A new highly efficient beta-glucosidase from the novel species, Aspergillus saccharolyticus

A new highly efficient beta-glucosidase from the novel species, Aspergillus saccharolyticus

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A new highly efficient beta-glucosidase from the novel species, Aspergillus saccharolyticus
A new highly efficient beta-glucosidase from the novel species, Aspergillus saccharolyticus


Sørensen, Annette



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In a biorefinery concept, biomass polymers of cellulose and hemicellulose are converted into sugars, which can be used for production of biofuels and biochemicals, which could act as platform molecules serving as building blocks in the synthesis of chemicals and polymeric materials. This is a sustainable solution that is expected to replace today’s oil refineries. Main components of lignocellulosic biomass, primarily consisting of plant cell walls, are cellulose, hemicellulose, and lignin. Prior to enzymatic hydrolysis for generating sugar monomers, the biomass is pretreated. The pretreatment mainly opens up the cell wall structure and partly hydrolyzes hemicellulose, so that cellulose is the main target for enzyme hydrolysis. Beta-glucosidases (EC play an essential role in efficient hydrolysis of cellulose. By hydrolysis of cellobiose, beta-glucosidases relieve inhibiting conditions, allowing for increased hydrolysis of cellulose by cellobiohydrolases and endoglucanases. Efficient hydrolysis requires high conversion rates throughout the hydrolysis. The major factors influencing this are product inhibition and temperature stability. Traditionally, the commercial enzyme preparations Novozym 188 (mainly beta-glucosidase activity) and Celluclast 1.5L (mainly cellobiohydrolase and endoglucanase activity) (Novozymes A/S) have been used in combination for hydrolysis of pretreated biomass, and recently complete enzyme cocktails have been launched, Cellic CTec (Novozymes A/S) and AcceleraseDUET (Genencor A/S). The enzyme preparations by Novozymes A/S are used as benchmarks in the following research. Superior enzymes can be obtained either by discovery of new enzymes through different screening strategies or by improvement of known enzymes mainly by different molecular methods. Initially, we employed a screening strategy using different lignocellulosic materials for discovery of new enzymes to be used in an onsite enzyme production concept during bioethanol production. Different fungi were applied in this screening, mainly fungi isolated from different woody habitats. A low value stream of a cellulosic ethanol production was explored as enzyme production medium, finding Aspergillus niger as well as an unidentified strain AP as promising candidates for the utilization of the filter cake for growth and enzyme production. The filter cake inoculated with the respective fungi could, combined with Celluclast 1.5L, substitute the use of Novozym 188 in hydrolysis of pretreated biomass. In the wake of this, focus was placed on beta-glucosidases. A screening for beta-glucosidase activity was conducted, using wheat bran as substrate in simple submerged fermentation, testing selected strains from the previous screening as well as new isolated strains and strains from our in-house strain collection, including A. niger. The strain AP showed significantly greater potential in beta-glucosidase activity than all other fungi screened. The beta-glucosidase activity of a solid state fermentation extract of strain AP was compared with Novozym 188 and Cellic CTec. In terms of cellobiose hydrolysis, the extract of strain AP was found to be a valid substitute for Novozym 188, corresponding to the previous result where filter cake inoculated with the fungus was directly used in hydrolysis of pretreated biomass. The Michaelis Menten kinetics affinity constants of strain AP extract and Novozym 188 were approximately the same, and the two preparations performed equally well in cellobiose hydrolysis with regard to product inhibition. However, the extract of strain AP showed higher specific activity (U/total protein) as well as increased thermostability. The significant thermostability of strain AP beta-glucosidases was further confirmed when compared with Cellic CTec. The extract of strain AP facilitated hydrolysis of cellodextrins with an exo-acting approach, and was, when combined with Celluclast 1.5L, able to contribute to the generation of a sugar platform from pretreated bagasse, by hydrolyzing the biomass to monomeric sugars. Strain AP was from a polyphasic taxonomic approach identified as a yet undescribed uniseriate Aspergillus species belonging to section Nigri. Morphologically, at first glance strain AP resembles A. japonicus. However, in detailed phenotypic analysis, strain AP distinguished itself from the other uniseriate aspergilli, both in terms of growth characteristics on different media as well as temperature tolerance. Futhermore, the extrolite production of strain AP differed significantly from other known aspergilli in section Nigri, as several well-known compounds from this series were not present, and the peaks detected did not match the approximately 13500 fungal extrolites in the natural product chemist’s database, Antibase2010. Genotypic analysis of the ITS region, partial beta-tubuline gene, and partial calmodulin gene placed strain AP on a separate branch in phylogenetic trees prepared with other black aspergilli, and universally primed PCR furthermore readily distinguished strain AP data from other black aspergilli. We named the novel species A. saccharolyticus, referring to its ability to hydrolyze cellobiose and cellodextrins, and it was deposited in the strain collection of Centraalbureau voor Schimmelcultures (CBS 127449T). Identification, isolation, and characterization of the most prominent beta-glucosidase from A. saccharolyticus were undertaken. The extract from this fungus, mentioned above, was fractionated by ion exchange chromatography, obtaining fractions pure enough that a specific SDS-page gel protein band of high beta-glucosidase activity could be excised and analyzed by LC-MS/MS. Using the peptide matches for design of degenerate primers, the beta-glucosidase gene, bgl1, of A. saccharolyticus was cloned. The 2919 bp genomic sequence encodes a 680 aa polypeptide which has 91% and 82% identity with BGL1 from A. aculeatus and BGL1 from A. niger, respectively. BGL1 of A. saccharolyticus was identified as belonging to GH family 3. A three dimentsional structure was proposed through homology modeling,finding retaining enzyme characteristics and, interestingly, a more open catalytic pocket compared to other beta-glucosidases. A cloning vector for heterologous expression of bgl1 in Trichoderma reesei was constructed, combining promoter, gene, and terminator of different eukaryotic origin. Codons coding for histidine residues were included in the 3’end of the gene to assist purification. The purified BGL1 was assayed for beta-glucosidase activity, studying enzyme kinetics, temperature and pH profiles, glucose tolerance, and cellodextrin hydrolysis. A striking similarity was found when comparing the data of purified BGL1 to the data previously reported of the crude extract, indicating that we have successfully cloned and expressed the protein mainly contributing to the beta-glucosidase activity observed in the crude extract, thus the most prominent beta-glucosidase of A. saccharolyticus.