Peroxidase from peach fruit: Thermal stability

Peroxidase from peach fruit was purified 28.9-fold by DEAE-cellulose, Sephadex G-100 and hydroxylapatite chromatography. The purified enzyme showed only one peak of activity with an optimum pH of 5.0 and temperature of 40 degreesC. The calculated activation energy (Ea) for the reaction was 7.97 kcal/mol. The enzyme was heat-labile in the temperature range of 60 to 80 degreesC with a fast inactivation at 80 degreesC. PAGE of the inactivation course at 70 degreesC showed only one band of activity. Different sugars increased the heat stability of the activity in the following order: sucrose>lactose>glucose>fructose. Measurement of residual activity showed a stabilizing effect of sucrose at various temperature/sugar concentrations (10 to 40%, w/w) with the Ea for inactivation increasing with sucrose concentration from 0 to 20% (w/w). After inactivation at 70 degreesC and 75 degreesC the enzyme was able to be reactivated by up to 40% of the initial activity when stared at 30 degreesC.

Purification and characterization of beta-fructosidase with inulinase activity from Aspergillus niger-245

Aspergillus niger - 245 a strain isolated from soil samples showed good beta -fructosidase activity when inoculated in medium formulated with dahlia extract tubers. The enzyme was purified by precipitation in ammonium sulphate and percolated in DEAE-Sephadex A-50 and CM-cellulose columns, witch showed a single peack in all the purification steps, maintaining the I/S ratio between 0.32 to, 0.39. Optimum pH for inulinase activity (I) was between 4.0 - 4.5 and for invertase activity (S) between 2.5 and 50. The optimum temperature was 60 degrees .C for both activities and no loss in activity was observed when it was maintained at this temperature for 30 min. The K-m value was 1.44 and 5.0 respectively, for I and S and V-m value 10.48 and 30.55 respectively. The I activity was strongly inhibited by Hg2+ and Ag+ and 2 x 10(-3) M of glucose, but not by fructose at the same concentration. The enzyme showed an exo-action mechanism acting on the inulin of different origins. In assay conditions total hydrolysis of all the frutans was obtained although it has shown larger activity on the chicory inulin than that one from artichoke Jerusalem and dahlia, in the first 30 min. The obtained results suggested that the enzyme presented good potential for industrial application in the preparing the fructose syrups.

Production of extracellular alkaline proteases by Aspergillus clavatus

The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37degreesC. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25degreesC for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH4NO3 showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40degreesC and the half-lives at 40, 45 and 50degreesC were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.

Partial purification and characterization of pectin methylesterase from orange (Citrus sinensis) cv. Pera-rio

The enzyme pectin methylesterase (PME) from orange was extracted and partially purified by filtration on Sephadex G-100. The extraction buffer for orange PME was borate-acetate containing 0.4 M NaCl. Orange PME showed optimum pH at 8.0 and optimum temperature at 50C. The PME enzyme was completely inactivated after 1 min of incubation at 90C. The specific activity increased in the presence of 0.15 M NaCl or 0.025 M Na2SO4, 0.10 M KCl, 0.025 M K2SO4, 0.05 and 0.1 M NH4Cl. Lithium chloride and Li(2)SO(4)inhibited the enzymatic activity at all concentrations studied. The K-m and V(max)value of PME were 0.36 mg/mL and 5.26 mu mol/mL-mg protein, respectively.

Purification and characterization of a cyclomaltodextrin glucanotransferase from Paenibacillus campinasensis strain H69-3

A cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19) from a newly isolated alkalophilic and moderately thermophilic Paenibacillus campinasensis strain H69-3 was purified as a homogeneous protein from culture supernatant. Cyclomaltodextrin glucanotransferase was produced during submerged fermentation at 45 degrees C and purified by gel filtration on Sephadex G50 ion exchange using a Q-Sepharose column and ion exchange using a Mono-Q column. The molecular weight of the purified enzyme was 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the pI was 5.3. The optimum pH for enzyme activity was 6.5, and it was stable in the pH range 6.0-11.5. The optimum temperature was 65 degrees C at pH 6.5, and it was thermally stable up to 60 degrees C without substrate during 1 h in the presence of 10 mm CaCl2. The enzyme activity increased in the presence of Co2+, Ba2+, and Mn2+. Using maltodextrin as substrate, the K-m and K-cat were 1.65 mg/mL and 347.9 mu mol/mg.min, respectively.

Purification and characterization of two beta-glucosidases from the thermophilic fungus Thermoascus aurantiacus

beta-Glucosidase from the fungus Thermoascus aurantiacus grown oil semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps - ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized. After the first anion exchange chromatography, beta-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3). Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix. Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively. The temperature optimum of both glucosidases was at 75-80 degreesC. The pH stability of Gl-2 (4.0-9.0) was higher than Gl-3 (5.5-8.5); both enzyme activities showed similar patterns of thermostability. Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively. Using 4-nitrophenyl beta-D-glucopyranoside as substrate, K-m, values of 1.17 +/- 0.35 and 1.38 +/- 0.86 mmol/L were determined for Gl-2 and Gl-3, respectively. Both enzymes were inhibited by Ag+ and stimulated by Ca2+.

Photodegradation of dichloroacetic acid and 2,4-dichlorophenol by ferrioxalate/H2O2 system

The photo-Fenton process using potassium ferrioxalate as a mediator in the photodegradation reaction of organochloride compounds in an aqueous medium was investigated. The influence of parameters such as hydrogen peroxide and ferrioxalate concentrations and initial pH, was evaluated using dichloroacetic acid (DCA) as a model compound under black-light lamp irradiation. An upflow annular photoreactor, operating in a single pass or recirculating mode was used during photodegradation experiments with artificial light. The extent of the release of chloride ions was used to evaluate the photodegradation reaction. The optimum pH range observed was 2.5-2.8. The efficiency of DCA dechlorination increased with increasing concentrations of H2O2 and potassium ferrioxalate, reaching a plateau after the addition of 6 and 1.5 mmol/L of those reagents, respectively. The total organic carbon (TOC) content in DCA and 2,4-dichlorophenol (DCP) solutions was compared with the chloride released after photodegradation. The influence of natural solar light intensity, measured at 365 nm, was evaluated for the dechlorination of DCA on typical summer's days showing a linear dependency. The photodegradation of DCA using black-light lamp and solar irradiation was compared.

Purification and characterization of two β-glucosidases from the thermophilic fungus Thermoascus aurantiacus

β-Glucosidase from the fungus Thermoascus aurantiacus grown on semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps-ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized. After the first anion exchange chromatography, β-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3). Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix. Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively. The temperature optimum of both glucosidases was at 75-80°C. The pH stability of Gl-2 (4.0-9.0) was higher than Gl-3 (5.5-8.5); both enzyme activities showed similar patterns of thermostability. Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively. Using 4-nitrophenyl β-D-glucopyranoside as substrate, Km values of 1.17 ± 0.35 and 1.38 ± 0.86 mmol/L were determined for Gl-2 and Gl-3, respectively. Both enzymes were inhibited by Ag+ and stimulated by Ca2+.

Effect of Bacillus circulans D1 thermostable xylanase on biobleaching of eucalyptus kraft pulp

The alkalophilic Bacillus circulans D1 was isolated from decayed wood. It produced high levels of extracellular cellulase-free xylanase. The enzyme was thermally stable up to 60°C, with an optimal hydrolysis temperature of 70°C. It was stable over a wide pH range (5.5-10.5), with an optimum pH at 5.5 and 80% of its activity at pH 9.0. This cellulase-free xylanase preparation was used to biobleach kraft pulp. Enzymatic treatment of kraft pulp decreased chlorine dioxide use by 23 and 37% to obtain the same kappa number (κ number) and brightness, respectively. Separation on Sephadex G-50 isolated three fractions with xylanase activity with distinct molecular weights.

The dermatophyte Trichophyton rubrum secretes an EDTA-sensitive alkaline phosphatase on high-phosphate medium

In this communication, we show that the growth of isolate H6 of the dermatophyte Trichophyton rubrum on non-buffered medium and under saturating phosphate conditions is dependent on the initial growth pH, with an apparent optimum at pH 4.0. In addition, irrespective of the initial growth pH, the pH of the medium altered during cultivation reaching values that ranged from 8.3 to 8.9. Furthermore, this isolate synthesized and secreted almost the same levels of an alkaline phosphatase with an apparent optimum pH ranging from 9.0 to 10.0 when grown on both low- and high-phosphate medium. Also, this alkaline phosphatase is activated by Mg2+ and is EDTA-sensitive. On the other hand, the very low levels of the enzyme retained by the mycelium grown on buffered medium at pH 5.0-5.2 suggest that this enzyme is encoded by an alkaline gene, i.e., a gene responsive to ambient pH signaling.

Purification and characterization of xylanases from Aspergillus giganteus

A strain of Aspergillus giganteus cultivated in a medium with xylan produced two xylanases (xylanase I and II) which were purified to homogeneity. Their molar mass, estimated by SDS-PAGE, were 21 and 24 kDa, respectively. Both enzymes are glycoproteins with 50°C temperature optimum; optimum pH was 6.0-6.5 for xylanase I and 6.0 for xylanase II. At 50°C xylanase I exhibited higher thermostability than xylanase II. Hg2+, Cu 2+ and SDS were strong inhibitors, 1,4-dithiothreitol stimulated the reaction of both enzymes. Both xylanases are xylan-specific; kinetic parameters indicated higher efficiency in the hydrolysis of oat spelts xylan. In hydrolysis of this substrate, xylotriose, xylotetraose and larger xylooligosaccharides were released and hence the enzymes were classified as endoxylanases.

Production of CGTase by a bacillus Alkalophilic CGII strain isolated from wastewater of a manioc flour industry

GCTase production by a new strain of Bacillus alkalophillc CGII isolated from Brazilian wastewater of manioc flour industry was examined. The growth medium used was composed by 1.5% starch, 1.5% nitrogen and 1% Na 2CO3. Higher activity was obtained with starch, maltodextrin and galactose. When glucose was added to the medium, no enzyme production was observed. High enzyme activity and growth were reached when aeration was increased (88.6 U/mL). The enzyme characterization showed an optimum pH and temperature 8.0 and 55°C for starch hydrolyses, respectively. Mg+ and Ca++ showed small activation; however, Hg + and Cu+ showed a strong enzyme inhibition.

Production of cellulolytic and hemicellulolytic enzymes from Aureobasidium pulluans on solid state fermentation

This article investigates a strain of the yeast Aureobasidium pullulans for cellulase and hemicellulase production in solid state fermentation. Among the substrates analyzed, the wheat bran culture presented the highest enzymatic production (1.05 U/mL endoglucanase, 1.3 U/mL β-glucosidase, and 5.0 U/mL xylanase). Avicelase activity was not detected. The optimum pH and temperature for xylanase, endoglucanase and β-glucosidase were 5.0 and 50, 4.5 and 60, 4.0 and 75°C, respectively. These enzymes remained stable between a wide range of pH. The β-glucosidase was the most thermostable enzyme, remaining 100% active when incubated at 75°C for 1 h. © 2007 Humana Press Inc.

Purification of an exopolygalacturonase from Penicillium viridicatum RFC3 produced in submerged fermentation

An exo-PG obtained from Penicillium viridicatum in submerged fermentation was purified to homogeneity. The apparent molecular weight of the enzyme was 92 kDa, optimum pH and temperature for activity were pH 5 and 50-55°C. The exo-PG showed a profile of an exo-polygalacturonase, releasing galacturonic acid by hydrolysis of pectin with a high degree of esterification (D.E.). Ions Ca 2+ enhanced the stability of enzyme and its activity by 30%. The K m was 1.30 in absence of Ca 2+ and 1.16mg mL -1 in presence of this ion. In relation to the Vmax the presence of this ion increased from 1.76 to 2.07 μmol min -1mg -1. Copyright © 2009 Eleni Gomes et al.

Production of pectate lyase by penicillium viridicatum RFC3 in solid-state and submerged fermentation

Pectate lyase (PL) was produced by the filamentous fungus Penicillium viridicatum RFC3 in solid-state cultures of a mixture of orange bagasse and wheat bran (1 : 1 w/w), or orange bagasse, wheat bran and sugarcane bagasse (1 : 1 : 0.5 w/w), and in a submerged liquid culture with orange bagasse and wheat bran (3%) as the carbon source. PL production was highest (1,500U mL -1 or 300Ug -1 of substrate) in solid-state fermentation (SSF) on wheat bran and orange bagasse at 96 hours. PL production in submerged fermentation (SmF) was influenced by the initial pH of the medium. With the initial pH adjusted to 4.5, 5.0, and 5.5, the peak activity was observed after 72, 48, and 24 hours of fermentation, respectively, when the pH of the medium reached the value 5.0. PL from SSF and SmF were loaded on Sephadex-G75 columns and six activity peaks were obtained from crude enzyme fromSSF and designated PL I, II, III, IV, V, andVI, while five peaks were obtained fromcrude enzyme fromSmF and labeled PL I', II', III', IV', and VII'. Crude enzyme and fraction III from each fermentative process were tested further. The optimum pH for crude PL from either process was 5.5, while that for PL III was 8.0. The maximum activity of enzymes from SSF was observed at 35°C, but crude enzyme was more thermotolerant than PL III, maintaining its maximum activity up to 45°C. Crude enzyme from SmF and PL III' showed thermophilic profiles of activity, with maximum activity at 60 and 55°C, respectively. In the absence of substrate, the crude enzyme from SSF was stable over the pH range 3.0-10.0 and PL III was most stable in the pH range 4.0-7.0. Crude enzyme from SmF retained 70%-80% of its maximum activity in the acid-neutral pH range (4.0-7.0), but PIII showed high stability at alkaline pH (7.5-9.5). PL from SSF was more thermolabile than that from SmF. The latter maintained 60% of its initial activity after 1 h at 55°C. The differing behavior of the enzymes with respect to pH and temperature suggests that they are different isozymes. Copyright © 2010 Viviani Ferreira et al.