RESUMO
At the end of the summer season, grapevine buds (Vitis vinifera L) grown in temperate climates enter a state of winter recess or endodormancy (ED), which is induced by the shortening of the photoperiod, and during this period, the buds accumulate sucrose. In this study, we investigated whether the shortening of the photoperiod regulates the accumulation of sucrose in the buds in the same way as it regulates its entry into the ED. Because sucrose accumulation is regulated by genes that control its transport and degradation, the effect of the SD photoperiod and the transition of buds from paradormancy (PD) to ED on the expression of sucrose transporter (VvSUTs) and invertase genes (VvINVs) was studied. To analyze the possible role of sucrose during ED development, its effect on bud swelling and sprouting was studied on dormant and nondormant buds under forced growth conditions. The results showed that the SD photoperiod upregulates the expression of the VvSUT genes and downregulates that of the VvINV genes in grapevine buds. Additionally, during the transition of buds from PD to ED, the sucrose content increased, the expression of the VvINV genes decreased, and the expression of the VvSUT genes did not change significantly. Sucrose delayed bud swelling and sprouting when applied to dormant buds but had no effect when applied to nondormant buds. Therefore, we concluded that ED development and sucrose accumulation were synchronized events induced by the SD photoperiod and that a sucrose peak marks the end of ED development in grapevine buds.
Assuntos
Fotoperíodo , Vitis , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sacarose/metabolismo , Vitis/metabolismo , beta-Frutofuranosidase/metabolismoRESUMO
AIMS: The objective of this study was to determine the best conditions to produce invertase by Cunninghamella echinulata PA3S12MM and to immobilize and apply the enzyme. METHODS AND RESULTS: The maximum production was verified in 8 days of cultivation at 28°C supplemented with 10 g L-1 apple peel, reaching 1054.85 U ml-1 . The invertase was purified from the DEAE-Sephadex column. The derivative immobilized in alginate-gelatin-calcium phosphate showed reusability >50% for 19 cycles. The derivative immobilized in glutaraldehyde-chitosan showed greater thermostability and at a different pH. The hydrolysis of 15 ml of sucrose 500 g L-1 in a fixed bed reactor (total volume of 31 ml) produced 24.44 µmol min-1 of glucose and fructose at a residence time of 30 min and a conversion factor of 0.5. CONCLUSIONS: The new wild strain C. echinulata PA3S12MM presents high invertase production in medium supplemented with an agro-industrial residue and the immobilized enzyme showed high thermal stability and resistance at a different pH. SIGNIFICANCE AND IMPACT OF THE STUDY: The fungus C. echinulata PA3S12MM is an excellent producer of invertases in Vogel medium supplemented with apple peel. The enzyme is promising for industrial application since it has good performance in reusability and inverted sugar production.
Assuntos
Cunninghamella , beta-Frutofuranosidase , Cunninghamella/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas , Frutose , Concentração de Íons de Hidrogênio , Temperatura , beta-Frutofuranosidase/metabolismoRESUMO
Yeasts involved in the spontaneous fermentation of traditional beverages like chicha (indigenous Andean beer) may have the potential to be used as starter cultures to improve the quality and microbiological safety of these products, but also as non-conventional alternatives to other food alcoholic fermentations. In this research, we isolated, identified and characterised yeast strains from four Ecuadorian chichas made by using four different raw materials: rice (RC), oat (OC), grape (GC) and a mixture of seven corn varieties (yamor, YC). Finally, 254 yeast isolates were obtained and identified by molecular methods. Eleven yeast genera and 16 yeast species were identified with relatively few isolates belonging to Saccharomyces cerevisiae (9.1% belonging to 6 strains) and Torulaspora delbrueckii (18.6% belonging to 2 strains). In order to select good candidates for fermentative starter production, different analyses were performed. The results of the stress response tests showed a wide variability between species and strains, and identified some yeasts displaying high stress tolerance, similarly to commercial wine strains. Amylase production was screened as being indicative of the capacity to degrade and ferment starch-rich substrates. A Cryptococcus sp. isolate showed the highest amylase activity. The growth rate and fermentative capacity in molasses medium was measured for three S. cerevisiae, T. delbrueckii and Candida sp. strains as tests for yield and performance in biomass production. Based on their excellent behaviour, three S. cerevisiae strains and one T. delbrueckii strain were selected for further analyses, including dehydration tolerance and invertase activity as additional desired traits for chicha starters. All the S. cerevisiae strains exhibited high invertase activity and one also displayed high resistance to dehydration. The yeasts selected in this study can thus be suitably used as dry starters for the microbiologically controlled production of traditional beverages, and also for other alcoholic fermentations.
Assuntos
Cerveja/microbiologia , Fermentação/fisiologia , Saccharomyces cerevisiae/metabolismo , Torulaspora/metabolismo , beta-Frutofuranosidase/metabolismo , Avena/microbiologia , Equador , Indústria Alimentícia , Oryza/microbiologia , Saccharomyces cerevisiae/isolamento & purificação , Torulaspora/isolamento & purificação , Vitis/microbiologia , Vinho/microbiologia , Leveduras/classificação , Leveduras/isolamento & purificação , Leveduras/metabolismo , Zea mays/microbiologiaRESUMO
Eight different Aspergillus strains were tested for their ability to produce ß-fructofuranosidase (FFase) by Solid-State Fermentation. The Aspergillus tamarii URM4634 strain was selected as the most performant and tested on six different agroindustrial by-products. Soy, wheat and oat brans, which allowed for the highest hydrolytic (UH) and transfructosylating (UTF) activities, were tested individually or in mixtures according to a simplex-centroid mixture design in order to investigate their effects on FFase production at different times. The best results in terms of both enzyme activities were obtained with only soy bran. The influence of substrate, moisture and sucrose levels on FFase production was evaluated, and the highest UH and UTF activities were 229.43 ± 4.88 and 66.93 ± 3.02 U/mL, respectively. The obtained results indicate that A. tamarii FFase may be a biocatalyst with great potential for industrial applications such as sugar inversion and fructo-oligosaccharides production.
Assuntos
Aspergillus/enzimologia , beta-Frutofuranosidase/metabolismo , Aspergillus/crescimento & desenvolvimento , Fermentação , Frutose/metabolismo , Glucose/metabolismo , Hidrólise , Oligossacarídeos/metabolismo , Especificidade por SubstratoRESUMO
Aspergillus thermomutatus produces an extracellular ß-D-fructofuranosidase when cultured in Khanna medium with sucrose as additional carbon source at 30°C under agitation for 72 hr. Addition of glucose and fructose in the culture medium affected the production of the enzyme negatively. The optimum hydrolytic activity was achieved at 60°C and pH 5.0, with half-life (T50) of 30 hr at 50°C and 62% of its activity maintained at pH 5.0 for 48 hr. The extracellular extract containing ß-D-fructofuranosidase was effective in producing fructooligosaccharides (FOS), mainly 1-kestose. The highest concentration of FOS was obtained at 30°C and 60°C, indicating the existence of at least two enzymes with transfructosylating activity. At 30°C, the maximal FOS concentration was obtained from 48 to 72 hr, while at 60°C, it was achieved only at 72 hr. The best production of FOS (86.7 g/L) was obtained using 500 g/L sucrose as substrate. PRACTICAL APPLICATION: Fructooligosaccharides (FOS) are linear oligomers of fructose units with important applications in the food industry as sweetening agents and biopreservatives. Due to the presence of ß-glycosidic bonds, they cannot be hydrolyzed by human enzymes, allowing the use of FOS-containing products by diabetics. FOS used in the preparation of dairy products imparts humectancy to soft baked products, lowers the freezing point of frozen desserts, provides crispness to low-fat cookies, and provides many other advantages. Diets containing FOS can reduce the levels of triglycerides and cholesterol and improve the absorption of ions, such as Ca2+ and Mg2+ . FOS also exhibit bifidogenic effect on Bifidobacterium and Lactobacillus strains in the colon. Industrially, FOS is produced during the transfructosylation reaction of sucrose catalyzed by ß-D-fructofuranosidase. Identifying new sources of ß-D-fructofuranosidase is an important challenge to meet its industrial demand.
Assuntos
Aspergillus/enzimologia , Oligossacarídeos/química , Oligossacarídeos/metabolismo , beta-Frutofuranosidase/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
This study reports on the biochemical characterization as well as the kinetic and thermodynamic study of Aspergillus tamarii URM4634 ß-fructofuranosidase (FFase) with transfructosylating activity. Conditions for FFase activity were optimized by means of a central composite rotational design using pH and temperature as the independent variables, while residual activity tests carried out in the temperature range of 45-65°C enabled us to investigate FFase thermostability and estimate the kinetic and thermodynamic parameters of enzyme denaturation. Optimal conditions for sucrose hydrolysis and fructosyl transfer catalyzed by crude FFase were 50°C, and pH 6.0 and 7.4, respectively. The thermodynamic properties of irreversible enzyme inactivation were found to be activation energy of 293.1 kJ mol-1 , and activation enthalpy, entropy, and Gibbs free energy in the ranges 290.3-290.4 kJ mol-1 , 568.7-571.0 J mol-1 K-1 , and 97.9-108.8 kJ mol-1 , respectively. The results obtained in this study point out satisfactory enzyme activity and thermostability at temperatures commonly used for industrial fructo-oligosaccharide (FOS) synthesis; therefore, this novel FFase appears to be a promising biocatalyst with great potential for long-term FOS synthesis and invert sugar production. To the best of our knowledge, this is the first report on kinetic and thermodynamic parameters of an A. tamarii FFase.
Assuntos
Aspergillus/enzimologia , Frutose/metabolismo , Termodinâmica , beta-Frutofuranosidase/metabolismo , Concentração de Íons de Hidrogênio , Cinética , TemperaturaRESUMO
ß-fructofuranosidase (invertase) and ß-D-fructosyltransferase (FTase) are enzymes used in industrial processes to hydrolyze sucrose aiming to produce inverted sugar syrup or fructooligosaccharides. In this work, a black Aspergillus sp. PC-4 was selected among six filamentous fungi isolated from canned peach syrup which were initially screened for invertase production. Cultivations with pure carbon sources showed that invertase and FTase were produced from glucose and sucrose, but high levels were also obtained from raffinose and inulin. Pineapple crown was the best complex carbon source for invertase (6.71 U/mL after 3 days of cultivation) and FTase production (14.60 U/mL after 5 days of cultivation). Yeast extract and ammonium chloride nitrogen sources provided higher production of invertase (6.80 U/mL and 6.30 U/mL, respectively), whereas ammonium nitrate and soybean protein were the best nitrogen sources for FTase production (24.00 U/mL and 24.90 U/mL, respectively). Fermentation parameters for invertase using yeast extract were Y P/S = 536.85 U/g and P P = 1.49 U/g/h. FTase production showed values of Y P/S = 2,627.93 U/g and P P = 4.4 U/h using soybean protein. The screening for best culture conditions showed an increase of invertase production values by 5.10-fold after 96 h cultivation compared to initial experiments (fungi bioprospection), while FTase production increased by 14.60-fold (44.40 U/mL) after 168 h cultivation. A. carbonarius PC-4 is a new promising strain for invertase and FTase production from low cost carbon sources, whose synthesized enzymes are suitable for the production of inverted sugar, fructose syrups, and fructooligosaccharides.
Assuntos
Aspergillus/enzimologia , Alimentos em Conserva/microbiologia , Proteínas Fúngicas/metabolismo , Hexosiltransferases/metabolismo , beta-Frutofuranosidase/metabolismo , Aspergillus/efeitos dos fármacos , Carbono/metabolismo , Carbono/farmacologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Fermentação , Proteínas Fúngicas/isolamento & purificação , Hexosiltransferases/isolamento & purificação , Xarope de Milho Rico em Frutose , Microbiologia Industrial/métodos , Nitrogênio/metabolismo , Nitrogênio/farmacologia , Prunus persica/química , Prunus persica/microbiologia , beta-Frutofuranosidase/isolamento & purificaçãoRESUMO
Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like ß-fructofuranosidase. Gene sacA from Bacillus licheniformis ATCC 14580 codifies a sucrose-6-phosphate hydrolase, a ß-fructofuranosidase (E.C. 3.1.2.26, protein BlsacA), which has no crystallographic structure available. In this study, we report the results from numerous biochemical and biophysical techniques applied to the investigation of BlsacA in solution. BlsacA was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Results showed that the optimum activity of BlsacA occurred at 30 °C around neutrality (pH 6.0-7.5) with a tendency to alkalinity. Circular dichroism spectrum confirmed that BlsacA contains elements of a ß-sheet secondary structure at the optimum pH range and the maintenance of these elements is related to BlsacA enzymatic stability. Dynamic light scattering and small-angle X-ray scattering measurements showed that BlsacA forms stable and elongated homodimers which displays negligible flexibility in solution at optimum pH range. The BlsacA homodimeric nature is strictly related to its optimum activity and is responsible for the generation of biphasic curves during differential scanning fluorimetry analyses. The homodimer is formed through the contact of the N-terminal ß-propeller domain of each BlsacA unit. The results presented here resemble the key importance of the homodimeric form of BlsacA for the enzyme stability and the optimum enzymatic activity.
Assuntos
Bacillus licheniformis/enzimologia , Sacarose/análogos & derivados , Fosfatos Açúcares/metabolismo , beta-Frutofuranosidase/química , beta-Frutofuranosidase/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Especificidade por Substrato , Sacarose/metabolismo , Difração de Raios XRESUMO
Invertases are used for several purposes; one among these is the production of fructooligosaccharides. The aim of this study was to biochemically characterize invertase from industrial Saccharomyces cerevisiae CAT-1 and Rhodotorula mucilaginosa isolated from Cerrado soil. The optimum pH and temperature were 4.0 and 70 °C for Rhodotorula mucilaginosa invertase and 4.5 and 50 °C for Saccharomyces cerevisiae invertase. The pH and thermal stability from 3.0 to 10.5 and 75 °C for R. mucilaginosa invertase, respectively. The pH and thermal stability for S. cerevisiae CAT-1 invertase from 3.0 to 7.0, and 50 °C, respectively. Both enzymes showed good catalytic activity with 10% of ethanol in reaction mixture. The hydrolysis by invertases occurs predominantly when sucrose concentrations are ≤5%. On the other hand, the increase in the concentration of sucrose to levels above 10% results in the highest transferase activity, reaching about 13.3 g/L of nystose by S. cerevisiae invertase and 12.6 g/L by R. mucilaginosa invertase. The results demonstrate the high structural stability of the enzyme produced by R. mucilaginosa, which is an extremely interesting feature that would enable the application of this enzyme in industrial processes.
Assuntos
Oligossacarídeos/biossíntese , Rhodotorula/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , beta-Frutofuranosidase/biossíntese , beta-Frutofuranosidase/metabolismo , Catálise , Estabilidade Enzimática , Etanol/metabolismo , Indústria Alimentícia/métodos , Concentração de Íons de Hidrogênio , Hidrólise , Indústrias , Especificidade da Espécie , Sacarose/metabolismo , Temperatura , beta-Frutofuranosidase/químicaRESUMO
KEY MESSAGE: The recent release of the maize genome (AGPv4) contains annotation errors of invertase genes and therefore the enzymes are bestly curated manually at the protein level in a comprehensible fashion The synthesis, transport and degradation of sucrose are determining factors for biomass allocation and yield of crop plants. Invertase (INV) is a key enzyme of carbon metabolism in both source and sink tissues. Current releases of the maize genome correctly annotates only two vacuolar invertases (ivr1 and ivr2) and four cell wall invertases (incw1, incw2 (mn1), incw3, and incw4). Our comprehensive survey identified 21 INV isogenes for which we propose a standard nomenclature grouped phylogenetically by amino acid similarity: three vacuolar (INVVR), eight cell wall (INVCW), and ten alkaline/neutral (INVAN) isogenes which form separate dendogram branches due to distinct molecular features. The acidic enzymes were curated for the presence of the DPN tripeptide which is coded by one of the smallest exons reported in plants. Particular attention was placed on the molecular role of INV in vascular tissues such as the nodes, internodes, leaf sheath, husk leaves and roots. We report the expression profile of most members of the maize INV family in nine tissues in two developmental stages, R1 and R3. INVCW7, INVVR2, INVAN8, INVAN9, INVAN10, and INVAN3 displayed the highest absolute expressions in most tissues. INVVR3, INVCW5, INVCW8, and INVAN1 showed low mRNA levels. Expressions of most INVs were repressed from stage R1 to R3, except for INVCW7 which increased significantly in all tissues after flowering. The mRNA levels of INVCW7 in the vegetative stem correlated with a higher transport rate of assimilates from leaves to the cob which led to starch accumulation and growth of the female reproductive organs.