Douglas Chodi Masui

Optimization of β-Glucosidase, β-Xylosidase and Xylanase Production by Colletotrichum graminicola under Solid-State Fermentation and Application in Raw Sugarcane Trash Saccharification

Efficient, low-cost enzymatic hydrolysis of lignocellulosic residues is essential for cost-effective production of bioethanol. The production of β-glucosidase, β-xylosidase and xylanase by Colletotrichum graminicola was optimized using Response Surface Methodology (RSM). Maximal production occurred in wheat bran. Sugarcane trash, peanut hulls and corncob enhanced β-glucosidase, β-xylosidase and xylanase production, respectively. Maximal levels after optimization reached 159.3 ± 12.7 U g−1, 128.1 ± 6.4 U g−1 and 378.1 ± 23.3 U g−1, respectively, but the enzymes were produced simultaneously at good levels under culture conditions optimized for each one of them. Optima of pH and temperature were 5.0 and 65 °C for the three enzymes, which maintained full activity for 72 h at 50 °C and for 120 min at 60 °C (β-glucosidase) or 65 °C (β-xylosidase and xylanase). Mixed with Trichoderma reesei cellulases, C. graminicola crude extract hydrolyzed raw sugarcane trash with glucose yield of 33.1% after 48 h, demonstrating good potential to compose efficient cocktails for lignocellulosic materials hydrolysis.

Purification and biochemical properties of a glucose-stimulated β-D-glucosidase produced by Humicola grisea var. thermoidea grown on sugarcane bagasse.

The effect of several carbon sources on the production of mycelial-bound β-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated β-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The β-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50°C, respectively. The purified enzyme was thermostable up to 60 min in water at 55°C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60°C. The enzyme hydrolyzed p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-Dgalactopyranoside, p-nitrophenyl-β-D-fucopyranoside, p-nitrophenyl-β-D-xylopyranoside, o-nitrophenyl-β-Dgalactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-β-Dfucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude β-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea β-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.

Production of a xylose-stimulated β-glucosidase and a cellulase-free thermostable xylanase by the thermophilic fungus Humicola brevis var. thermoidea under solid state fermentation

Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of β-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2xa0±xa0411.2xa0Uxa0g−1, while β-glucosidase production was increased about 2.6-fold, reaching 20.7xa0±xa01.5xa0Uxa0g−1. Cellulase levels were negligible. Biochemical characterization of H. brevis β-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for β-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80xa0% of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The β-glucosidase maintained about 95xa0% of its activity after 26xa0h in water at 55xa0°C, with half-lives of 15.7xa0h at 60xa0°C and 5.1xa0h at 65xa0°C. The presence of xylose during heat treatment at 65xa0°C protected β-glucosidase against thermal inactivation. Xylanase maintained about 80xa0% of its activity after 200xa0h in water at 60xa0°C. Xylose stimulated β-glucosidase activity up to 1.7-fold, at 200xa0mmolxa0L−1. The notable features of both xylanase and β-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.

Adsorption kinetics and dilatational rheological studies for the soluble and anchored forms of alkaline phosphatase at the air/water interface

This work presents equilibrium and dynamic aspects for the adsorption at the air/liquid interface of two rat osseous plate alkaline phosphatase forms: DSAP, solubilized by a surfactant, C12E9, and containing a glycosylphosphatidylinositol (GPI) anchor; and PLSAP, resulting from phospholipase-C cleavage of the hydrophobic portion of the GPI anchor. Dynamic surface tension, gdyn, and surface elasticity modulus, e, were determined for PLSAP, DSAP and pure C12E9 solutions using harmonic oscillation and axisymmetric drop shape analysis Adsorption kinetics studies revealed that DSAP adsorbs thirty times faster than PLSAP, presenting a minimum in the curve. For DSAP/ C12E9 mixed system, e increases with concentration and a maximum appears at the critical aggregation concentration (CAC). For PLSAP, a continuous decreasing with concentration for gdyn and e was observed. For pure C12E9 solution, the elasticity modulus increases with concentration and e values are higher when compared to the mixed system. A model based on the influence of the GPI anchor is proposed.

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

We investigated modulation by ATP, Mg2+, Na+, K+ and NH4+ and inhibition by ouabain of (Na+,K+)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na+,K+)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (KMxa0=xa00.09xa0±xa00.01xa0mmolxa0L−1) of the decapodid III (Na+,K+)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na+,K+)-ATPase activity by K+ also revealed a threefold greater affinity for K+ (K0.5xa0=xa00.91xa0±xa00.04xa0mmolxa0L−1) in decapodid III than in other stages; NH4+ had no modulatory effect. The affinity for Na+ (K0.5xa0=xa013.2xa0±xa00.6xa0mmolxa0L−1) of zoea I (Na+,K+)-ATPase was fourfold less than other stages. Modulation by Na+, Mg2+ and NH4+ obeyed cooperative kinetics, while K+ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg2+ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg2+-stimulated ATPases other than (Na+,K+)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na+-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH4+-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.

Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na,K-ATPase activity by mammalian FXYD2 peptide

This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (γC(33)) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K(+), ATP and NH(4)(+). K(0.5) for Na(+) was unaffected. Exogenous pig FXYD2 increased the V(max) for stimulation of gill Na,K-ATPase activity by Na(+), K(+) and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na,K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity.

A Novel β-Glucosidase from Humicola insolens with High Potential for Untreated Waste Paper Conversion to Sugars

Humicola insolens produced a new β-glucosidase (BglHi2) under solid-state fermentation. The purified enzyme showed apparent molecular masses of 116xa0kDa (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and 404xa0kDa (gel-filtration), suggesting that it is a homotetramer. Mass spectrometry analysis showed amino acid sequence similarity with a β-glucosidase from Chaetomium thermophilum. Optima of pH and temperature were 5.0 and 65xa0°C, respectively, and the enzyme was stable for 60xa0min at 50xa0°C, maintaining 71xa0% residual activity after 60xa0min at 55xa0°C. BglHi2 hydrolyzed p-nitrophenyl-β-d-glucopyranoside and cellobiose. Cellobiose hydrolysis occurred with high apparent affinity (KMu2009=u20090.24u2009±u20090.01xa0mmolxa0L−1) and catalytic efficiency (kcat/KMu2009=u20091,304.92u2009±u200953.32xa0Lxa0mmol−1xa0s−1). The activity was insensitive to Fe+3, Cr+2, Mn+2, Co+2, and Ni2+, and 50–60xa0% residual activities were retained in the presence of Pb2+, Hg2+, and Cu2+. Mixtures of pure BglHi2 or H. insolens crude extract (CE) with crude extracts from Trichoderma reesei fully hydrolyzed Whatman no. 1 paper. Mixtures of H. insolens CE with T. reesei CE or Celluclast 1.5xa0L fully hydrolyzed untreated printed office paper, napkin, and magazine papers after 24–48xa0h, and untreated cardboard was hydrolyzed by a H. insolens CE/T. reesei CE mixture with 100xa0% glucose yield. Data revealed the good potential of BglHi2 for the hydrolysis of waste papers, promising feedstocks for cellulosic ethanol production.

Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae).

Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine>spermidine>putrescine. Spermidine affected K(0.5) values for Na(+) with minor alterations in K(0.5) values for K(+) and NH(4)(+), causing a decrease in maximal velocities under saturating Na(+), K(+) and NH(4)(+) concentrations. Phosphorylation measurements in the presence of 20 microM ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na(+), both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na(+), the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na(+) at the Na(+)-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments.

Structural and kinetic alterations of constitutive conidial alkaline phosphatase from the osmotically-sensitive mutant ofNeurospora crassa

The osmotically-sensitiveos-1 mutant ofNeurospora crassa overproduced conidial alkaline phosphatase. The enzyme was purified by Phenyl-Sepharose CL-4B chromatography and Sephadex G-200 gel filtration. PAGE analysis of the purified enzyme suggested the occurrence of aggregation and/or disaggregation phenomena. The enzyme is a glycoprotein containing 16 % saccharide, with apparent molar mass of 137 kDa. Two protein bands (36 and 62 kDa) were observed in SDS-PAGE, suggesting that the native enzyme was a trimer. The pI was estimated to be 2.7, and optima of pH and temperature were 9.5 and 65 °C, respectively. The enzyme showed broad substrate specificity, hydrolyzing preferentially 4-nitrophenyl phosphate,O-phosphoamino-acids and 2-phosphoglycerate. The hydrolysis of 4-nitrophenyl phosphate was stimulated by CoII (26 %), NiII (23 %) and MgII ions (80 %). The enzyme was stable for up to 6 months at 4 °C in 5 mmol/L Tris-HCl buffer and also upon storage at 25 °C for 10 d. The kinetic and structural properties of the conidial enzyme purified from theos-1 mutant were quite different from those of the wild type strain. The enzyme overproduction observed in the mutant may be related to cell wall alterations that affect the process of enzyme secretion.

Production and secretion of a multifunctional ß-glucosidase by Humicola grisea var. thermoidea: effects of L-sorbose

The effects of L-sorbose on growth, morphology and production of a multifunctional ß-glucosidase by the thermophilic fungus Humicola grisea var. thermoidea were investigated. Sorbose increased the lag phase period 3-fold and drastically altered the morphology of the fungal hyphae. Cellobiose and lactose were good inducers of the enzyme. The addition of 5xa0% sorbose to cultures containing 1xa0% cellobiose enhanced the extracellular levels of the ß-glucosidase 3.3-fold with constant cytosolic and cell-wall bound levels, demonstrating stimulation of both enzyme synthesis and secretion. The stimulation of enzyme production by sorbose was dependent on the presence of cellobiose as inducer, since 2- to 3-fold inhibition was observed in lactose and glucose. Production and secretion of phosphatases and endoglucanases was not stimulated by sorbose, which did not affect the subcellular distribution of the ß-glucosidase also. However, it reduced the uptake rates of glucose and cellobiose. Taken together, the results discarded increased non-specific enzyme secretion and/or increased release of the enzyme from the cell-wall as possible molecular mechanisms for the effects of sorbose on the production of the multifunctional ß-glucosidase by H. grisea. An alternative mechanism, based on a prolonged action of cellobiose as inducer associated with a decreased catabolic repression by glucose, was discussed.