Luis Henrique Souza Guimarães

Screening of filamentous fungi for production of enzymes of biotechnological interest

Many enzymes produced by fungi have relevant biotechnological applications in several industrial areas. The purpose of this study was to collect and isolate filamentous fungi from soil and humus, plants and sugar cane bagasse of different regions of the Sao Paulo state. Forty isolates were examined for their ability to produce xylanase, glucose-oxidase, alkaline phosphatase, acid phosphatase, phytase, pectinase and amylase. Among these, twenty three isolates exhibited enzymatic potential. The xylanases produced by two of these isolates (Aspergillus caespitosus and A. phoenicis) showed good potential for pulp bleaching. Among seventeen isolates, at least three produced high levels of glucose-oxidase, being Rhizopus stolonifer and A. versicolor the best producer strains. A. caespitosus, Mucor rouxii, and nine others still not identified were the best producers of phosphatases in submerged fermentation. Pectinase was best produced by IF II and C-8 belong R. stolonifer. Significant levels of amylase were produced by Paecilomyces variotii and A. phoenicis. A remarkable enzyme producer was Rhizopus microsporus var. rhizopodiformis that produced high levels of amylase, alkaline and acid phosphatases, and pectinase. Some morphological structures of this fungus were illustrated using light microscopy (LM) and scanning electron microscopy (SEM). This study contributes to catalogue soil fungi isolated in the state of Sao Paulo, and provides additional information to support future research about the industrial potential of these microorganisms that may produce enzymes and, eventually, also secondary metabolites with anti-microbial or anti-parasitic activities.

Production of thermostable invertases by Aspergillus caespitosus under submerged or solid state fermentation using agroindustrial residues as carbon source

The filamentous fungus Aspergillus caespitosus was a good producer of intracellular and extracellular invertases under submerged (SbmF) or solid-state fermentation (SSF), using agroindustrial residues, such as wheat bran, as carbon source. The production of extracellular enzyme under SSF at 30°C, for 72h, was enhanced using SR salt solution (1:1, w/v) to humidify the substrate. The extracellular activity under SSF using wheat bran was around 5.5-fold higher than that obtained in SbmF (Khanna medium) with the same carbon source. However, the production of enzyme with wheat bran plus oat meal was 2.2-fold higher than wheat bran isolated. The enzymatic production was affected by supplementation with nitrogen and phosphate sources. The addition of glucose in SbmF and SSF promoted the decreasing of extracellular activity, but the intracellular form obtained in SbmF was enhanced 3-5-fold. The invertase produced in SSF exhibited optimum temperature at 50°C while the extra- and intracellular enzymes produced in SbmF exhibited maximal activities at 60°C. All enzymatic forms exhibited maximal activities at pH 4.0-6.0 and were stable up to 1 hour at 50°C.

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.

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.

Thermostable invertases from Paecylomyces variotii produced under submerged and solid-state fermentation using agroindustrial residues

The filamentous fungus Paecylomices variotii was able to produce high levels of cell extract and extracellular invertases when grown under submerged fermentation (SbmF) and solid-state fermentation, using agroindustrial products or residues as substrates, mainly soy bran and wheat bran, at 40°C for 72xa0h and 96xa0h, respectively. Addition of glucose or fructose (≥1%; w/v) in SbmF inhibited enzyme production, while the addition of 1% (w/v) peptone as organic nitrogen source enhanced the production by 3.7-fold. However, 1% (w/v) (NH4)2HPO4 inhibited enzyme production around 80%. The extracellular form was purified until electrophoretic homogeneity (10.5-fold with 33% recovery) by DEAE-Fractogel and Sephacryl S-200 chromatography. The enzyme is a monomer with molecular mass of 102xa0kDa estimated by SDS–PAGE with carbohydrate content of 53.6%. Optima of temperature and pH for both, extracellular and cell extract invertases, were 60°C and 4.0–4.5, respectively. Both invertases were stable for 1xa0h at 60°C with half-lives of 10xa0min at 70°C. Mg2+, Ba2+ and Mn2+ activated both extracellular and cell extract invertases from P. variotii. The kinetic parameters Km and Vmax for the purified extracellular enzyme corresponded to 2.5xa0mM and 481xa0U/mg prot−1, respectively.

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 °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 °C and 5.5 respectively. The extra‐ and intra‐cellular acid phosphatases were stable for more than 60 min at 60 °C. The extracellular acid phosphatase was slightly inhibited by NaF, in contrast with the significant inhibition of the intracellular form. KH2PO4 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, K0.5=0.082 mM and Vmax=4.43 units/mg, whereas the intracellular enzyme exhibited Michaelian kinetics with Km=0.029 mM and Vmax=0.082 unit/mg. Phytase activity was also observed for both the enzymes, suggesting that they could be useful for biotechnological applications.

Thermostable conidial and mycelial alkaline phosphatases from the thermophilic fungus Scytalidium thermophilum

An extracellular (conidial) and an intracellular (mycelial) alkaline phosphatase from the thermophilic fungus Scytalidium thermophilum were purified by DEAE-cellulose and Concanavalin A-Sepharose chromatography. These enzymes showed allosteric behavior either in the presence or absence of MgCl2, BaCl2, CuCl2, and ZnCl2. All of these ions increased the maximal velocity of both enzymes. The molecular masses of the conidial and mycelial enzymes, estimated by gel filtration, were 162 and 132 kDa, respectively. Both proteins migrated on SDS-PAGE as a single polypeptide of 63 and 58.5 kDa, respectively, suggesting that these enzymes were dimers of identical subunits. The best substrate for the conidial and mycelial phosphatases was p-nitrophenylphosphate, but β-glycerophosphate and other phosphorylated compounds also served as substrates. The optimum pH for the conidial and mycelial alkaline phosphatases was 10.0 and 9.5 in the presence of AMPOL buffer, and their carbohydrate contents were about 54% and 63%, respectively. The optimum temperature was 70–75°C for both activities. The enzymes were fully stable up to 1 h at 60°C. These and other properties suggested that the alkaline phosphatases of S. thermophilum might be suitable for biotechnological applications. Journal of Industrial Microbiology & Biotechnology (2001) 27, 265–270.

Effect of glycosylation on the biochemical properties of β-xylosidases from Aspergillus versicolor

Aspergillus versicolor grown on xylan or xylose produces two β-xylosidases with differences in biochemical properties and degree of glycosylation. We investigated the alterations in the biochemical properties of these β-xylosidases after deglycosylation with Endo-H or PNGase F. After deglycosylation, both enzymes migrated faster in PAGE or SDS-PAGE exhibiting the same Rf. Temperature optimum of xylan-induced and xylose-induced β-xylosidases was 45°C and 40°C, respectively, and 35°C after deglycosylation. The xylan-induced enzyme was more active at acidic pH. After deglycosylation, both enzymes had the same pH optimum of 6.0. Thermal resistance at 55°C showed half-life of 15 min and 9 min for xylose- and xylan-induced enzymes, respectively. After deglycosylation, both enzymes exhibited half-lives of 7.5 min. Native enzymes exhibited different responses to ions, while deglycosylated enzymes exhibited identical responses. Limited proteolysis yielded similar polypeptide profiles for the deglycosylated enzymes, suggesting a common polypeptide core with differential glycosylation apparently responsible for their biochemical and biophysical differences.

A high-throughput method for GMO multi-detection using a microfluidic dynamic array

The ever-increasing production of genetically modified crops generates a demand for high-throughput DNA-based methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the number of GMOs that is potentially present in an individual sample. The present work presents the results of an innovative approach in genetically modified crops analysis by DNA based methods, which is the use of a microfluidic dynamic array as a high throughput multi-detection system. In order to evaluate the system, six test samples with an increasing degree of complexity were prepared, preamplified and subsequently analysed in the Fluidigm system. Twenty-eight assays targeting different DNA elements, GM events and species-specific reference genes were used in the experiment. The large majority of the assays tested presented expected results. The power of low level detection was assessed and elements present at concentrations as low as 0.06xa0% were successfully detected. The approach proposed in this work presents the Fluidigm system as a suitable and promising platform for GMO multi-detection.

Purification and Biochemical Characterization of Thermostable Alkaline Phosphatases Produced by Rhizopus microsporus var. rhizopodiformis

The biochemical properties of the alkaline phosphatases (AlPs) produced by Rhizopus microsporus are described. High enzymic levels were produced within 1–2 d in agitated cultures with 1 % wheat bran. Intra- and extracellular AlPs were purified 5.0 and 9.3×, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and 120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for both forms. Mn2+, Na+ and Mg2+ stimulated the activity, while Al3+ and Zn2+ activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 °C and pH 8.0, respectively. The enzymes were stable at 50 °C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a Km 0.28 and 0.22 mmol/L, with υlim 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable for biotechnological application.