Estudio de las β-glucosidasas del complejo celulolítico de Talaromyces amestolkiae: Caracterización y aplicaciones biotecnológicas.

Abstract

Introduction Cellulose is the most abundant biopolymer on Earth and it has a great potential as a renewable biomass and energy source. Biological conversion of cellulosic biomass is a promising green alternative for the production of second-generation (2G) ethanol and other chemicals. First generation (1G) biofuels are directly related to a generally edible biomass so that 2G biofuels appear to end the food-versus-fuel debate. Chemically, cellulose is a linear β-(1,4)-glucose polymer containing amorphous and crystalline regions depending on their chains order degree. This heterogeneous nature requires the action of several enzymes, acting synergistically, to be fully converted into its building blocks: cellulases can be divided into three major enzymes: i) endoglucanases (EG), acting on amorphous regions, ii) cellobiohydrolases (CBH), preferring crystalline substrates and iii) β-glucosidases (BGL) acting on cellobiose and cello-oligosaccharides. BGLs are especially attractive for two reasons: they are defective in current commercial cocktails and they can also be applied to synthesize oligomers and other complex molecules, such as alkyl-glucosides, by transglycosylation reactions. Filamentous fungi are the main source of commercial cellulases. In recent years, cellulase systems of Penicillium strains (or its perfect state Talaromyces) deserve significant attention in the production of cellulases. Specifically, several researchers have pointed out high BGL activity released by Penicillium species. Aims In this context, the aims of the present Thesis are: i) To identify a new cellulolytic fungus, according to their morphological characteristics and using molecular techniques. ii) To analyze the ability of the isolated fungus to produce cellulases in the presence of different carbon sources. iii) To purify and characterize BGLs secreted by the fungus, from a biochemical and molecular point of view. iv) To use these enzymes and/or the fungal enzyme crude in biotechnological applications.

Results and Discussion Identification of the isolated strain In a previous work, a high-cellulase-producing fungal strain was isolated from cereal samples. Sequence analysis of genetic markers such as ITS rDNA, RPB1, and Bt2, as well as morphological studies of this strain revealed its identity with Talaromyces amestolkiae. Optimization of cellulases production To select a suitable carbon source for cellulase production, T. amestolkiae was cultivated in Mandels´ medium containing different carbon sources: Avicel, wheat straw, wheat bran, oat bran, and wheat straw pretreated by acidic steam explosion (slurry). Among these, Avicel was found to be the best inducer for cellulase production, leading to avicelase, EG, and BGL activities of 0.4, 3.5, and 1.9 U/mL when cultured in 250 mL Erlenmeyer flasks with 50 mL of medium. Avicel is microcrystalline cellulose, being entirely metabolized by the fungus, whereas the other carbon sources tested contain also hemicellulose and lignin, hindering the access to cellulose. It should be noted that the production of cellulase by T. amestolkiae dramatically increased after the scale-up of fermentations to both higher capacity flasks and a laboratory-scale fermenter. In this last case, cultures in a 1-L fermentor gave two-fold values of the three cellulase activities tested (1.0, 7.0 and 4.0 U/mL of avicelase, EG and BGL, respectively). Purification of BGLs Three BGLs were purified to homogeneity from the culture supernatant of T. amestolkiae grown on Avicel using anion exchange and gel filtration chromatography. All of them were first loaded into a HiTrap Capto Adhere cartridge, a multimodal anion exchanger. After this step, BGL-1 was subjected to an additional anion exchange chromatography using a Resource Q column. For purification of BGL-2, the Capto Adhere eluate was subsequently loaded into Mono Q column and then submitted to size exclusion chromatography (SEC) in a Superdex 75 matrix. Regarding BGL-3, only a Mono Q separation was needed. The overall enzymes yields were 12.6, 6.1, and 18.1% for BGL-1, BGL-2, and BGL-3, respectively. The purified enzymes appeared as single protein bands on SDS-PAGE with approximate molecular mass of 60 kDa for BGL-1 and 100 kDa for BGL-2 and BGL-3.

Biochemical characterization of BGLs BGL-1 and BGL-2 are monomeric proteins while BGL-3 is a functional dimer, as observed by SEC. Maximal activity of these enzymes were obtained at pH 4.0 and 50-60° C, being stable over a pH range between 4-7 and at 50 °C. All three enzymes showed different behavior depending on the substrate used (p-nitrophenyl-β-D-glucopyranoside -pNPG- or cellobiose). The three enzymes showed superior activity against pNPG. Being a dimer, BGL3 displayed the best catalytic efficacy (kcat/Km) for both substrates. The effect of certain chemical compounds and inhibitors in BGL activity was also assayed. All of them were strongly inhibited by Pb2+, while Cu2+, Hg2+ and Fe3+ and Fe2+ acted differentially on BGL-3, where inhibition was lower. BGLs of T. amestolkiae have the ability to hydrolyze cellooligosaccharides with different length, decreasing their efficiency when the polymerization degree of the substrate increases, and they are not active against polysaccharides. In this sense, BGL-2 was able to depolymerize longer chain cellooligosacharides (up to 6 glucose units) with higher efficiency. The three enzymes showed transglycosylation activity, being able to transfer glucose from pNPG to aliphatic alcohols as MeOH, EtOH, PrOH, and BuOH to form the corresponding alkyl-glucosides. Furthermore, these enzymes catalyzed the incorporation of additional glucose residues to small celloligosaccharides, detecting cellodecaose as the product with the highest polymerization degree when BGL-1 and BGL-2 were used as catalysts. Identification and sequencing of BGLs BGL-1, BGL-2, and BGL-3 of T. amestolkiae were identified by peptide mass fingerprinting and peptide fragmentation (MALDI-TOF/TOF), showing that they were different enzymes. According to the results, maximum identity was obtained with putative -glucosidases from Talaromyces and Penicillium species, as expected. Based on this high homology with other phylogenetically related fungal BGLs, specific primers were designed that allowed sequencing of the genes encoding each enzyme, and introns were also predicted. Analysis of the amino acid sequences showed that BGL-1 is a member of the family 1 of glycosyl hydrolases, while BGL-2 and BGL-3 belong to family 3. In spite of the fact that there are few fungal BGLs crystallized, structural models of BGL-2 and BGL-3 were constructed on the basis of their identity (~ 65% homology) with BGLs from Hypocrea jecorina (the anamorph of the cellulolytic fungus Trichoderma reesei) and Aspergillus aculeatus, respectively. BGL-2 and BGL-3 exhibited the N-terminal, C-terminal and type III fibronectin domains described in other fungal BGLs. In addition, it should be highlighted the presence of a cellulose-binding domain (CBD) in BGL-2 attached to the catalytic domain through a flexible linker rich in serin and threonin. This characteristic could explain the higher activity of this particular enzyme when hydrolyzing longer-chain cellooligosaccharides.

Saccharification of slurry from wheat straw Since the efficient hydrolysis of a complex lignocellulosic biomass is a key step toward the conversion of agricultural residues into value-added products, the activity of T. amestolkiae crudes (Talarozyme) on saccharification of wheat straw slurry from acidic steam-explosion was investigated. Either alone or in combination with other commercial cocktails used for saccharification, the slurry was efficiently hydrolyzed. In the first case, Talarozyme yielded better results than N50010 and Ultraflo L (more than six-fold sugars were released from the slurry when Talarozyme was used). When it was added as a supplement of Celluclast 1,5 L or Ultraflo L cocktails, saccharification increased 50 and 75%, respectively. These results indicated that the fungus secretes, besides cellulases, some additional enzymes that could enhance fermentable sugars release. Secretome analysis In order to analyze in depth the enzymes produced by the fungus in the presence of different lignocellulosic sources, the secretome from cultures with either Avicel or slurry as carbon sources was analyzed by two dimensional SDS-PAGE and massive peptide mass analysis by nanoLC-MS/MS. In both secretomes, CBHs showed to be the most abundant enzymes, followed by BGLs. However, in the slurry-induced culture, the proportion of BGL and enzymes other than cellulases increased. This indicates that greater enzymatic diversity is necessary for degradation of a complex substrate, such as slurry. However, since slurry comes from the steam explosion pretreatment from wheat straw, polysaccharides are more available for enzyme saccharification, and more enzymes are induced. Besides cellulases and hemicellulases, other accessory enzymes like swollenins were detected in slurry-induced secretomes. The results obtained open new routes for the development of effective enzyme cocktails in the context of degradation of lignocellulosic biomass. Conclusions In this work, T. amestolkiae has been identified and described for the first time as a cellulolytic fungus. This species produces an enzymatic cocktail rich on BGLs, which are the limiting enzymes in the hydrolysis of cellulose. Three extracellular BGLs secreted by the fungus have been purified and biochemically characterized. Their different properties could be of interest for bioethanol production. In addition, all of them showed high transglycosylation activity, allowing the synthesis of several alkyl-glucosides and cellooligosaccharides. The enzymatic crude of T. amestolkiae has been used for supplementing BGL-deficient commercial cocktails, enhancing glucose release from wheat straw slurry obtained by acidic steam explosion. The results obtained suggest that BGLs from T. amestolkiae, as well as other accessory proteins present in the new enzyme cocktail, could be of interest for biotechnological plant biomass applications.