Comprehensive analysis of the GALACTINOL SYNTHASE (GolS) gene family in citrus and the function of CsGolS6 in stress tolerance.

Galactinol synthase (GolS) catalyzes the first and rate-limiting step in the synthesis of raffinose family of oligosaccharides (RFOs), which serve as storage and transport sugars, signal transducers, compatible solutes and antioxidants in higher plants. The present work aimed to assess the potential functions of citrus GolS in mechanisms of stress response and tolerance. By homology searches, eight GolS genes were found in the genomes of Citrus sinensis and C. clementina. Phylogenetic analysis showed that there is a GolS ortholog in C. clementina for each C. sinensis GolS, which have evolved differently from those of Arabidopsis thaliana. Transcriptional analysis indicated that most C. sinensis GolS (CsGolS) genes show a low-level tissue-specific and stress-inducible expression in response to drought and salt stress treatments, as well as to 'Candidatus Liberibacter asiaticus' infection. CsGolS6 overexpression resulted in improved tobacco tolerance to drought and salt stresses, contributing to an increased mesophyll cell expansion, photosynthesis and plant growth. Primary metabolite profiling revealed no significant changes in endogenous galactinol, but different extents of reduction of raffinose in the transgenic plants. On the other hand, a significant increase in the levels of metabolites with antioxidant properties, such as ascorbate, dehydroascorbate, alfa-tocopherol and spermidine, was observed in the transgenic plants. These results bring evidence that CsGolS6 is a potential candidate for improving stress tolerance in citrus and other plants.

Impact of protein blocking on enzymatic saccharification of bagasse from sugarcane clones.

Lignin plays an important functional and structural role in plants, but also contributes to the recalcitrance of lignocellulosic biomass to hydrolysis. This study addresses the influence of lignin in hydrolysis of sugarcane bagasse from conventional bred lines (UFV260 and UFV204) that were selected from 432 field-grown clones. In addition to higher sugar production, bagasse clone UFV204 had a small, but statistically significant, lower insoluble lignin content compared with clone UFV260 (15.5% vs, 16.6%) and also exhibited a significantly higher cellulose conversion to glucose (81.3% vs. 63.3%) at a cellulase loading of 5 (filter paper unit) FPU/g of glucan or 3 FPU/g total solids for liquid hot water pretreated bagasse (200°C, 10 min). The enzyme loading was further decreased by 50% to 2.5 FPU/g glucan and resulted in a similar glucan conversion (88.5%) for clone UFV204 when the bagasse was preincubated with bovine serum albumin at pH 4.8 and nonproductive binding of cellulase components was blocked. Comparison of Langmuir adsorption isotherms and differential adsorption of the three major cellulolytic enzyme components endoglucanase, cellobiohydrolase, and ß-glucosidase help to explain differences due to lignin content.

Inhibitors Compounds on Sugarcane Bagasse Saccharification: Effects of Pretreatment Methods and Alternatives to Decrease Inhibition.

Considering bioethanol production, extensive research has been performed to decrease inhibitors produced during pretreatments, to diminish energy input, and to decrease costs. In this study, sugarcane bagasse was pretreated with NaOH, H2SO4, and water. The higher concentration of phenols, 3.3 g/L, was observed in biomass liquid fraction after alkaline pretreatment. Acid pretreatment was responsible to release considerable acetic acid concentration, 2.3 g/L, while water-based pretreatment was the only to release formic acid, 0.02 g/L. Furans derivatives were not detected in liquid fractions regardless of pretreatment. Furthermore, washing step removed most of the phenols from pretreated sugarcane bagasse. Saccharification of alkali-pretreated biomass plus polyethylene glycol (PEG) at 0.4% (w/v) enhanced 8 and 26% the glucose and the xylose release, respectively, while polyvinylpyrrolidone (PVP) also at 0.4% (w/v) increased the release by 10 and 31% of these sugars, respectively, even without washing and filtration steps. Moreover, these polymers cause above 50% activation of endoglucanase and xylanase activities which are crucial for biomass hydrolysis.

Penicillium citrinum UFV1 ß-glucosidases: purification, characterization, and application for biomass saccharification.

BACKGROUND: ß-Glucosidases are components of the cellulase system, a family of enzymes that hydrolyze the ß-1,4 linkages of cellulose. These proteins have been extensively studied due to the possibility of their use in various biotechnological processes. They have different affinities for substrates (depending on their source) and their activities can be used for saccharification of different types of biomass. In this context, the properties and the synergistic capacity of ß-glucosidases from different organisms, to supplement the available commercial cellulase cocktails, need a comprehensive evaluation. RESULTS: Two ß-glucosidases belonging to GH3 family were secreted by Penicillium citrinum UFV. PcßGlu1 (241 kDa) and PcßGlu2 (95 kDa) presented acidic and thermo-tolerant characteristics. PcßGlu1 showed Michaelis-Menten kinetics for all substrates tested with Km values ranging from 0.09 ± 0.01 (laminarin) to 1.7 ± 0.1 mM (cellobiose, C2) and kcat values ranging from 0.143 ± 0.005 (laminarin) to 8.0 ± 0.2 s-1 (laminaribiose, Lb). PcßGlu2 showed substrate inhibition for 4-methylumbelliferyl-ß-d-glucopyranoside (MUßGlu), p-nitrophenyl-ß-d-glucopyranoside (pNPßGlu), cellodextrins (C3, C4, and C5), N-octil-ß-d-glucopyranoside, and laminaribiose, with Km values ranging from 0.014 ± 0.001 (MUßGlu) to 0.64 ± 0.06 mM (C2) and kcat values ranging from 0.49 ± 0.01 (gentiobiose) to 1.5 ± 0.2 s-1 (C4). Inhibition constants (Ki) for PcßGlu2 substrate inhibition ranged from 0.69 ± 0.07 (MUßGlu) to 10 ± 1 mM (Lb). Glucose and cellobiose are competitive inhibitors of PcßGlu1 and PcßGlu2 when pNPßGlu is used as a substrate. For PcßGlu1 inhibition, Ki = 1.89 ± 0.08 mM (glucose) and Ki = 3.8 ± 0.1 mM (cellobiose); for PcßGlu2, Ki = 0.83 ± 0.05 mM (glucose) and Ki = 0.95 ± 0.07 mM (cellobiose). The enzymes were tested for saccharification of different biomasses, individually or supplementing a Trichoderma reesei commercial cellulose preparation. PcßGlu2 was able to hydrolyze banana pseudostem and coconut fiber with the same efficiency as the T. reesei cocktail, showing significant synergistic properties with T. reesei enzymes in the hydrolysis of these alternative biomasses. CONCLUSIONS: The ß-glucosidases from P. citrinum UFV1 present different enzymatic properties from each other and might have potential application in several biotechnological processes, such as hydrolysis of different types of biomass.

Deactivation and activation of lignocellulose degrading enzymes in the presence of laccase.

Cellulase and hemicellulase activities in a 1:1 ratio of enzymes extracted from Chrysoporthe cubensis and Penicillium pinophilum were evaluated in the presence of known monocomponent phenolic inhibitors and also with phenol mixtures derived from alkali pretreated sugarcane bagasse. The cellulolytic activities from C. cubensis:P. pinophilum displayed a much higher tolerance to phenolic inhibitors than equivalent enzyme activities obtained from Trichoderma reesei and Aspergillus niger. Enzymes from T. reesei and A. niger were deactivated at 0.3 and 1.5mg phenols/mg protein, respectively, as reported previously, while enzymes from C. cubensis:P. pinophilum resisted deactivation at 35mg phenols/mg protein. However, tolerance of xylanase with respect to phenols required the presence of laccase. Removal of laccase (enzyme) activity using sodium azide resulted in a 2x higher xylanase deactivation (from 40% to 80%). This paper identifies enzymes that are phenol tolerant, and whose adoption for lignocellulose hydrolysis could contribute to reductions in enzyme loading needed to hydrolyze alkali pretreated lignocellulosic substrates in the presence of lignin derived phenols.

Characterization and biotechnological application of recombinant xylanases from Aspergillus nidulans.

Two xylanases from Aspergillus nidulans, XlnB and XlnC, were expressed in Pichia pastoris, purified and characterized. XlnB and XlnC achieved maximal activities at 60°C and pH 7.5 and at 50°C and pH 6.0, respectively. XlnB showed to be very thermostable by maintaining 50% of its original activity after 49h incubated at 50°C. XlnB had its highest activity against wheat arabinoxylan while XlnC had the best activity against beechwood xylan. Both enzymes were completely inhibited by SDS and HgCl2. Xylotriose at 1mg/ml also totally inibited XlnB activity. TLC analysis showed that the main product of beechwood xylan hydrolysis by XlnB and XlnC was xylotetraose. An additive effect was shown between XlnB and XlnC and the xylanases of two tested commercial cocktails. Sugarcane bagasse saccharification results showed that these two commercial enzymatic cocktails were able to release more glucose and xylose after supplementation with XlnB and XlnC.

Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance.

An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The K M for sodium phytate hydrolysis was 30.9 mM, while the k cat and k cat / K M were 1.46 ×10 (5) s (-1) and 4.7 × 10 (6) s (-1) .M (-1) , respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg (2+) , Cd (2+) , K (+) and Ca (2+) , and it was drastically inhibited by F (-) . The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t 1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 µmol phosphate/mL after 2.5 h of treatment.

Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance

An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The KM for sodium phytate hydrolysis was 30.9 mM, while the kcat and kcat/KM were 1.46 ×105 s−1 and 4.7 × 106 s−1.M−1, respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg2+, Cd2+, K+ and Ca2+, and it was drastically inhibited by F−. The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment.

Production and characterization of ß-glucanase secreted by the yeast Kluyveromyces marxianus.

An extracellular ß-glucanase secreted by Kluyveromyces marxianus was identified for the first time. The optimal conditions for the production of this enzyme were evaluated by response surface methodology. The optimal conditions to produce ß-glucanase were a glucose concentration of 4% (w/v), a pH of 5.5, and an incubation temperature of 35 °C. Response surface methodology was also used to determine the pH and temperature required for the optimal enzymatic activity. The highest enzyme activity was obtained at a pH of 5.5 and a temperature of 55 °C. Furthermore, the enzyme was partially purified and sequenced, and its specificity for different substrates was evaluated. The results suggest that the enzyme is an endo-ß-1,3(4)-glucanase. After optimizing the conditions for ß-glucanase production, the culture supernatant was found to be effective in digesting the cell wall of the yeast Saccharomyces cerevisiae, showing the great potential of ß-glucanase in the biotechnological production of soluble ß-glucan.

Optimization of endoglucanase and xylanase activities from Fusarium verticillioides for simultaneous saccharification and fermentation of sugarcane bagasse.

Enzymatic hydrolysis is an important but expensive step in the production of ethanol from biomass. Thus, the production of efficient enzymatic cocktails is of great interest for this biotechnological application. The production of endoglucanase and xylanase activites from F. verticillioides were optimized in a factorial design (2(5)) followed by a CCDR design. Endoglucanase and xylanase activities increased from 2.8 to 8.0 U/mL and from 13.4 to 114 U/mL, respectively. The optimal pH and temperature were determined for endoglucanase (5.6, 80 °C), cellobiase (5.6, 60 °C), FPase (6.0, 55 °C) and xylanase (7.0, 50 °C). The optimized crude extract was applied in saccharification and fermentation of sugarcane bagasse from which 9.7 g/L of ethanol was produced at an ethanol/biomass yield of 0.19.

Hydrolysis of soybean isoflavones by Debaryomyces hansenii UFV-1 immobilised cells and free ß-glucosidase.

An intracellular ß-glucosidase from Debaryomyceshansenii UFV-1 was produced in an YP medium with cellobiose as the carbon source. This enzyme was purified, characterised and presented a Mr of 65.15kDa. Yeast cells containing the intracellular ß-glucosidase were immobilised in calcium alginate. The free ß-glucosidase and immobilised cells containing the enzyme presented optima values of pH and temperature of 6.0 and 45°C and 5.5 and 50°C, respectively. The free enzyme maintained 62% and 47% of its original activity after 90days at 4°C and after 15days at room temperature, respectively. The immobilisation process resulted in higher enzyme thermostability at 45 and 50°C. Soy molasses treatment with the free enzyme and the immobilised cells containing ß-glucosidase, for 2h at 40°C, promoted efficient hydrolysis of isoflavone glicosides to their aglycon forms. The results suggest that this enzyme could be used in the food industry, in the free or immobilised forms, for a safe and efficient process to hydrolyse isoflavone glycosides in soy molasses.

Direct ethanol production from glucose, xylose and sugarcane bagasse by the corn endophytic fungi Fusarium verticillioides and Acremonium zeae.

Production of ethanol with two corn endophytic fungi, Fusarium verticillioides and Acremonium zeae, was studied. The yield of ethanol from glucose, xylose and a mixture of both sugars were 0.47, 0.46 and 0.50g/g ethanol/sugar for F. verticillioides and 0.37, 0.39 and 0.48g/g ethanol/sugar for A. zeae. Both fungi were able to co-ferment glucose and xylose. Ethanol production from 40g/L of pre-treated sugarcane bagasse was 4.6 and 3.9g/L for F. verticillioides and A. zeae, respectively, yielding 0.31g/g of ethanol per consumed sugar. Both fungi studied were capable of co-fermenting glucose and xylose at high yields. Moreover, they were able to produce ethanol directly from lignocellulosic biomass, demonstrating to be suitable microorganisms for consolidated bioprocessing.

Characteristics of free endoglucanase and glycosidases multienzyme complex from Fusarium verticillioides.

A novel multienzyme complex, E1C, and a free endoglucanase, E2 (GH5), from Fusarium verticillioides were purified. The E1C contained two endoglucanases (GH6 and GH10), one cellobiohydrolase (GH7) and one xylanase (GH10). Maximum activity was observed at 80 °C for both enzymes and they were thermostable at 50 and 60 °C. The activation energies for E1C and E2 were 21.3 and 27.5 kJ/mol, respectively. The KM for E1C was 10.25 g/L while for E2 was 6.58 g/L. Both E1C and E2 were activated by Mn(2+) and CoCl2 while they were inhibited by SDS, CuSO4, FeCl3, AgNO4, ZnSO4 and HgCl2. E1C and E2 presented endo-ß-1,3-1,4-glucanase activity. E1C presented crescent activity towards cellopentaose, cellotetraose and cellotriose. E2 hydrolyzed the substrates cellopentaose, cellotetraose and cellotriose with the same efficiency. E1C showed a higher stability and a better hydrolysis performance than E2, suggesting advantages resulting from the physical interaction between proteins.

Activity of Debaryomyces hansenii UFV-1 α-galactosidases against α-D-galactopyranoside derivatives.

α-D-Galactopyranosides were synthesized and their inhibitory activities toward the Debaryomyces hansenii UFV-1 extracellular and intracellular α-galactosidases were evaluated. Methyl α-D-galactopyranoside was the most potent inhibitor compared to the others tested, with K(i)(') values of 0.82 and 1.12 mmolL(-1), for extracellular and intracellular enzymes, respectively. These results indicate that the presence of a hydroxyl group in the C-6 position of α-D-galactopyranoside derivatives is important for the recognition by D. hansenii UFV-1 α-galactosidases.

Characterization of an exoinulinase produced by Aspergillus terreus CCT 4083 grown on sugar cane bagasse.

Exoinulinase (beta-d-fructan fructohydrolase, EC 3.2.1.80) secreted by Aspergillus terreus CCT4083 was obtained using sugar cane bagasse, an agroindustrial residue, as a carbon source. It was further purified from the supernatant culture in a rapid procedure. The enzyme presented 57 kDa on SDS-PAGE and 56 kDa on gel filtration chromatography. Inulin was hydrolyzed by the purified enzyme, yielding d-fructose as the main product. This enzyme showed maximum activity at pH 4.0 and 60 degrees C and maintained more than 90 and 75% of its original activity at 40 and 50 degrees C, respectively, after 3.5 h of preincubation. The K(M) values for inulin, sucrose, and raffinose were 11, 4.20, and 27.89 mM, respectively, and d-fructose was a competitive inhibitor (K(i) = 47.55 mM). The activation energies for sucrose, raffinose, and inulin were 10.4, 5.61, and 4.44 kcal/mol, respectively. The characteristics of A. terreus exoinulinase were compared to those of inulinases isolated from other organisms. The exoinulinase traits presented especially good thermostability and the ability to produce pure d-fructose, suggesting its application to the production of high-fructose syrup.

Spectroscopic and thermodynamic properties of Debaryomyces hansenii UFV-1 alpha-galactosidases.

Spectroscopic and thermodynamic properties were determined for Debaryomyces hansenii UFV-1 extracellular and intracellular alpha-galactosidases. alpha-Galactosidases showed similar secondary structure compositions (alpha-helix, beta-sheet parallel and beta-turn). Effects of pH and temperature on the structure of alpha-galactosidases were investigated using circular dichroism spectroscopy. It was more pronounced at low pH. Microcalorimetry was employed for the determination of thermodynamic parameters. Immediate thermal denaturation reversibility was not observed for alpha-galactosidases; it occurred as a thermodynamically driven process. Extracellular alpha-galactosidase, at pH 5.5, showed lower T(m) when compared to the intracellular enzyme. The CD and DSC data suggest that D. hansenii alpha-galactosidases have different behaviors although they possess some similar secondary structures.

Debaryomyces hansenii UFV-1 intracellular alpha-galactosidase characterization and comparative studies with the extracellular enzyme.

Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular alpha-galactosidases, which showed 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDS-PAGE) and 5.15 and 4.15 pI values, respectively. The extracellular and intracellular deglycosylated forms presented 36 and 40 kDa molecular mass, with 40 and 34% carbohydrate content, respectively. The N-terminal sequences of the alpha-galactosidases were identical. Intracellular alpha-galactosidase showed smaller thermostability when compared to the extracellular enzyme. D. hansenii UFV-1 extracellular alpha-galactosidase presented higher kcat than the intracellular enzyme (7.16 vs 3.29 s-1, respectively) for the p-nitrophenyl-alpha-D-galactopyranoside substrate. The Km for hydrolysis of pNPalphaGal, melibiose, stachyose, and raffinose were 0.32, 2.12, 10.8, and 32.8 mM, respectively. The intracellular enzyme was a competitively inhibited by galactose (Ki = 0.70 mM), and it was inactivated by Cu(II) and Ag(I). Enzyme incubation with soy milk for 6 h at 55 degrees C reduced stachyose and raffinose amounts by 100 and 73%, respectively.

Processing of soybean products by semipurified plant and microbial alpha-galactosidases.

Galactooligosaccharides (GO) are responsible for intestinal disturbances following ingestion of legume-derived products. Enzymatic reduction of GO level in these products is highly desirable to improve their acceptance. For this purpose, plant and microbial semipurified alpha-galactosidases were used for GO hydrolysis in soybean flour and soy molasses. alpha-Galactosidases from soybean germinating seeds, Aspergillus terreus, and Penicillium griseoroseum presented maximal activities at pH 4.0-5.0 and 45-65 degrees C. The KM,app values determined for raffinose by the soybean, A. terreus, and P. griseoroseum alpha-galactosidases were 3.44, 19.39, and 20.67 mM, respectively. The enzymes were completely inhibited by Ag+ and Hg2+, whereas only soybean enzyme was inhibited by galactose. A. terreus alpha-galactosidase was more thermostable than the enzymes from the other two sources. This enzyme maintained about 100% of its original activity after 3 h at 60 C. The microbial alpha-galactosidases were more efficient for reducing GO in soybean flour and soy molasses than soybean enzyme.

Extracellular alpha-galactosidase from Debaryomyces hansenii UFV-1 and its use in the hydrolysis of raffinose oligosaccharides.

Raffinose oligosaccharides (RO) are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. ROs are hydrolyzed by alpha-galactosidases that cleave alpha-1,6-linkages of alpha-galactoside residues. The objectives of this study were the purification and characterization of extracellular alpha-galactosidase from Debaryomyces hansenii UFV-1. The enzyme purified by gel filtration and anion exchange chromatographies presented an Mr value of 60 kDa and the N-terminal amino acid sequence YENGLNLVPQMGWN. The Km values for hydrolysis of pNP alphaGal, melibiose, stachyose, and raffinose were 0.30, 2.01, 9.66, and 16 mM, respectively. The alpha-galactosidase presented absolute specificity for galactose in the alpha-position, hydrolyzing pNPGal, stachyose, raffinose, melibiose, and polymers. The enzyme was noncompetitively inhibited by galactose (Ki = 2.7 mM) and melibiose (Ki = 1.2 mM). Enzyme treatments of soy milk for 4 h at 60 degrees C reduced the amounts of stachyose and raffinose by 100%.