GH3 (glycoside hydrolase family 3) BGLs (β-glucosidases) from filamentous fungi have been widely and commercially used for the supplementation of cellulases. AaBGL1 (Aspergillus aculeatus BGL1) belongs to the GH3 and shows high activity towards cellooligosaccharides up to high degree of polymerization. In the present study we determined the crystal structure of AaBGL1. In addition to the substrate-free structure, the structures of complexes with glucose and various inhibitors were determined. The structure of AaBGL1 is highly glycosylated with 88 monosaccharides (18 N-glycan chains) in the dimer. The largest N-glycan chain comprises ten monosaccharides and is one of the largest glycans ever observed in protein crystal structures. A prominent insertion region exists in a fibronectin type III domain, and this region extends to cover a wide surface area of the enzyme. The subsite +1 of AaBGL1 is highly hydrophobic. Three aromatic residues are present at subsite +1 and are located in short loop regions that are uniquely present in this enzyme. There is a long cleft extending from subsite +1, which appears to be suitable for binding long cellooligosaccharides. The crystal structures of AaBGL1 from the present study provide an important structural basis for the technical improvement of enzymatic cellulosic biomass conversion.

To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes.

The alpha-amylase from Bacillus sp. no. 195 (BAA) consists of two domains: one is the catalytic domain similar to alpha-amylases from animals and Streptomyces in the N-terminal region; the other is the functionally unknown domain composed of an approx. 90-residue direct repeat in the C-terminal region. The gene coding for BAA was expressed in Streptomyces lividans TK24. Three active forms of the gene products were found. The pH and thermal profiles of BAAs, and their catalytic activities for p-nitrophenyl maltopentaoside and soluble starch, showed almost the same behaviours. The largest, 69 kDa, form (BAA-alpha) was of the same molecular mass as that of the mature protein estimated from the nucleotide sequence, and had raw-starch-binding and -degrading abilities. The second largest, 60 kDa, form (BAA-beta), whose molecular mass was the same as that of the natural enzyme from Bacillus sp. no. 195, was generated by proteolytic processing between the two repeat sequences in the C-terminal region, and had lower activities for raw starch binding and degrading than those of BAA-alpha. The smallest, 50 kDa, form (BAA-gamma) contained only the N-terminal catalytic domain as a result of removal of the C-terminal repeat sequence, which led to loss of binding and degradation of insoluble starches. Thus the starch adsorption capacity and raw-starch-degrading activity of BAAs depends on the existence of the repeat sequence in the C-terminal region. BAA-alpha was specifically adsorbed on starch or dextran (alpha-1,4 or alpha-1,6 glucan), and specifically desorbed with maltose or beta-cyclodextrin. These observations indicated that the repeat sequence of the enzyme was functional in the starch-binding domain (SBD). We propose the designation of the homologues to the SBD of glucoamylase from Aspergillus niger as family I SBDs, the homologues to that of glucoamylase from Rhizopus oryzae as family II, and the homologues of this repeat sequence of BAA as family III.

The cellobiose- and cellulose-responsive induction of the FIII-avicelase (cbhI), FII-carboxymethyl cellulase (cmc2), and FIa-xylanase (xynIa) genes is not regulated by XlnR in Aspergillus aculeatus, which suggests that this fungus possesses an unknown cellulase gene-activating pathway. To identify the regulatory factors involved in this pathway, we constructed a random insertional mutagenesis library using Agrobacterium tumefaciens-mediated transformation of A. aculeatus NCP2, which harbors a transcriptional fusion between the cbhI promoter (P ( CBHI )) and the orotidine 5'-phosphate decarboxylase gene (pyrG). Of the ~6,000 transformants screened, one 5-FOA-resistant transformant, S4-22, grew poorly on cellulose-containing media and exhibited reduced cellobiose-induced expression of cbhI. Southern blot analysis and nucleotide sequencing of the flanking regions of the T-DNA inserted in S4-22 indicated that the T-DNA was inserted within the coding region of a previously unreported Zn(II)(2)Cys(6)-transcription factor, which we designated the cellobiose response regulator (ClbR). The disruption of the clbR gene resulted in a significant reduction in the expression of cbhI and cmc2 in response to cellobiose and cellulose. Interestingly, the cellulose-responsive induction of FI-carboxymethyl cellulase (cmc1) and FIb-xylanase (xynIb) genes that are under the control of XlnR, was also reduced in the clbR-deficient mutant, but there was no effect on the induction of these genes in response to D-xylose or L-arabinose. These data demonstrate that ClbR participates in both XlnR-dependent and XlnR-independent cellobiose- and cellulose-responsive induction signaling pathways in A. aculeatus.

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