Different Covalent Immobilizations Modulate Lipase Activities of Hypocrea pseudokoningii.

Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application at large scale processes.

A High Redox Potential Laccase from Pycnoporus sanguineus RP15: Potential Application for Dye Decolorization.

Laccase production by Pycnoporus sanguineus RP15 grown in wheat bran and corncob under solid-state fermentation was optimized by response surface methodology using a Central Composite Rotational Design. A laccase (Lacps1) was purified and characterized and the potential of the pure Lacps1 and the crude culture extract for synthetic dye decolorization was evaluated. At optimal conditions (eight days, 26 °C, 18% (w/w) milled corncob, 0.8% (w/w) NH4Cl and 50 mmol·L(-1) CuSO4, initial moisture 4.1 mL·g(-1)), the laccase activity reached 138.6 ± 13.2 U·g(-1). Lacps1 was a monomeric glycoprotein (67 kDa, 24% carbohydrate). Optimum pH and temperature for the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) were 4.4 and 74.4 °C, respectively. Lacps1 was stable at pH 3.0-8.0, and after two hours at 55-60 °C, presenting high redox potential (0.747 V vs. NHE). ABTS was oxidized with an apparent affinity constant of 147.0 ± 6.4 µmol·L(-1), maximum velocity of 413.4 ± 21.2 U·mg(-1) and catalytic efficiency of 3140.1 ± 149.6 L·mmol(-1)·s(-1). The maximum decolorization percentages of bromophenol blue (BPB), remazol brilliant blue R and reactive blue 4 (RB4), at 25 or 40 °C without redox mediators, reached 90%, 80% and 60%, respectively, using either pure Lacps1 or the crude extract. This is the first study of the decolorization of BPB and RB4 by a P. sanguineus laccase. The data suggested good potential for treatment of industrial dye-containing effluents.

Co-cultivation of Aspergillus nidulans Recombinant Strains Produces an Enzymatic Cocktail as Alternative to Alkaline Sugarcane Bagasse Pretreatment.

Plant materials represent a strategic energy source because they can give rise to sustainable biofuels through the fermentation of their carbohydrates. A clear example of a plant-derived biofuel resource is the sugar cane bagasse exhibiting 60-80% of fermentable sugars in its composition. However, the current methods of plant bioconversion employ severe and harmful chemical/physical pretreatments raising biofuel cost production and environmental degradation. Replacing these methods with co-cultivated enzymatic cocktails is an alternative. Here we propose a pretreatment for sugarcane bagasse using a multi-enzymatic cocktail from the co-cultivation of four Aspergillus nidulans recombinant strains. The co-cultivation resulted in the simultaneous production of GH51 arabinofuranosidase (AbfA), GH11 endo-1,4-xylanase (XlnA), GH43 endo-1,5-arabinanase (AbnA) and GH12 xyloglucan specific endo-ß-1,4-glucanase (XegA). This core set of recombinant enzymes was more efficient than the alternative alkaline method in maintaining the cellulose integrity and exposing this cellulose to the following saccharification process. Thermogravimetric and differential thermal analysis revealed residual byproducts on the alkali pretreated biomass, which were not found in the enzymatic pretreatment. Therefore, the enzymatic pretreatment was residue-free and seemed to be more efficient than the applied alkaline method, which makes it suitable for bioethanol production.

Beauveria bassiana Lipase A expressed in Komagataella (Pichia) pastoris with potential for biodiesel catalysis.

Lipases (EC 3.1.1.3) comprise a biotechnologically important group of enzymes because they are able to catalyze both hydrolysis and synthesis reactions, depending on the amount of water in the system. One of the most interesting applications of lipase is in the biofuel industry for biodiesel production by oil and ethanol (or methanol) transesterification. Entomopathogenic fungi, which are potential source of lipases, are still poorly explored in biotechnological processes. The present work reports the heterologous expression and biochemical characterization of a novel Beauveria bassiana lipase with potential for biodiesel production. The His-tagged B. bassiana lipase A (BbLA) was produced in Komagataella pastoris in buffered methanol medium (BMM) induced with 1% methanol at 30°C. Purified BbLA was activated with 0.05% Triton X-100 and presented optimum activity at pH 6.0 and 50°C. N-glycosylation of the recombinant BbLA accounts for 31.5% of its molecular weight. Circular dichroism and molecular modeling confirmed a structure composed of α-helix and ß-sheet, similar to α/ß hydrolases. Immobilized BbLA was able to promote transesterification reactions in fish oil, demonstrating potential for biodiesel production. BbLA was successfully produced in K. pastoris and shows potential use for biodiesel production by the ethanolysis reaction.

Purification and biochemical properties of multiple xylanases from Aspergillus ochraceus tolerant to Hg2+ ion and a wide range of pH.

Production of multiple xylanases, in which each enzyme has a specific characteristic, can be one strategy to achieve the effective hydrolysis of xylan. Three xylanases (xyl 1, xyl 2, and xyl 3) from Aspergillus ochraceus were purified by chromatography using diethylaminoethyl (DEAE) cellulose, Biogel P-60, and Sephadex G-100 columns. These enzymes are glycoproteins of low molecular weight with an optimum temperature at 60 °C. The glycosylation presented is apparently not related to thermostability, since xyl 3 (20 % carbohydrate) was more thermostable than xyl 2 (67 % carbohydrate). Xyl 3 was able to retain most of its activity in a wide range of pH (3.5-8.0), while xyl 1 and xyl 2 presented optimum pH of 6.0. Xyl 1 and xyl 2 were activated by 5 and 10 mM MnCl2 and CoCl2, while xyl 3 was activated by 1 mM of the same compounds. Interestingly, xyl 2 presented high tolerance toward mercury ion. Xylanases from A. ochraceus hydrolyzed xylans of different origins, such as birchwood, oat spelt, larchwood, and eucalyptus (around 90 % or more), except xyl 2 and xyl 3 that hydrolyzed with lesser efficiency eucalyptus (66.7 %) and oat spelt (44.8 %) xylans.

A novel thermostable xylanase GH10 from Malbranchea pulchella expressed in Aspergillus nidulans with potential applications in biotechnology.

BACKGROUND: The search for novel thermostable xylanases for industrial use has intensified in recent years, and thermophilic fungi are a promising source of useful enzymes. The present work reports the heterologous expression and biochemical characterization of a novel thermostable xylanase (GH10) from the thermophilic fungus Malbranchea pulchella, the influence of glycosylation on its stability, and a potential application in sugarcane bagasse hydrolysis. RESULTS: Xylanase MpXyn10A was overexpressed in Aspergillus nidulans and was active against birchwood xylan, presenting an optimum activity at pH 5.8 and 80°C. MpXyn10A was 16% glycosylated and thermostable, preserving 85% activity after 24 hours at 65°C, and deglycosylation did not affect thermostability. Circular dichroism confirmed the high alpha-helical content consistent with the canonical GH10 family (ß/α)8 barrel fold observed in molecular modeling. Primary structure analysis revealed the existence of eight cysteine residues which could be involved in four disulfide bonds, and this could explain the high thermostability of this enzyme even in the deglycosylated form. MpXyn10A showed promising results in biomass degradation, increasing the amount of reducing sugars in bagasse in natura and in three pretreated sugarcane bagasses. CONCLUSIONS: MpXyn10A was successfully secreted in Aspergillus nidulans, and a potential use for sugarcane bagasse biomass degradation was demonstrated.

Optimization of ß-glucosidase, ß-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 ß-glucosidase, ß-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 ß-glucosidase, ß-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 (ß-glucosidase) or 65 °C (ß-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.

A novel xylan degrading ß-D-xylosidase: purification and biochemical characterization.

Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular ß-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. ß-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 °C and 3.0-5.5, respectively. ß-xylosidase was stable in acidic pH (3.0-6.0) and 70 °C for 1 h. The enzyme was activated by 5 mM MnCl2 (28 %) and MgCl2 (20 %) salts. The ß-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-ß-D-xylopyranoside, exhibiting apparent K(m) and V(max) values of 0.66 mM and 39 U (mg protein)⁻¹ respectively, and to a lesser extent p-nitrophenyl-ß-D-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel ß-D-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by ß-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.

Production of xylanase and ß-xylosidase from autohydrolysis liquor of corncob using two fungal strains.

Agroindustrial residues are materials often rich in cellulose and hemicellulose. The use of these substrates for the microbial production of enzymes of industrial interest is mainly due to their high availability associated with their low cost. In this work, corncob (CCs) particles decomposed to soluble compounds (liquor) were incorporated in the microbial growth medium through autohydrolysis, as a strategy to increase and undervalue xylanase and ß-xylosidase production by Aspergillus terricola and Aspergillus ochraceus. The CCs autohydrolysis liquor produced at 200 °C for 5, 15, 30 or 50 min was used as the sole carbon source or associated with untreated CC. The best condition for enzyme synthesis was observed with CCs submitted to 30 min of autohydrolysis. The enzymatic production with untreated CCs plus CC liquor was higher than with birchwood xylan for both microorganisms. A. terricola produced 750 total U of xylanase (144 h cultivation) and 30 total U of ß-xylosidase (96-168 h) with 0.75% untreated CCs and 6% CCs liquor, against 650 total U of xylanase and 2 total U of ß-xylosidase in xylan; A. ochraceus produced 605 total U of xylanase and 56 total U of ß-xylosidase (168 h cultivation) with 1% untreated CCs and 10% CCs liquor against 400 total U of xylanase and 38 total U of ß-xylosidase in xylan. These results indicate that the treatment of agroindustrial wastes through autohydrolysis can be a viable strategy in the production of high levels of xylanolytic enzymes.

Xylanase and ß-xylosidase production by Aspergillus ochraceus: new perspectives for the application of wheat straw autohydrolysis liquor.

The xylanase biosynthesis is induced by its substrate-xylan. The high xylan content in some wastes such as wheat residues (wheat bran and wheat straw) makes them accessible and cheap sources of inducers to be mainly applied in great volumes of fermentation, such as those of industrial bioreactors. Thus, in this work, the main proposal was incorporated in the nutrient medium wheat straw particles decomposed to soluble compounds (liquor) through treatment of lignocellulosic materials in autohydrolysis process, as a strategy to increase and undervalue xylanase production by Aspergillus ochraceus. The wheat straw autohydrolysis liquor produced in several conditions was used as a sole carbon source or with wheat bran. The best conditions for xylanase and ß-xylosidase production were observed when A. ochraceus was cultivated with 1% wheat bran added of 10% wheat straw liquor (produced after 15 min of hydrothermal treatment) as carbon source. This substrate was more favorable when compared with xylan, wheat bran, and wheat straw autohydrolysis liquor used separately. The application of this substrate mixture in a stirred tank bioreactor indicated the possibility of scaling up the process to commercial production.

Optimization of fibrolytic enzyme production by Aspergillus japonicus C03 with potential application in ruminant feed and their effects on tropical forages hydrolysis.

Fibrolytic enzyme production by Aspergillus japonicus C03 was optimized in a medium containing agro-industrial wastes, supplemented with peptone and yeast extract. A 2(3) full factorial composite and response surface methodology were used to design the experiments and analysis of results. Tropical forages were hydrolyzed by A. japonicus C03 enzymatic extract in different levels, and they were also tested as enzymatic substrate. Optimal production to xylanase was obtained with soybean bran added to crushed corncob (1:3), 0.01% peptone, and 0.2% yeast extract, initial pH 5.0, at 30 °C under static conditions for 5 days of incubation. Optimal endoglucanase production was obtained with wheat bran added to sugarcane bagasse (3:1), 0.01% peptone, and 0.2% yeast extract, initial pH 4.0, at 30 °C, for 6 days, under static conditions. Addition of nitrogen sources as ammonium salts either inhibited or did not influence xylanase production. This enzymatic extract had a good result on tropical forage hydrolyzes and showed better performance in the Brachiaria genera, due to their low cell wall lignin quantity. These results represent a step forward toward the use of low-cost agricultural residues for the production of valuable enzymes with potential application in animal feed, using fermentation conditions.

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii.

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60°C and 4.0, respectively. The enzyme was stable for 1 h at 55°C, showing a t50 of 53 min at 60°C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).

Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme.

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of approximately 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80-150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 degrees C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 degrees C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K(M) was 42 mM, and V(max) was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence.

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii.

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 degrees C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 degrees C, with a t (50) of 45 min at 60 degrees C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl alpha-D-maltoside, methyl-alpha-D-glucopyranoside, pullulan, alpha- and beta-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in alpha-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-alpha-D-glucan glucohydrolase).

Biochemical characterisation of the trehalase of thermophilic fungi: an enzyme with mixed properties of neutral and acid trehalase.

The trehalases from some thermophilic fungi, such as Humicola grisea, Scytalidium thermophilum, or Chaetomium thermophilum, possess mixed properties in comparison with those of the two main groups of trehalases: acid and neutral trehalases. Such as acid trehalases these enzymes are highly thermostable extracellular glycoproteins, which act at acidic pH. However, these enzymes are activated by calcium or manganese, and as a result inhibited by chelators and by ATP, properties typical of neutral trehalases. Here we extended the biochemical characterisation of these enzymes, by assaying their activity at acid and neutral pH. The acid activity (25-30% of total) was assayed in McIlvaine buffer at pH 4.5. Under these conditions the enzyme was neither activated by calcium nor inhibited by EDTA or ATP. The neutral activity was estimated in MES buffer at pH 6.5, after subtracting the activity resistant to EDTA inhibition. The neutral activity was activated by calcium and inhibited by ATP. On the other hand, the acid activity was more thermostable than the neutral activity, had a higher temperature optimum, exhibited a lower K(m), and different sensitivity to several ions and other substances. Apparently, these trehalases represent a new class of trehalases. More knowledge is needed about the molecular structure of this protein and its corresponding gene, to clarify the structural and evolutionary relationship of this trehalase to the conventional trehalases.

Phenoloxidase activity in Apis mellifera honey bee pupae, and ecdysteroid-dependent expression of the prophenoloxidase mRNA.

Phenoloxidase (monophenol, l-dopa: oxygen oxidoreductase, EC 1.14.18.1) is a multicopper oxidase, which plays an important role in melanin synthesis, necessary for defense against intruding microorganisms and parasites, wound healing and cuticle pigmentation. A phenoloxidase from the hemolymph of honey bee pupae exhibited an apparent molecular mass of 70 kDa, as estimated by gel filtration and SDS-PAGE. Optimal pH and temperature were 6.5 and 20 degrees C, respectively. Activity was fully stable for 30 min at 50 degrees C. Like phenoloxidases from the hemolymph of other insects, the honey bee enzyme was activated by trypsin and inhibited by protease inhibitors and phenylthiourea. Only high concentrations of sodium azide effectively inhibited the detected activity. A low concentration (5 microM) of Ca2+, Mg2+, and Mn2+ had a stimulatory effect on the activity. Single Michaelis-Menten curves were observed for l-dopa and dopamine oxidation, but the affinity of the enzyme for dopamine was greater than for L-dopa. Semiquantitative RT-PCR and Southern blot analysis using a 359 bp labeled probe, and quantification of the prophenoloxidase mRNA levels by real-time PCR showed increased amounts of transcripts in hemocytes and integument from young pupae injected with 20-hydroxyecdysone.

Characterization and properties of acid phosphatases with phytase activity produced by Aspergillus caespitosus.

High levels of thermostable acid phosphatases were produced by Aspergillus caespitosus in culture media supplemented with xylan birchwood or agricultural residues, as carbon sources. The optimal culture conditions for production of phosphatases were 40 degrees C and pH 6.0. Extra- and intra-cellular acid phosphatases were purified by chromatography on DEAE-cellulose, followed by concanavalin A-Sepharose affinity separation. Both extra- and intra-cellular enzymes were glycoproteins showing 63.0 and 58.3% of carbohydrate content respectively. Molecular masses estimated on Sepharose CL-6B column were 186 and 190+/-15 kDa, and 84 and 74+/-5 kDa according to SDS/PAGE, for extra- and intra-cellular acid phosphatases respectively. Taken together, these results suggest that both native enzymes were homodimers. Optimum temperature and pH for both phosphatase activities were 80 degrees C and 5.5 respectively. The extra- and intra-cellular acid phosphatases were stable for more than 60 min at 60 degrees C. The extracellular acid phosphatase was slightly inhibited by NaF, in contrast with the significant inhibition of the intracellular form. KH(2)PO(4) inhibited both activities equally. Both extra- and intracellular acid phosphatases were tartarate-resistant. Among several phosphorylated substrates used, the extracellular enzyme preferentially hydrolysed p-nitrophenyl phosphate. Kinetic parameters calculated for the hydrolysis of p-nitrophenyl phosphate by extracellular acid phosphatase were h (Hill coefficient)=1.2, K(0.5)=0.082 mM and V(max)=4.43 units/mg, whereas the intracellular enzyme exhibited Michaelian kinetics with K(m)=0.029 mM and V(max)=0.082 unit/mg. Phytase activity was also observed for both the enzymes, suggesting that they could be useful for biotechnological applications.

Influence of temperature on the properties of the xylanolytic enzymes of the thermotolerant fungus Aspergillus phoenicis.

This study reports on the effects of growth temperature on the secretion and some properties of the xylanase and beta-xylosidase activities produced by a thermotolerant Aspergillus phoenicis. Marked differences were observed when the organism was grown on xylan-supplemented medium at 25 degrees C or 42 degrees C. Production of xylanolytic enzymes reached maximum levels after 72 h of growth at 42 degrees C; and levels were three- to five-fold higher than at 25 degrees C. Secretion of xylanase and beta-xylosidase was also strongly stimulated at the higher temperature. The optimal temperature was 85 degrees C for extracellular and 90 degrees C for intracellular beta-xylosidase activity, independent of the growth temperature. The optimum temperature for extracellular xylanase increased from 50 degrees C to 55 degrees C when the fungus was cultivated at 42 degrees C. At the higher temperature, the xylanolytic enzymes produced by A. phoenicis showed increased thermostability, with changes in the profiles of pH optima. The chromatographic profiles were distinct when samples obtained from cultures grown at different temperatures were eluted from DEAE-cellulose and Biogel P-60 columns.

Rhizopus microsporus var. rhizopodiformis: a thermotolerant funguswith potential for production of thermostable amylases

The effect of several nutritional and environmental parameters on growth and amylase production from Rhizopus microsporus var. rhizopodiformis was analysed. This fungus was isolated from soil of the Brazilian «cerrado» and produced high levels of amylolytic activity at 45 degrees C in liquid medium supplemented with starch, sugar cane bagasse, oat meal or cassava flour. Glucose in the culture medium drastically repressed the amylolytic activity. The products of hydrolysis were analysed by thin layer chromatography, and glucose was detected as the main component. The amylolytic activity hydrolysed several substrates, such as amylopectin, amylase, glycogen, pullulan, starch, and maltose. Glucose was always the main end product detected by high-pressure liquid chromatography analysis. These results indicated that the amylolytic activity studied is a glucoamylase, but there were also low levels of alpha-amylase. As compared to other fungi, R. microsporus var. rhizopodiformis can be considered an efficient producer of thermostable amylases, using raw residues of low cost as substrates. This information is of technological value, considering the importance of amylases for industrial hydrolysis (AU)

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Rhizopus microsporus var. rhizopodiformis: a thermotolerant fungus with potential for production of thermostable amylases.

The effect of several nutritional and environmental parameters on growth and amylase production from Rhizopus microsporus var. rhizopodiformis was analysed. This fungus was isolated from soil of the Brazilian "cerrado" and produced high levels of amylolytic activity at 45 degrees C in liquid medium supplemented with starch, sugar cane bagasse, oat meal or cassava flour. Glucose in the culture medium drastically repressed the amylolytic activity. The products of hydrolysis were analysed by thin layer chromatography, and glucose was detected as the main component. The amylolytic activity hydrolysed several substrates, such as amylopectin, amylase, glycogen, pullulan, starch, and maltose. Glucose was always the main end product detected by high-pressure liquid chromatography analysis. These results indicated that the amylolytic activity studied is a glucoamylase, but there were also low levels of alpha-amylase. As compared to other fungi, R. microsporus var. rhizopodiformis can be considered an efficient producer of thermostable amylases, using raw residues of low cost as substrates. This information is of technological value, considering the importance of amylases for industrial hydrolysis.