Spray Drying of Coloring Extracts Produced by Fungi Isolated from Brazilian Caves

Abstract Pigments produced by submerged fermentation of three filamentous fungi isolated from Brazilian caves, namely Aspergillus keveii, Penicillium flavigenum, and Epicoccum nigrum, were submitted to spray drying in presence of the adjuvants maltodextrin, modified starch or gum arabic. Yellow fine powders with low moisture content and water activity, and high color retention (> 70%) were successfully generated with a high product recovery ratio (> 50%), independently of the adjuvant used. The dried products have enhanced stability and potential to might be used as a natural colorant in food and pharmaceutical applications.

Process optimization of the extraction condition of ß-amylase from brewer's malt and its application in the maltose syrup production.

ß-Amylase is of important biotechnological aid in maltose syrup production. In this study, the extraction condition of ß-amylase from brewer's malt and the optimal dosage of ß-amylase in maltose syrup production were optimized using response surface methodology and uniform design method. The optimal extraction condition of ß-amylase from brewer's malt was composed of 1:17 (g/v) material/liquid ratio, 44°C extraction temperature, pH 6.4 buffer pH, 2.3 H extraction time, and 1.64 g L-1 NaSO3 dosage with a predicted ß-amylase activity of 1,290.99 U g-1 , which was close to the experimental ß-amylase activity of 1,230.22 U g-1 . The optimal dosages of ß-amylase used in maltose syrup production were 455.67 U g-1 starch and its application in maltose syrup production led to a 68.37% maltose content in maltose syrup, which was 11.2% and 28.9% higher than those using ß-amylases from soybean and microbe (P < 0.01). Thus, ß-amylase from brewer's malt was beneficial for production of high maltose syrup.

13C labeling analysis of sugars by high resolution-mass spectrometry for metabolic flux analysis.

Metabolic flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography-high resolution mass spectrometry, without derivatized procedure, adapted for Metabolic flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic enrichments of free sugars compared to our previous methods [5, 6].

Characterization of the glucansucrase GTF180 W1065 mutant enzymes producing polysaccharides and oligosaccharides with altered linkage composition.

Exopolysaccharides produced by lactic acid bacteria are extensively used for food applications. Glucansucrase enzymes of lactic acid bacteria use sucrose to catalyze the synthesis of α-glucans with different linkage compositions, size and physico-chemical properties. Crystallographic studies of GTF180-ΔN show that at the acceptor binding sites +1 and +2, residue W1065 provides stacking interactions to the glucosyl moiety. However, the detailed functional roles of W1065 have not been elucidated. We performed random mutagenesis targeting residue W1065 of GTF180-ΔN, resulting in the generation of 10 mutant enzymes that were characterized regarding activity and product specificity. Characterization of mutant enzymes showed that residue W1065 is critical for the activity of GTF180-ΔN. Using sucrose, and sucrose (donor) plus maltose (acceptor) as substrates, the mutant enzymes synthesized polysaccharides and oligosaccharides with changed linkage composition. The stacking interaction of an aromatic residue at position 1065 is essential for polysaccharide synthesis.

Leuconostoc citreum SK24.002 glucansucrase: Biochemical characterisation and de novo synthesis of α-glucan.

The cell-associated glucansucrase from Leuconostoc citreum SK24.002 was isolated, purified, characterized and used for de novo synthesis of α-glucan and acceptor-products. The final specific glucansucrase activity was 1.4U/mg protein with 13.2-fold purification. The obtained glucansucrase had a molecular weight of 186kDa,Tm of 61.7 °C and △H of 176.7kJ/mol. The enzyme showed maximum activity at pH 5.0-6.0 and 45°C. The enzyme activity was enhanced by Ca(2+), Mn(2+) or Co(2+) ions, whereas the activity decreased as the methanol, ethanol, n-butanol, DMSO or isopropanol concentration increased. The chemical inhibitors including BD, DTNB, EDC or NBS also significantly inhibited enzyme activity. Km, Vmax and kcat of glucansucrase were 10.9mM, 3.6U/mg and 306.6 1/s, respectively. For de novo synthesis from sucrose, α-glucan polymer with molecular weight of 1.5×10(7)g/mol and maltose acceptor products (trisaccharide, tetrasaccharide and pentasaccharide) were obtained by glucansucrase via glucosyltransfer reactions, respectively.

Polyacrylamide Gel-Entrapped Maltase: An Excellent Design of Using Maltase in Continuous Industrial Processes.

Bacterial maltase catalyzes the hydrolysis of maltose and is known as one of the most significant hydrolases. It has several applications in different industrial processes but widely used in food fermentation technology and alcohol production. In the current study, entrapment technique was comprehensively examined using polyacrylamide gel as a matrix support to improve the stability and catalytic efficiency of maltase for continuous use. Maximum entrapment yield of maltase was achieved at 10 % polyacrylamide concentration with 3.0-mm bead size. Optimized conditions indicated an increase in the reaction temperature from 45 to 55 °C after maltase entrapment while no change was observed in the reaction time and pH. An increase in the K m value of entrapped maltase was attained whereas V max value decreased from 8411.0 to 6813.0 U ml(-1) min(-1) with reference to its free counterpart. Entrapped maltase showed remarkable thermal stability and retained 16 % activity at 70 °C even after 120.0 min. Entrapped maltase also exhibited excellent recycling efficiency up to eight consecutive reaction cycles. With respect to economic feasibility, entrapped maltase indicates its high potential to be used in various biotechnological applications.

Enhanced maltose production through mutagenesis of acceptor binding subsite +2 in Bacillus stearothermophilus maltogenic amylase.

Maltogenic amylases are used to decrease the maltotriose content of high maltose syrups. However, due to the interplay between the hydrolysis and transglycosylation activities of maltogenic amylases, the maltotriose contents of these syrups are still greater than that necessary for pure maltose preparation. In this study, the maltogenic amylase from Bacillus stearothermophilus was engineered to decrease its transglycosylation activity with the expectation that this would enhance maltose production. Site-directed mutagenesis was used to generate Trp 177 variants W177F, W177Y, W177L, W177N, and W177S. The transglycosylation activities of the mutant enzymes decreased as the hydrophilicity of the residue at position 177 increased. The mutant enzymes exhibited notable enhancements in maltose production, with a minimum of maltotriose contents of 0.2%, compared with 3.2% for the wild-type enzyme. Detailed characterization of the mutant enzymes suggests that the best of them, W177S, will deliver performance superior to that of the wild-type under industrial conditions.

Heterologous production of a feruloyl esterase from Pleurotus sapidus synthesizing feruloyl-saccharide esters.

The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal α-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 °C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides.

Effects of GLC7 and REG1 deletion on maltose metabolism and leavening ability of baker's yeast in lean dough.

Maltose metabolism and leavening ability of baker's yeast (Saccharomyces cerevisiae) in lean dough is negatively influenced by glucose repression. To improve maltose metabolism and leavening ability, it is necessary to alleviate glucose repression. In this study, we focus on the effects of regulators (GLC7 encoding the catalytic and REG1 encoding the regulatory subunits of protein phosphatase type 1) of glucose repression on maltose metabolism and leavening ability of baker's yeast in lean dough. To this end, GLC7 and/or REG1 deletions were constructed and characterized in terms of the growth characteristics, maltose metabolism, leavening ability, and enzyme activities. The results suggest that GLC7 and/or REG1 deletions increased maltose metabolism and leavening ability at different level with glucose derepression and increased enzymes (maltase and maltose permease) activities. In a medium containing glucose and maltose, at the point of glucose exhaustion the maltose metabolized and the leavening ability were increased 59.3% and 23.1%, respectively, in the case of a REG1 single gene deletion.

Production and biochemical characterization of a high maltotetraose (G4) producing amylase from Pseudomonas stutzeri AS22.

Amylase production and biochemical characterization of the crude enzyme preparation from Pseudomonas stutzeri AS22 were evaluated. The highest α-amylase production was achieved after 24 hours of incubation in a culture medium containing 10 g/L potato starch and 5 g/L yeast extract, with initial pH 8.0 at 30°C under continuous agitation at 200 rpm. The optimum temperature and pH for the crude α -amylase activity were 60°C and 8.0, respectively. The effect of different salts was evaluated and it was found that both α -amylase production and activity were Ca(2+)-dependent. The amylolytic preparation was found to catalyze exceptionally the formation of very high levels of maltotetraose from starch (98%, w/w) in the complete absence of glucose since the initial stages of starch hydrolysis (15 min) and hence would have a potential application in the manufacturing of maltotetraose syrups.

Thermal stability and starch degradation profile of α-amylase from Streptomyces avermitilis.

Amylases from Streptomyces are useful in the production of maltooligosaccharides, but they have weak thermal stability at temperatures higher than 40 °C. In this study, α-amylase (SAV5981 gene of Streptomyces avermitilis) was expressed from Streptomyces lividans 1326 and purified by ammonium sulfate fractionation followed by anionic chromatography (Q-HP sepharose). The properties of the purified SAV5981 amylase were determined by the starch-iodine method. The effect of metal ions on amylase activity was investigated. The optimal temperature shifted from 25 to 50 °C with the addition of the Ca(2+) ion. The thermal stability of SAV5981 was also dramatically enhanced by the addition of 10 mM CaCl2. Improvement of the thermal stability of SAV5981 was examined by CD spectra in the presence and the absence of the Ca(2+) ion. Thin-layer chromatography (TLC) analysis and HPLC analysis of starch degradation revealed that SAV5981 mainly produced maltose and maltotriose, not glucose. The maltoorigosaccharide-producing amylase examined in this study has the potential in the industrial application of oligosaccharide production.

Characteristics of a high maltose-forming, acid-stable, and Ca(2+)-independent α-amylase of the acidophilic Bacillus acidicola.

The purified acidic α-amylase of Bacillus acidicola is a monomer of 66.0 kDa, optimally active at pH 4.0 and 60 °C. The enzyme is Ca2+ independent with T1/2 for 18 min at 80 °C. The Km, Vmax, and catalytic efficiency (kcat/Km) of the enzyme are 1.6 mg mL(−1), 23.8 µmol mg(−1) min(−1), and 981 µmol s(−1), respectively. Among detergents, Tween 20, 40, and 80 stimulated enzyme activity, whereas sodium dodecyl sulfate and Triton X-100 inhibited even at low concentration. EGTA has not affected the activity, whereas EDTA ß-mercaptoethanol, iodoacetic acid, and Dithiothreitol exhibited a slight inhibitory action. Phenylmethanesulfonyl fluoride, N-bromosuccinimide, and Hg2+ strongly inhibited enzyme activity. The experimental activation energy and temperature quotient are 50.12 kJ mol(−1) and 1.37. When thermodynamic parameters (ΔH and ΔS) of the enzyme have been determined at different temperatures, ΔG is positive suggesting that the enzyme is thermostable. The enzyme hydrolyzes raw starches, and therefore, the enzyme finds application in raw starch saccharification at sub-gelatinization temperatures that saves energy needed for gelatinization of raw starch at 105 °C.

Structure of a novel α-amylase AmyB from Thermotoga neapolitana that produces maltose from the nonreducing end of polysaccharides.

An intracellular α-amylase, AmyB, has been cloned from the hyperthermophilic bacterium Thermotoga neapolitana. AmyB belongs to glycoside hydrolase family 13 and liberates maltose from diverse substrates, including starch, amylose, amylopectin and glycogen. The final product of AmyB is similar to that of typical maltogenic amylases, but AmyB cleaves maltose units from the nonreducing end, which is a unique property of this α-amylase. In this study, the crystal structure of AmyB from T. neapolitana has been determined at 2.4 Šresolution, revealing that the monomeric AmyB comprises domains A, B and C like other α-amylases, but with structural variations. In the structure, a wider active site and a putative extra sugar-binding site at the top of the active site were found. Subsequent biochemical results suggest that the extra sugar-binding site is suitable for recognizing the nonreducing end of the substrates, explaining the unique activity of this enzyme. These findings provide a structural basis for the ability of an α-amylase that has the common α-amylase structure to show a diverse substrate specificity.

Identification of Bacillus selenitireducens MLS10 maltose phosphorylase possessing synthetic ability for branched α-D-glucosyl trisaccharides.

We discovered an inverting maltose phosphorylase (Bsel2056) belonging to glycoside hydrolase family 65 from Bacillus selenitireducens MLS10, which possesses synthetic ability for α-D-glucosyl disaccharides and trisaccharides through the reverse phosphorolysis with ß-D-glucose 1-phosphate as the donor. Bsel2056 showed the flexibility for monosaccharide acceptors with alternative C2 substituent (2-amino-2-deoxy-D-glucose, 2-deoxy-D-arabino-hexose, 2-acetamido-2-deoxy-D-glucose, D-mannose), resulting in production of 1,4-α-D-glucosyl disaccharides with strict regioselectivity. In addition, Bsel2056 synthesized two maltose derivatives possessing additional D-glucosyl residue bound to C2 position of the D-glucose residue at the reducing end, 1,4-α-D-glucopyranosyl-[1,2-α-D-glucopyranosyl]-D-glucose and 1,4-α-D-glucopyranosyl-[1,2-ß-D-glucopyranosyl]-D-glucose, from 1,2-α-D-glucopyranosyl-D-glucose (kojibiose) and 1,2-ß-D-glucopyranosyl-D-glucose (sophorose), respectively, as the acceptors. These results suggested that Bsel2056 possessed a binding space to accommodate the bulky C2 substituent of D-glucose.

Gene cloning, heterologous expression, and characterization of a high maltose-producing α-amylase of Rhizopus oryzae.

A putative α-amylase gene, designated as RoAmy, was cloned from Rhizopus oryzae. The deduced amino acid sequence showed the highest (42.8%) similarity to the α-amylase from Trichoderma viride. The RoAmy gene was successfully expressed in Pichia pastoris GS115 under the induction of methanol. The molecular weight of the purified RoAmy determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was approximately 48 kDa. The optimal pH and temperature were 4-6 and 60 °C, respectively. The enzyme was stable at pH ranges of 4.5-6.5 and temperatures below 50 °C. Purified RoAmy had a K(m) and V(max) of 0.27 mg/ml and 0.068 mg/min, respectively, with a specific activity of 1,123 U/mg on soluble starch. Amylase activity was strongly inhibited by 5 mM Cu(2+) and 5 mM Fe(2+), whereas 5 mM Ca(2+) showed no significant effect. The RoAmy hydrolytic activity was the highest on wheat starch but showed only 55% activity on amylopectin relative to soluble corn starch, while the pullulanase activity was negligible. The main end products of the polysaccharides tested were glucose and maltose. Maltose reached a concentration of 74% (w/w) with potato starch as the substrate. The enzyme had an extremely high affinity (K(m) = 0.22 mM) to maltotriose. A high ratio of glucose/maltose of 1:4 was obtained when maltotriose was used at an initial concentration of 40 mM.

Lipase-catalyzed selective synthesis of monolauroyl maltose using continuous stirred tank reactor.

Monolauroyl maltose was synthesized by an immobilized lipase that catalyzed condensation of maltose and lauric acid in acetone using a batch reactor or a continuous stirred tank reactor. Mono- and di-lauroyl maltoses were identified by FT-IR, (1)H NMR, (13)C NMR and MS. Monolauroyl maltose was selectively synthesized in a continuous stirred tank reactor and no diester was detected. The highest concentration of monolauroyl maltose at 28 mmol/l was obtained in 250 ml acetone when maltose was added at 4 g/d and the molar ratio of lauric acid to maltose was fixed at 4:1 at a flow rate of 0.15 ml/min for both influx and effluent without supplement of fresh molecular sieve.

A novel cyclic isomaltooligosaccharide (cycloisomaltodecaose, CI-10) produced by Bacillus circulans T-3040 displays remarkable inclusion ability compared with cyclodextrins.

Cyclodextrans (CIs) are cyclic isomaltooligosaccharides and only CI-7, CI-8, and CI-9 were known. CI-7, CI-8, and CI-9, consisting of seven, eight, and nine glucoses, respectively, bound by alpha-(1-->6) linkages, are known to be produced by T-3040 strain of Bacillus circulans. However, we have found, using 13C NMR and mass spectrometry, that this strain also produces CI-10, CI-11 and CI-12. These large CIs are very soluble in water and inhibit the glucan synthesis of glucansucrases to the same degree as do the smaller CIs. The CIs were thought to be poor at forming inclusion complexes with chemical compounds, due to their flexible alpha-(1-->6)-glucosidic structure. Among these six CIs, CI-10 was much better at forming an inclusion complex, and it ability to do so was as good as cyclodextrins, as determined by its ability to stabilize the color of Victoria blue B. Therefore, CI-10 may be the most commercially useful CI.

Synthesis of linoleoyl disaccharides through lipase-catalyzed condensation and their surface activities.

Monolinoleoyl trehalose, maltose and cellobiose were synthesized by Candida antarctica lipase-catalyzed condensation in an organic solvent with a low water content. The use of a mixture of pyridine and tert-butanol as the reaction medium resulted in a high product concentration on the order of mmol/l for the synthesis of linoleoyl trehalose and maltose. The highest product concentration was achieved with the 0.4 volumetric fraction of pyridine. Linoleoyl cellobiose was also synthesized although its concentration was approximately one tenth the concentrations of linoleoyl trehalose and maltose. The surfactant properties of linoleoyl trehalose, maltose and cellobiose were measured. Among the esters, linoleoyl trehalose showed the strongest surface activity.