Characterization of a thermostable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33

<p>Abstract</p> <p>Background</p> <p>Recently, the increased demand of energy has strongly stimulated the research on the conversion of lignocellulosic biomass into reducing sugars for the subsequent production, and β-glucosidases have been the focus because of their important roles in a variety fundamental biological processes and the synthesis of useful β-glucosides. Although the β-glucosidases of different sources have been investigated, the amount of β-glucosidases are insufficient for effective conversion of cellulose. The goal of this work was to search for new resources of β-glucosidases, which was thermostable and with high catalytic efficiency.</p> <p>Results</p> <p>In this study, a thermostable native β-glucosidase (nBgl3), which is secreted by the lignocellulose-decomposing fungus <it>Aspergillus fumigatus </it>Z5, was purified to electrophoretic homogeneity. Internal sequences of nBgl3 were obtained by LC-MS/MS, and its encoding gene, <it>bgl3</it>, was cloned based on the peptide sequences obtained from the LC-MS/MS results. <it>bgl</it>3 contains an open reading frame (ORF) of 2622 bp and encodes a protein with a predicted molecular weight of 91.47 kDa; amino acid sequence analysis of the deduced protein indicated that nBgl3 is a member of the glycoside hydrolase family 3. A recombinant β-glucosidase (rBgl3) was obtained by the functional expression of <it>bgl</it>3 in <it>Pichia pastoris </it>X33. Several biochemical properties of purified nBgl3 and rBgl3 were determined - both enzymes showed optimal activity at pH 6.0 and 60°C, and they were stable for a pH range of 4-7 and a temperature range of 50 to 70°C. Of the substrates tested, nBgl3 and rBgl3 displayed the highest activity toward 4-Nitrophenyl-β-D-glucopyranoside (pNPG), with specific activities of 103.5 ± 7.1 and 101.7 ± 5.2 U mg^-1, respectively. However, these enzymes were inactive toward carboxymethyl cellulose, lactose and xylan.</p> <p>Conclusions</p> <p>An native β-glucosidase nBgl3 was purified to electrophoretic homogeneity from the crude extract of <it>A. fumigatus </it>Z5. The gene <it>bgl</it>3 was cloned based on the internal sequences of nBgl3 obtained from the LC-MS/MS results, and the gene <it>bgl3 </it>was expressed in <it>Pichia pastoris </it>X33. The results of various biochemical properties of two enzymes including specific activity, pH stability, thermostability, and kinetic properties (Km and Vmax) indicated that they had no significant differences.</p

Effect of CBM1 and linker region on enzymatic properties of a novel thermostable dimeric GH10 xylanase (Xyn10A) from filamentous fungus Aspergillus fumigatus Z5

Abstract Xylanase with a high thermostability will satisfy the needs of raising the temperature of hydrolysis to improve the rheology of the broth in industry of biomass conversion. In this study, a xylanase gene (xyn10A), predicted to encode a hydrolase domain of GH10, a linker region and a CBM1 domain, was cloned from a superior lignocellulose degrading strain Aspergillus fumigatus Z5 and successfully expressed in Pichia pastoris X33. Xyn10A has a specific xylanase activity of 34.4 U mg−1, and is optimally active at 90 °C and pH 6.0. Xyn10A shows quite stable at pHs ranging from 3.0 to 11.0, and keeps over 40% of xylanase activity after incubation at 70 °C for 1 h. Removal of CBM1 domain has a slight negative effect on its thermostability, but the further cleavage of linker region significantly decreased its stability at high temperature. The transfer of CBM1 and linker region to another GH10 xylanase can help to increase the thermostability. In addition, hydrolase domains between the two Xyn10A proteins naturally formed a dimer structure, which became more thermostable after removing the CBM1 or/and linker region. This thermostable Xyn10A is a suitable candidate for the highly efficient fungal enzyme cocktails for biomass conversion

MOESM1 of Effect of CBM1 and linker region on enzymatic properties of a novel thermostable dimeric GH10 xylanase (Xyn10A) from filamentous fungus Aspergillus fumigatus Z5

Additional file 1: Table S1. Primers used for enzyme expressions

Guttation capsules containing hydrogen peroxide: an evolutionarily conserved NADPH oxidase gains a role in wars between related fungi

When resources are limited, the hypocrealean fungus Trichoderma guizhouense can overgrow another hypocrealean fungus Fusarium oxysporum, cause sporadic cell death and arrest growth. A transcriptomic analysis of this interaction shows that T. guizhouense undergoes a succession of metabolic stresses while F. oxysporum responded relatively neutrally but used the constitutive expression of several toxin-encoding genes as a protective strategy. Because of these toxins, T. guizhouense cannot approach it is potential host on the substrate surface and attacks F. oxysporum from above. The success of T. guizhouense is secured by the excessive production of hydrogen peroxide (H2 O2 ), which is stored in microscopic bag-like guttation droplets hanging on the contacting hyphae. The deletion of NADPH oxidase nox1 and its regulator, nor1 in T. guizhouense led to a substantial decrease in H2 O2 formation with concomitant loss of antagonistic activity. We envision the role of NOX proteins in the antagonism of T. guizhouense as an example of metabolic exaptation evolved in this fungus because the primary function of these ancient proteins was probably not linked to interfungal relationships. In support of this, F. oxysporum showed almost no transcriptional response to T. guizhouense Δnox1 strain indicating the role of NOX/H2 O2 in signalling and fungal communication

Utilizing Fokker-Planck-Eddington approximation in modeling light transport in tissues-like media

Neighbor-joining phylogenetic tree based on partial gyrA (A) and cheA (B) nucleotide sequences. The consensus tree was reconstructed from 1,000 trees according to the extended majority rule (SEQBOOT program). Bootstrap values >50 % (1,000 repetitions) are indicated at branch points. (DOCX 136 kb

Additional file 17: Figure S8. of Whole transcriptomic analysis of the plant-beneficial rhizobacterium Bacillus amyloliquefaciens SQR9 during enhanced biofilm formation regulated by maize root exudates

Schematic representation of genes in the pks4 cluster in Bacillus amyloliquefaciens SQR9. Non-ribosomal polyketide synthetase/polyketide synthetase is marked in red, transporter genes in blue, accessory genes in green, and hypothetical genes in yellow. (DOCX 27 kb

Additional file 4: Figure S2. of Whole transcriptomic analysis of the plant-beneficial rhizobacterium Bacillus amyloliquefaciens SQR9 during enhanced biofilm formation regulated by maize root exudates

Comparison of the genomes of Bacillus amyloliquefaciens strains SQR9 and FZB42 and Bacillus subtilis 168. (A and B) Matches of the SQR9 genome with those of B. subtilis 168 (A) and B. amyloliquefaciens FZB42 (B). Synteny plots show the comparison at the nucleotide level of the B. amyloliquefaciens SQR9 genome (a and b, vertical axis) with the genomes of B. subtilis 168 (A, horizontal axis) and B. amyloliquefaciens FZB42 (B, horizontal axis). Forward matches are plotted in red and reverse matches in blue. (C) Global alignment of the three strains built by the M-GCAT program. (DOCX 295 kb