Marli Camassola

Characterization of the Cellulase Complex of Penicillium echinulatum

New cellulases from a strain of Penicillium echinulatum were characterized for their filter paper activity and β-glucosidase activity. Both activities showed maximum values between pH 4 and 5. With citrate buffer, activities were slightly higher than in acetate buffer of the same pH. Thermal stability of both activities was good up to 55°C. Filter paper activity was significantly reduced at higher temperatures.

Aqueous extracts of Lentinula edodes and Pleurotus sajor-caju exhibit high antioxidant capability and promising in vitro antitumor activity

Mushroom extracts are increasingly sold as dietary supplements because of several of their properties, including the enhancement of immune function and antitumor activity. We hypothesized that soluble polar substances present in mushroom extracts may show antioxidant and anticancer properties. This report shows that Brazilian aqueous extracts of Lentinula edodes and Pleurotus sajor-caju exert inhibitory activity against the proliferation of the human tumor cell lines laryngeal carcinoma (Hep-2) and cervical adenocarcinoma (HeLa). Cell viability was determined after using 3 different temperatures (4°C, 22°C, and 50°C) for mushroom extraction. Biochemical assays carried out in parallel indicated higher amounts of polyphenols in the L edodes extracts at all extraction temperatures investigated. The scavenging ability of the 2,2-diphenyl-1-picrylhydrazyl radical showed higher activity for L edodes extracts. Superoxide dismutase-like activity showed no statistically significant difference among the groups for the 2 tested extracts, and catalase-like activity was increased with the L edodes extracts at 4°C. The results for the cytotoxic activity from P sajor-caju extracts at 22°C revealed the half maximal inhibitory concentration values of 0.64% ± 0.02% for Hep-2 and 0.25% ± 0.02% for HeLa. A higher cytotoxic activity was found for the L edodes extract at 22°C, with half maximal inhibitory concentration values of 0.78% ± 0.02% for Hep-2 and 0.57% ± 0.01% for HeLa. Substantial morphological modifications in cells were confirmed by Giemsa staining after treatment with either extract, suggesting inhibition of proliferation and induction of apoptosis with increasing extract concentrations. These results indicate that the aqueous extracts of Brazilian L edodes and P sajor-caju mushrooms are potential sources of antioxidant and anticancer compounds. However, further investigations are needed to exploit their valuable therapeutic uses and to elucidate their modes of action.

A new Penicillium echinulatum strain with faster cellulase secretion obtained using hydrogen peroxide mutagenesis and screening with 2-deoxyglucose.

Aims:  The aim of this study is to improve cellulase production and secretion by Penicillium echinulatum using mutagenesis and selection in association with microfermentation and microanalysis methods.

The Penicillium echinulatum Secretome on Sugar Cane Bagasse

Plant feedstocks are at the leading front of the biofuel industry based on the potential to promote economical, social and environmental development worldwide through sustainable scenarios related to energy production. Penicillium echinulatum is a promising strain for the bioethanol industry based on its capacity to produce large amounts of cellulases at low cost. The secretome profile of P. echinulatum after grown on integral sugarcane bagasse, microcrystalline cellulose and three types of pretreated sugarcane bagasse was evaluated using shotgun proteomics. The comprehensive chemical characterization of the biomass used as the source of fungal nutrition, as well as biochemical activity assays using a collection of natural polysaccharides, were also performed. Our study revealed that the enzymatic repertoire of P. echinulatum is geared mainly toward producing enzymes from the cellulose complex (endogluganases, cellobiohydrolases and β-glucosidases). Glycoside hydrolase (GH) family members, important to biomass-to-biofuels conversion strategies, were identified, including endoglucanases GH5, 7, 6, 12, 17 and 61, β-glycosidase GH3, xylanases GH10 and GH11, as well as debranching hemicellulases from GH43, GH62 and CE2 and pectinanes from GH28. Collectively, the approach conducted in this study gave new insights on the better comprehension of the composition and degradation capability of an industrial cellulolytic strain, from which a number of applied technologies, such as biofuel production, can be generated.

Increased production of cellulases and xylanases by Penicillium echinulatum S1M29 in batch and fed-batch culture.

The development of more productive strains of microorganisms and processes that increase enzyme levels can contribute to the economically efficient production of second generation ethanol. To this end, cellulases and xylanases were produced with the S1M29 mutant strain of Penicillium echinulatum, using different concentrations of cellulose (20, 40, and 60 g L(-1)) in batch and fed-batch processes. The highest activities of FPase (8.3 U mL(-1)), endoglucanases (37.3 U mL(-1)), and xylanases (177 U mL(-1)) were obtained in fed-batch cultivation with 40 g L(-1) of cellulose. The P. echinulatum enzymatic broth and the commercial enzyme Cellic CTec2 were tested for hydrolysis of pretreated sugar cane bagasse. Maximum concentrations of glucose and xylose were achieved after 72 h of hydrolysis. Glucose yields of 28.0% and 27.0% were obtained using the P. echinulatum enzymatic extract and Cellic CTec2, respectively.

Effect of methylxanthines on production of cellulases by Penicillium echinulatum

Aim:  In this work, the effect of supplementing liquid cellulase production media (CPM) with methylxanthines (aminophylline, caffeine and theophylline), with and without the addition of glucose, on the secretion of cellulases by Penicillium echinulatum strain 2HH (wild‐type) and the derived mutant strain 9A02S1 was studied.

Application of cellulases from Acrophialophora nainiana and Penicillium echinulatum in textile processing of cellulosic fibres

New cellulases from the fungi Acrophialophora nainiana and Penicillium echinulatum were used in the finishing of knitted cotton fabrics (biopolishing) and compared with the well established enzymes from Trichoderma reesei. Both cellulases reduced the pilling tendency with a lower weight loss than T. reesei cellulases. Cellulases from P. echinulatum were also studied in stonewashing of denim fabrics to obtain the fashionable aged look in indigo dyed jeans ware and were found to remove more colour from denim fabrics and produce less indigo dye redeposition (back-staining) than commercial acid or neutral cellulases under the test conditions. Efficiency was found to be influenced by pH during textile processing and the substrate used for the production of cellulases. Cellulases produced by P. echinulatum grown on cellulose showed better stonewashing results (higher colour removal and less back-staining) than cellulases produced on sugar cane bagasse. The substrate used during enzyme production of P. echinulatum cellulases seems to have a significant influence on cellulose composition, which affects textile processing results.

Cellulase and Xylanase Production by Penicillium echinulatum in Submerged Media Containing Cellulose Amended with Sorbitol

The present work investigated the use of sorbitol as a soluble carbon source, in association with cellulose, to produce cellulases and xylanases in submerged cultures of Penicillium echinulatum 9A02S1. Because cellulose is an insoluble carbon source, in cellulase production, there are some problems with rheology and oxygen transfer. The submerged fermentations containing media composed of 0, 0.25, 0.5, 0.75, and 1% (w/v) sorbitol and cellulose that were added at different times during the cultivation; 0.2% (w/v) soy bran; 0.1% (w/v) wheat bran; and a solution of salts. The highest filter paper activity (FPA) (1.95  ±  0.04 IU·mL−1) was obtained on the seventh day in the medium containing 0.5% (w/v) sorbitol and 0.5% (w/v) cellulose added 24 h after the start of cultivation. However, the CMCases showed an activity peak on the sixth day (9.99 ± 0.75 IU·mL−1) in the medium containing 0.75% (w/v) sorbitol and 0.75% (w/v) cellulose added after 12 h of cultivation. The xylanases showed the highest activity in the medium with 0.75% (w/v) sorbitol and 0.25% (w/v) cellulose added 36 h after the start of cultivation. This strategy enables the reduction of the cellulose concentration, which in high concentrations can cause rheological and oxygen transfer problems.

High-pressure carbon dioxide/water pre-treatment of sugarcane bagasse and elephant grass: Assessment of the effect of biomass composition on process efficiency

The performance of two lignocellulosic biomasses was studied in high-pressure carbon dioxide/water pre-treatment. Sugarcane bagasse and elephant grass were used to produce C5-sugars from hemicellulose and, simultaneously, to promote cellulose digestibility for enzymatic saccharification. Different pre-treatment conditions, with combined severity factor ranging from -1.17 to -0.04, were evaluated and maximal total xylan to xylose yields of 59.2wt.% (34.4wt.% xylooligomers) and 46.4wt.% (34.9wt.% xylooligomers) were attained for sugarcane bagasse and elephant grass, respectively. Furthermore, pre-treated biomasses were highly digestible, with glucan to glucose yields of 77.2mol% and 72.4mol% for sugarcane bagasse and elephant grass, respectively. High-pressure carbon dioxide/water pre-treatment provides high total C5-sugars and glucose recovery from both lignocellulosic biomasses; however it is highly influenced by composition and intrinsic features of each biomass. The obtained results confirm this approach as an effective and greener alternative to conventional pre-treatment processes.

Enzymes for second generation ethanol: exploring new strategies for the use of xylose

Considering that the cost of cellulases and hemicellulases substantially contributes to the price of 2nd-generation ethanol, reducing the cost of the substances used as inducers of cellulose and xylose is essential to reducing the cost of these enzymes. Penicillium echinulatum is able to secrete most hemicellulolytic and cellulolytic enzymes. In this context, the aim of this work was to evaluate the use of xylose for the production of cellulases and xylanases by two mutant strains of P. echinulatum (9A02S1 and S1M29) and the parental 2HH. Xylose acts as an inducer for the production of xylanases and cellulases, especially endoglucanases. These data indicate the possibility of using the pentose xylose, which is not used by the majority of yeast for ethanol production, as a total or partial substitute in cellulose-based processes for the production of enzymes for enzymatic hydrolysis.