Secretome data from Trichoderma reesei and Aspergillus niger cultivated in submerged and sequential fermentation methods

The cultivation procedure and the fungal strain applied for enzyme production may influence levels and profile of the proteins produced. The proteomic analysis data presented here provide critical information to compare proteins secreted by Trichoderma reesei and Aspergillus niger when cultivated through submerged and sequential fermentation processes, using steam-explosion sugarcane bagasse as inducer for enzyme production. The proteins were organized according to the families described in CAZy database as cellulases, hemicellulases, proteases/peptidases, cell-wall-protein, lipases, others (catalase, esterase, etc.), glycoside hydrolases families, predicted and hypothetical proteins. Further detailed analysis of this data is provided in “Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation process: enzyme production for sugarcane bagasse hydrolysis” C. Florencio, F.M. Cunha, A.C Badino, C.S. Farinas, E. Ximenes, M.R. Ladisch (2016) [1].


Value of the data
This data set will be of value to the scientific community aiming to analyze the identified proteins secreted by T. reesei and A. niger under different cultivation methods.
The data can be a useful tool to effectively select fungal strain and cultivation procedure for the production of proteins of interest.
The data provided here identify key enzymes from T. reesei and A. niger for combined use to effectively degrade lignocellulose substrates, and therefore provide an opportunity to help researchers in the field to formulate enzyme cocktails in according to characteristics of lignocellulose biomass and enzyme activities found in the secretome.

Data
In Table 1, the proteins identified by proteomic analysis of enzymatic cocktails from Trichoderma reesei and Aspergillus niger, cultivated on pretreated sugarcane bagasse by either submerged or sequential fermentation processes, are presented according to the families classification from CAZy database.
The enzymatic hydrolysis of pretreated sugarcane bagasse was performed with combined extracts from T. reesei Rut C30 and A. niger A12, and the data of proteomic analysis of this combination of identified proteins is shown in Table 2. The indicated enzyme loadings were applied for steamexplosion sugarcane bagasse saccharification as described by Florencio et al. [1].

Fungal strains
The strains used for enzyme production were T. reesei Rut-C30 and A. niger wild type A12 obtained from Centre for Agricultural Bioscience International (CABI) culture collection (United Kingdom) and Embrapa Food Technology collection (Rio de Janeiro, Brazil), respectively. The conditions in which strains were maintained are described in Florencio et al. [1].

Cultivation conditions
Submerged and sequential fermentations carried out to obtain the enzymatic cocktails from T. reesei and A. niger are described in detail in Florencio et al. [1]. Briefly, the submerged fermentation was initiated with a 48 h pre-culture that contained a final conidia concentration of 10 7 spores/mL in Table 1 Major proteins identified in the secretome of Trichoderma reesei and Aspergillus niger cultivated under submerged (A) and sequential (B) fermentation methods. Alpha-galactosidase extracellular -

Gene ID
Xylanase GH11 x 83638302 Xylanase GH11 x 380293098 Xylanase II GH11 x x Pepsinogen Aminopeptidase - Aspartic endopeptidase x 145254317 Aspartic-type endopeptidase opsB - Aspartic-type endopeptidase opsB - Tripeptidyl peptidase precursor - Cell-wall protein Cell wall protein x 47028077 Cell-wall protein -CwpA x 145252266 GPI anchored cell wall protein GH64 x x 589109601 Ceramidase family protein x 145255556 Alkaline nonlysosomal ceramidase - Triacylglycerol lipase precursor - Carbohydrate esterase family 15 protein CBM15 x x 572279065 Carboxylesterase - Cholinesterase Catalase/peroxidase x Hypothetical protein ASPNIDRAFT_53540 x Table 2 Major proteins identified in the submerged (A) and sequential (B) fermentation enzymatic extracts from Trichoderma reesei þ Aspergillus niger, which were used in the hydrolysis process of the pretreated sugarcane bagasse at a 1:5 ratio, respectively. Hypothetical protein M419DRAFT_97005 x 100 mL of nutrient medium with 30 g/L of glucose, as described initially from Mandels and Stenberg [2] and adapted by Cunha et al. [3].

Gene ID
In the sequential fermentation, solid state fermentation was initiated using 5 g of dry sugarcane bagasse as solid substrate, and substrate moisture was adjusted through the addition of 12 mL of nutrient medium. The inoculum was added for a final concentration of 10 7 spores/g of dry bagasse in the pre-culture, which was maintained under static conditions for 24 h. Then, the pre-culture step was continued as a submerged fermentation after the addition of 100 mL of nutrient medium enriched with 30 g/L of glucose per 5 g of dry bagasse. After 48 h for both submerged and sequential fermentation, a volume of pre-culture suspension corresponding to 10% (v/v) was transferred to 100 mL of culture medium for enzyme production, which was supplemented with 10 g/L of glucose and 1% (w/v) of steam-exploded non-washed sugarcane bagasse. All cultivation experiments were carried out in triplicate, and the averaged data presented with standard deviations.

Sample preparation
Sequence grade Lys-C/Trypsin (Promega) was used to enzymatically digest the samples. Acetone precipitation was performed prior to sample digestion. The protein samples were reduced with a 10 mM dithiothreitol (DTT)/25 mM ammonium bicarbonate solution at 37°C for 1 h and alkylated at 37°C also for 1 h using a solution of 97% acetonitrile (ACN), 2% iodoethanol, and 0.5% triethylphosphine (v/v). Samples were dried before adding Lys-C/trypsin to them in a 25:1 ratio of protease to protein. Digestions were carried out in a barocycler NEP2320 (PBI) at 50°C and 20 kpsi for 2 h. The samples were cleaned over C18 columns (MicroSpin, Nest Group), dried and resuspended in 97% purified water/3% ACN/0.1% formic acid (FA). A volume of 1 mL was used for LC-MS/MS analysis.

LC-MS/MS analysis
A nanoLC system (1100 Series LC, Agilent Technologies, Santa Clara, CA) was used to separate the peptides for downstream MS analysis using a C18 reversed phase ZORBAX 300SB-C18 analytical column (0.75 μm Â 150 mm, 3.5 um) from Agilent. The column was directly connected to New Objective's emission tip coupled to the nano-electrospray ionization (ESI) source of the high resolution hybrid ion trap mass spectrometer LTQ-Orbitrap XL (Thermo Scientific). Elution was conducted using an ACN/0.1% FA (mobile phase B) linear gradient. The column was equilibrated initially for 5 min with 95% H 2 O /0.1% FA (mobile phase A) followed by the linear gradient of 5-40% B for 85 min at 0.3uL/min, then from 40-95% B for 12 min. Blank injections were performed in between experimental runs. The resulting eluents were analyzed by a data-dependent positive acquisition mode at full MS scan (30,000 resolution) where the eight most abundant molecular ions were selected and fragmented by collision induced dissociation (CID) using a normalized collision energy of 35% to acquire the data for the LTQ-Orbitrap XL.

Data analysis
Database search analyses were done using Mascot Daemon version 2.4.0 (Matrix Science) against an all fungal protein database from the NCBI database. Peptide and spectral count data were performed on the searches. For protein identification, at least two peptides detected were considered, and the false discovery rate (FDR) was set to 1%.