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High Levels of Β-Xylosidase in Thermomyces Lanuginosus

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High Levels of Β-Xylosidase in Thermomyces Lanuginosus b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 680–690 ht tp://www.bjmicrobiol.com.br/ Industrial Microbiology High levels of ␤-xylosidase in Thermomyces lanuginosus: potential use for saccharification a,∗ a b c Juliana Moc¸o Corrêa , Divair Christi , Carla Lieko Della Torre , Caroline Henn , b b b José Luis da Conceic¸ão-Silva , Marina Kimiko Kadowaki , Rita de Cássia Garcia Simão a Centro de Ciências Exatas e Tecnológicas b Centro de Ciências Médicas e Farmacêuticas, UNIOESTE, Cascavel, PR, Brazil c Central Hidrelétrica de Itaipu, Itaipu Binacional, Foz do Iguac¸u, PR, Brazil a r t a b i c l e i n f o s t r a c t Article history: A new strain of Thermomyces lanuginosus was isolated from the Atlantic Forest biome, and Received 5 November 2015 its ␤-xylosidases optimization in response to agro-industrial residues was performed. Using Accepted 20 February 2016 statistical approach as a strategy for optimization, the induction of ␤-xylosidases activity Available online 27 April 2016 was evaluated in residual corn straw, and improved so that the optimum condition achieved ␤ Associate Editor: Solange Ines high -xylosidases activities 1003 U/mL. According our known, this study is the first to show Mussatto so high levels of ␤-xylosidases activities induction. In addition, the application of an experi- mental design with this microorganism to induce ␤-xylosidases has not been reported until ◦ Keywords: the present work. The optimal conditions for the crude enzyme extract were pH 5.5 and 60 C ◦ showing better thermostability at 55 C. The saccharification ability of ␤-xylosidase in the Experimental design Saccharification presence of hemicellulose obtained from corn straw raw and xylan from beechwood sub- ␤-Xylosidase strates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, ◦ at 50 C. Our data strongly indicated that the ␤-xylosidases activities was not subjected to the Thermomyces lanuginosus effects of potential enzyme inhibitors often produced during fermentation process. These Atlantic Forest data suggest the application of this enzyme studied for saccharification of hemicellulose, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production. © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). 1 frequently isolated from organic waste. Several strains of Introduction Themomyces lanuginosus have been shown to be potential 2–4 producers of enzymes with different applications. In gen- Thermomyces lanuginosus, previously known as Humicola lanug- eral, microbial enzymes have received attention due to being inosa, is a thermophilic fungus widely distributed and ∗ Corresponding author. E-mail: [email protected] (J.M. Corrêa). http://dx.doi.org/10.1016/j.bjm.2016.04.028 1517-8382/© 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 680–690 681 precursors to clean technology for the production of indus- The generated sequence was deposited in GenBank under trial and commercially important compounds using residual the access number KJ934703.1. In both analyses used to iden- biomass as substrates. tify the fungus, the isolate was classified as belonging to the T. ◦ The biomass present in agro-industrial residues is com- lanuginosus species. The microorganism was grown at 42 C for posed of hemicellulose, and the enzymatic depolymerization 7 days in tubes containing solid PDA medium (20% potato infu- of its structure is commercially interesting due to its soft sion (w/v), 1.5% dextrose (w/v) and 1.5% agar (w/v)) and was ◦ condition and no formation of toxic compounds during the subsequently stored under refrigeration at 4 C with periodic degradation of the products. The complete deconstruction of maintenance every 30 days. the hemicellulose structure requires the synergistic action of several enzymes, including xylanases (EC 3.2.1.8) and Preparation of agro-industrial residues and plant biomass ␤-xylosidases (EC 3.2.1.37). ␤-Xylosidase cleaves ␤-1,4-xylo- oligosaccharides released by xylanases producing xylose, a The following carbon sources tested for use in the experiments monosaccharide that can be used by different microorganisms of fungal growth and induction of ␤-xylosidases enzyme activ- in the process of saccharification and production compounds ities were selected according to availability: corn straw, orange 1,5 of the biotechnological application. peels, banana peels and passion fruit peels. The plant biomass ◦ Currently, market analyses indicate that global maize pro- residues were dried at 70 C for 24 h followed by trituration duction is abundant, and the US is the largest producer of the (SL30 slicer Willey) using a 20 mesh sieve, and the residues crop followed by Argentina and Brazil totaling approximately were then stored in glass vials at room temperature. 53.2 million tons per harvest. Corn is mainly used for animal feed production but is also used to a lesser extent for the pro- Cultivation conditions of T. lanuginosus duction of oils, flours and breakfast cereals. Thus, the residual biomass of the crop, such as straw and corncobs, should be Newly grown fungal conidia were used to prepare a suspen- 5 −1 considered for the production of energy and other products of sion (1 × 10 conidia mL ) in sterile water, which was then 1,2 biotechnological interest. inoculated into 25 mL of mineral medium (distilled water, 0.3 g Brazilian indices related to corn crops show that only 5% of of NaNO3, 0.1 g of K2HPO4, 0.05 g of MgSO4·7H2O, 0.05 g of KCl, production is intended for human consumption. The residues 0.001 g of FeSO4·7H2O, and 0.1 g of yeast extract; pH 6.0) supple- from the processing of this crop make up 58% of the produced mented with 1% (w/v) different selected carbon sources. The biomass, and this portion is still neglected and could alter- submerged fermentation (SMF) cultivations were performed natively contribute as a source of renewable energy sources to two different ways as follows: stationary liquid and agitated ◦ generate wealth and to minimize the accumulation of residues liquid (150 rpm) at 42 C for 7 days in biological duplicates. The 1,2 that can be harmful to the environment. carbon source most effective in inducing the highest enzyme In the present report, an isolate from the Brazilian Atlantic activity was used for further optimization experiments. ␤ Forest biome was optimized for the production total of - The enzyme production was monitored, by collecting the xylosidases. The optimized enzyme crude extract was used mycelial cells of T. lanuginosus by vacuum filtration of the cul- for testing the saccharification of xylan from beechwood ture to sterile Whatman paper. The obtained mycelial cells and hemicellulose derived from corn straw emphasizing the were frozen, and they were then macerated with 1 g of glass potential biotechnological role for the enzyme. beads, resuspended in 5 mL of ice-cold distilled water and cen- ◦ trifuged at 8000 × g and 4 C for 5 min. The supernatant was collected and used in the analytical tests. Materials and methods Determination of fungal biomass Isolation, identification and maintenance of culture The samples used for the determination of biomass were fil- ® The fungus cataloged under the ID of PC-7S-1-T was isolated tered through 5 ␮m filter paper (Whatman ), dried for 48 h ◦ from a soil sample obtained from the Atlantic Forest biome at 105 C and weighed on an analytical balance. The assay ◦ of the Beautiful View Refuge (latitude 24 55 16 S and lon- for measure the fungal biomass was conducted using a con- ◦ gitude 53 54 35 W) Foz do Iguac¸u, Paraná, Brazil (collection trol for determination of biomass. In the control flask was authorized by Itaipu Bi-national). The isolate was subjected to added the residue in the absence of inoculum. The biomass morphological identification followed by molecular identifica- was quantified by the difference of weight obtained in the test tion by nucleotide sequence analysis of the ITS region of DNA experiments containing residue and fungus, and, the control corresponding to the rRNA of the microorganism. This analy- containing only the residue. sis was performed at the Laboratory of Molecular Biochemistry The optimal parameters determined for growth of the at the State University of West Paraná (Brazil) by extracting the fungus under different conditions were applied to the total DNA of the fungus followed by replication of the target experimental design based on the following three factors: amplicon using specific primers. The amplicons obtained were concentration of the residue as a carbon source (corn straw), sequenced by the sequencing service provided by HELIXXA temperature and concentration of the nitrogen source (yeast (Sequencing Service, Brazil), and the sequences obtained extract) during 5 days of agitation at 150 rpm in liquid culture. 3 were analyzed using sequence alignment tools (Blast-n The 2 experimental matrix consisted of three central points from the National Center for Biotechnology Information; and six axial points where the two factors combined with three NCBI). levels totaling 17 tests. We selected the combination of 0.25 g 682 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 680–690 Table 1 – Levels of factors used in CCRD.
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