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℃, respectively. The purified enzyme was thermostable up to 60 min in water at 55℃ and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60℃. The enzyme hydrolyzed p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-D-galactopyranoside, p-nitrophenyl-β-D-fucopyranoside, p-nitrophenyl-β-D-xylopyranoside, o-nitrophenyl-beta-β-galactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-β-D-fucopyranoside 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.
SigrafW: An easy‐to‐use program for fitting enzyme kinetic data Leone, Francisco Assis; Baranauskas, José Augusto; Furriel, Rosa Prazeres Melo ...
Biochemistry and molecular biology education,
November 2005, 2005-11-00, 2005-Nov, 20051101, Volume:
33, Issue:
6
Journal Article
Peer reviewed
Open access
SigrafW is Windows‐compatible software developed using the Microsoft® Visual Basic Studio program that uses the simplified Hill equation for fitting kinetic data from allosteric and Michaelian ...enzymes. SigrafW uses a modified Fibonacci search to calculate maximal velocity (V), the Hill coefficient (n), and the enzyme‐substrate apparent dissociation constant (K). The estimation of V, K, and the sum of the squares of residuals is performed using a Wilkinson nonlinear regression at any Hill coefficient (n). In contrast to many currently available kinetic analysis programs, SigrafW shows several advantages for the determination of kinetic parameters of both hyperbolic and nonhyperbolic saturation curves. No initial estimates of the kinetic parameters are required, a measure of the goodness‐of‐the‐fit for each calculation performed is provided, the nonlinear regression used for calculations eliminates the statistical bias inherent in linear transformations, and the software can be used for enzyme kinetic simulations either for educational or research purposes.
Persons interested in receiving a free copy of the software should contact Dr. F. A. Leone.
Xylose isomerase catalyzes the isomerization of D-xylose to D-xylulose with promiscuous activity for other saccharides including D-glucose, D-allose, and L-arabinose. The xylose isomerase from the ...fungus Piromyces sp. E2 (PirE2_XI) is used to engineer xylose usage by the fermenting yeast Saccharomyces cerevisiae, but its biochemical characterization is poorly understood with divergent catalytic parameters reported. We have measured the kinetic parameters of the PirE2_XI and analyzed its thermostability and pH-dependence towards different substrates. The PirE2_XI shows promiscuous activity towards D-xylose, D-glucose, D-ribose and L-arabinose with variable effects depending on different divalent ions and epimerizes D-xylose at C3 to produce D-ribulose in a substrate/product dependent ratio. The enzyme follows Michaelis-Menten kinetics for the substrates used and although KM values for D-xylose are comparable at 30 and 60 °C, the kcat/KM is three-fold greater at 60 °C. The purified PirE2_XI shows maximal activity at 65 °C in the pH range of 6.5–7.5 and is a thermostable enzyme, maintaining full activity over 48 h at 30 °C or 12 h at 60 °C. This is the first report demonstrating epimerase activity of the PirE2_XI and its ability to isomerize D-ribose and L-arabinose, and provides a comprehensive in vitro study of substrate specificity, effect of metal ions and temperature on enzyme activity and these findings advance the knowledge of the mechanism of action of this enzyme.
•Kinetic parameters measured for the xylose isomerase from Piromyces sp. E2 (PirE2_XI).•The PirE2_XI isomerizes D-xylose, D-glucose, D-ribose and L-arabinose.•The PirE2_XI epimerizes D-xylose to D-ribulose via a D-xylulose intermediate.•Biophysical characterization shows the PirE2_XI is thermostable at 30 and 60 °C.
Rat osseous plate alkaline phosphatase, a glycosylphosphatidylinositol (GPI)-anchored phosphomonohydrolase, was immobilized on Langmuir–Blodgett (LB) films. Enzyme solubilization either with ...polyoxyethylene-9-lauryl ether or with a glycosylphosphatidylinositol-specific phospholipase C resulted in a GPI-anchor-containing and a GPI-anchor-depleted form, respectively. Both forms were adsorbed on dimyristoylphosphatidic acid LB films and restricted to the outermost layer. The surface density and enzyme activity were determined using a quartz crystal microbalance and
p-nitrophenylphosphatase activity, respectively. The detergent-solubilized form was co-spread with dimyristoylphosphatidic acid on the air/water interface and transferred to solid supports, providing an enzyme maximum surface density of 530 ng/cm
2. Maximal phosphohydrolytic activity, corresponding to 43% of that observed in homogeneous medium, was obtained at a surface density of 179 ng/cm
2. The phospholipase C-solubilized form was adsorbed directly from solution, reaching a maximum surface density of 1541 ng/cm
2, although the phosphomonohydrolase activity was 10 times lower than that obtained for the anchor-containing form. The combined analysis of surface density and enzymatic activity suggests that the alignment of the protein molecules on the LB lipid films induced by the glycosylphosphatidylinositol anchor facilitates the access of the substrate to the active site. This access is hampered by increasing enzyme surface densities and depends on a specific orientation of the adsorbed enzyme.
The kinetics and the adsorption isotherms of the surfactant-solubilized alkaline phosphatase from rat osseous plate adsorbed by dip-coating on dimyristoyl phosphatidic acid (DMPA) Langmuir–Blodgett ...(LB) films were studied. The phosphomonohydrolase activity of the enzyme on the LB film was estimated by the hydrolysis of
p-nitrophenylphosphate (PNPP). Films prepared from solutions containing 0.30 μg ml
−1 of protein showed maximum activity for the supported enzyme above the critical micellar concentration of the non-ionic surfactant (polyoxyethylene-9-lauryl ether) used for enzyme solubilization. The surface density of the enzyme on DMPA LB films was determined from quartz crystal microbalance measurements. A consistent explanation concerning the maximum enzymatic activity is supported by data of surface tension for the mixed non-ionic surfactant–enzyme system.
The morphology of phospholipid Langmuir–Blodgett films containing a glycosylphosphatidylinositol-anchored rat osseous plate alkaline phosphatase was studied by atomic force microscopy. Two forms of ...the enzyme were used in this study: an anchor-containing detergent-solubilized form, and a phospholipase-solubilized form, without the hydrophobic moiety. Direct measurements of catalytic activity in enzyme/phospholipid mixed films, and nanogravimetric estimations by quartz crystal microbalance confirmed the adsorption of both forms of alkaline phosphatase. Atomic force microscopy analysis showed that both enzyme forms originated aggregates when adsorbed on phospholipid Langmuir–Blodgett films. However, the phospholipase-solubilized form showed a higher roughness when compared to the detergent solubilized alkaline phosphatase. A model suggesting different alignments of the major axis of the ellipsoid polypeptide moiety relative to the film surface is proposed. This model, supported by surface density and catalytic activity data, might explain the difference of roughness observed for films containing each enzyme form adsorbed onto a phospholipid-modified solid support.
Aiming to clarify the mechanism of inhibition of (Na super(+), K super(+))-ATPase activity by polyamines, we examined the effects of exogenous putrescine, spermidine, and spermine on the kinetic ...behavior of phosphoenzyme-linked partial reactions using a microsomal gill (Na super(+), K super(+))-ATPase from juvenile and adult M. amazonicum, a freshwater palaemonid shrimp. The time course of phosphointermediate formation is greater (0.089 plus or minus 0.006 s super(-1)) in adults than in juveniles (0.053 plus or minus 0.003 s super(-1)) for spermidine, but similar to juveniles (0.059 plus or minus 0.004 s super(-1)) for putrescine. Maximum phosphointermediate formation for the (Na super(+), K super(+))-ATPase from juveniles decreased by 46% and 32% with spermidine and putrescine, respectively. In adults, maximum phosphointermediate levels decreased by 50% and 8%, respectively. For both spermidine and putrescine, dephosphorylation rates were higher for adults than for juveniles, and were higher than in controls without polyamines. Spermine had a negligible effect (<10%) on phosphorylation/dephosphorylation rates of both juvenile and adult enzymes. This is the first report on the effects of polyamines on phosphoenzyme-linked partial reactions in juvenile and adult M. amazonicum gill (Na super(+), K super(+))-ATPases. Our findings suggest that the phosphorylation/dephosphorylation steps of this gill enzyme may be regulated by polyamines during ontogenetic development.
Aiming to clarify the mechanism of inhibition of (Na
+
, K
+
)-ATPase activity by polyamines, we examined the effects of exogenous putrescine, spermidine, and spermine on the kinetic behavior of ...phosphoenzyme-linked partial reactions using a microsomal gill (Na
+
, K
+
)-ATPase from juvenile and adult
M. amazonicum
, a freshwater palaemonid shrimp. The time course of phosphointermediate formation is greater (0.089 ± 0.006 s
−1
) in adults than in juveniles (0.053 ± 0.003 s
−1
) for spermidine, but similar to juveniles (0.059 ± 0.004 s
−1
) for putrescine. Maximum phosphointermediate formation for the (Na
+
, K
+
)-ATPase from juveniles decreased by 46% and 32% with spermidine and putrescine, respectively. In adults, maximum phosphointermediate levels decreased by 50% and 8%, respectively. For both spermidine and putrescine, dephosphorylation rates were higher for adults than for juveniles, and were higher than in controls without polyamines. Spermine had a negligible effect (<10%) on phosphorylation/dephosphorylation rates of both juvenile and adult enzymes. This is the first report on the effects of polyamines on phosphoenzyme-linked partial reactions in juvenile and adult
M. amazonicum
gill (Na
+
, K
+
)-ATPases. Our findings suggest that the phosphorylation/dephosphorylation steps of this gill enzyme may be regulated by polyamines during ontogenetic development.
A mycelial β-glucosidase from the thermophilic mold
Humicola insolens was purified and biochemically characterized. The enzyme showed carbohydrate content of 21% and apparent molecular mass of 94
...kDa, as estimated by gel filtration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a single polypeptide band of 55
kDa, suggesting that the native enzyme was a homodimer. Mass spectrometry analysis showed amino acid sequence similarity with a β-glucosidase from
Humicola grisea var.
thermoidea, with about 22% coverage. Optima of temperature and pH were 60
°C and 6.0–6.5, respectively. The enzyme was stable up to 1
h at 50
°C and showed a half-life of approximately 44
min at 55
°C. The β-glucosidase hydrolyzed cellobiose, lactose, p-nitrophenyl-β-
d-glucopyranoside, p-nitrophenyl-β-
d-fucopyranoside, p-nitrophenyl-β-
d-xylopyranoside, p-nitrophenyl-β-
d-galactopyranoside, o-nitrophenyl-β-
d-galactopyranoside, and salicin. Kinetic studies showed that p-nitrophenyl-β-
d-fucopyranoside and cellobiose were the best enzyme substrates. Enzyme activity was stimulated by glucose or xylose at concentrations up to 400
mM, with maximal stimulatory effect (about 2-fold) around 40
mM. The high catalytic efficiency for the natural substrate, good thermal stability, strong stimulation by glucose or xylose, and tolerance to elevated concentrations of these monosaccharides qualify this enzyme for application in the hydrolysis of cellulosic materials.
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