by Mn(2+), Mg(2+) and Co(2+), and inhibited by Cu(2+), Hg(2+) and EDTA. The enzyme hydrolyzed sucrose, inulin and raffinose. K(d) and V(max) values were 18 mM and 189 U/mg protein using sucrose as substrate.

A fructan: fructan fructosyltransferase activity from Lolium rigidum

Fructan:fructan fructosyltransferase (FFT) activity was purified about 300-fold from leaves of Lolium rigidura Gaudin by a combination of affinity chromatography, gel filtration, anion exchange and isoelectric focusing. The FFT activity was free of sucrose:sucrose fructosyltransferase and invertase activities. It had an apparent pI of 4.7 as determined by isoelectric focusing, and a molecular mass of about 50000 (gel filtration). The FFT activity utilized the trisaccharides 1-kestose and 6(G)-kestose as sole substrates, but was not able to use 6-kestose as sole substrate. The FFT activity was not saturated when assayed at concentrations of 1-kestose, 6(G)-kestose or (1,1)-kestotetraose of up to 400 mM The rate of reaction of the FFT activity was most rapid when assayed with 1-kestose and was less rapid when assayed with 6(G)-kestose, (1,1)-kestotetraose or (1,1,1)-kestopentaose. The FFT activity when assayed at a relatively high concentration of enzyme activity (approximately equivalent to about half the activity in crude extracts per gram fresh mass) did not synthesize fructan of degree of polymerization > 6, even during extended assays of up to 10 h. When assayed with a combination of 1-kestose and uniformly labelled [C-14]sucrose as substrates, the major reaction was the transfer of a fructosyl residue from 1-kestose to sucrose resulting in the re-synthesis of 1-kestose. Tetrasaccharide and 6(G)-kestose were also synthesized. When assayed with 6(G)-kestose and [C-14]sucrose as substrates, the major reaction of the FFT activity was the synthesis of tetrasaccharide. However, some synthesis of 1-kestose and re-synthesis of 6(G)-kestose also occurred. When 6, kestose was the sole substrate for the FFT activity, synthesis of tetrasaccharide was 2.7 to 3.4-fold slower than when 1-kestose was used as the sole substrate. Owing to differences in the fructan:sucrose fructosyltransferase activity of the FFT with each of the trisaccharides, net synthesis of tetrasaccharide by the FFT was altered significantly in the presence of sucrose. The magnitude of this effect depended on the concentration of the trisaccharides. In the presence of sucrose, 6(G)-kestose could be a substrate of equivalent importance to 1-kestose for synthesis of tetrasaccharide.

Synthesis of fructans by partially purified fructosyltransferase activities from Lolium rigidum

Sucrose:sucrose fructosyltransferase (SST) activity was partially purified from whole shoots of Lolium rigidum by a combination of affinity chromatography, gel filtration and anion-exchange chromatography. The SST activity co-eluted with some fructan:fructan fructosyltransferase (FFT) and invertase activities and consequently the partially purified preparation was termed the fructosyltransferase (FT) preparation. The SST-like activity in the FT preparation was purified 214-fold and had an apparent molecular mass of 84 000. The FT preparation contained several peptides with an apparent pI of 4.6-4.7. When assayed with sucrose concentrations up to 600 mM, the FT preparation synthesized 1-kestose at all concentrations, and synthesized 6-kestose at concentrations of 150 mM and greater. The K-m of 1-kestose production was 0.2 M. When the FT preparation was assayed at a concentration of activity approximately half that measured in fresh tissue with 100 mM sucrose, 1-kestose, or 6(G)-kestose as substrates, fructans of degree of polymerization (DP) less than or equal to 5 were synthesized. A partially purified FFT activity, free of SST and invertase activities, which synthesized beta-2,1-glycosidic linked oligofructans of DP less than or equal to 6, was combined in vitro with the FT preparation (FFT-FT preparation) to give a ratio of SST:FFT activities similar to that measured in crude enzyme extracts from L. rigidum. The FFT-FT preparation synthesized oligofructans when assayed with 100 mM concentrations of sucrose, 1-kestose or 6(G)-kestose, but was not able to synthesize fructans of DP greater than or equal to 6 even after extended assays of up to 10 h. The FFT-FT preparation was also assayed with 100 mM sucrose with small amounts of concentrated sucrose added periodically during the assay to maintain the substrate concentration. In this assay, the FFT-FT preparation synthesized fructans up to an apparent DP of 17 or greater. The fructans of DP greater than or equal to 6 synthesized in the assay appeared to form two molecular series containing both beta-2,1- and beta-2,6-glycosidic linked fructosyl residues with terminal or internal glucosyl residues. The apparent rate of SST activity in the assay of the FFT-FT preparation was greater than that measured in a similar assay of the FT preparation alone which did not result in fructans with DP greater than or equal to 6. It was concluded that the FFT-FT preparation, when assayed with a continual supply of sucrose, contained a factor which promoted synthesis of fructans of DP greater than or equal to 6 and synthesis of beta-2,B-glycosidic linkages between fructosyl residues.

Estudos preliminares sobre as invertases de cafeeiros atacados por Hemileia vastatrix

No presente trabalho, que o autor considera exploratório, foram estudadas as invertases presentes em folhas de cafeeiros das variedades Geisha (SH1 SH5) e Kent (SH2 SH5), inoculados com as raças III (n1 n5) e XXIV (n2 n4) de Hemileia vastatrix. Devido ao genótipo do material utilizado as reações são opostas, ou seja, o cafeeiro Kent é resistente à raça III e suscetível à raça XXIV e o Geisha o contrário. Em cada par de folhas, uma foi inoculada e outra mantida como controle e a determinação das invertases foi feita aos 6 e 16 dias após a inoculação. Os resultados preliminares obtidos mostraram que, independente da variedade de cafeeiro ou da raça de Hemileia vastatrix, nas combinações suscetíveis não houve alteração das invertases presentes, iguais às de plantas sadias, com o máximo de atividade em pH 4,5. Já nas combinações resistentes no 6.º dia após a inoculação houve o aparecimento de uma "invertase ácida pH 4,0", ausente tanto nas plantas sadias como nas combinações suscetíveis das mesmas variedades. O aparecimento da "invertase ácida pH 4,0" está relacionado com a diminuição do teor de sacarose nas folhas. Quanto ao significado desta "invertase ácida pH 4,0" na patogenese da ferrugem do cafeeiro, o autor aventa 3 hipóteses que poderiam ocorrer isolada ou simultaneamente. 1. A sacarose seria desviada para a formação de defesa, tais como fenóis, fitoalexinas e outras. 2. A sacarose seria desviada para outras partes da planta, num processo fisiológico semelhante ao das folhas em senectude. 3. Haveria um aumento da respiração e metabolismo dos tecidos doentes, com o implícito consumo da energia armazenada.