A multiple xylanase system with high levels of xylanase activity produced from Penicillium oxalicum GZ-2 using agricultural waste as a substrate has been previously reported. However, the eco-physiological properties and origin of the multiplicity of xylanases remain unclear. In the present study, eight active bands were detected using zymography, and all bands were identified as putative xylanases using MALDI-TOF-MS/MS. These putative xylanases are encoded by six different xylanase genes. To evaluate the functions and eco-physiological properties of xylanase genes, xyn10A, xyn11A, xyn10B and xyn11B were expressed in Pichia pastoris. The recombinant enzymes xyn10A and xyn10B belong to the glycoside hydrolase (GH) family 10 xylanases, while xyn11A and xyn11B belong to GH11 xylanases. Biochemical analysis of the recombinant proteins revealed that all enzymes exhibited xylanase activity against xylans but with different substrate specificities, properties and kinetic parameters. These results demonstrated that the production of multiple xylanases in P. oxalicum GZ-2 was attributed to the genetic redundancy of xylanases and the post-translational modifications, providing insight into a more diverse xylanase system for the efficient degradation of complex hemicelluloses.

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Luteolin-7-O-glucoside (LUTG) was isolated from the plants of Dracocephalum tanguticum Maxim. Previous research has showed that LUTG pretreatment had a significant protective effect against doxorubicin (DOX)-induced cardiotoxicity by reducing intracellular calcium overload and leakage of creatine kinase and lactate dehydrogenase. But the underlying mechanisms have not been completely elucidated. In the present study, we investigated the effects of LUTG on H9c2 cell morphology, viability, apoptosis, reactive oxygen species generation, and the mitochondrial transmembrane potentials. The expression of p-PTEN, p-Akt, p-ERK, p-mTOR, and p-GSK-3β were detected by Western blotting. Compared with DOX alone treatment group, the morphological injury and apoptosis of the cells in groups treated by DOX plus LUTG were alleviated, cell viability was increased, ROS generation was lowered remarkably, and mitochondrial depolarization was mitigated. In DOX group, the expression of p-PTEN was lower than normal group and the expression of p-Akt and p-ERK was higher than normal group. In the groups treated with LUTG (20 μM), the expression of p-PTEN was upregulated and the expression of p-Akt, p-ERK, p-mTOR, and p-GSK-3β was downregulated. These results indicated that the protective effects of LUTG against DOX-induced cardiotoxicity may be related to anti-apoptosis through PTEN/Akt and ERK pathway.

BackgroundAgricultural residue is more efficient than purified cellulose at inducing lignocellulolytic enzyme production in Penicillium oxalicum GZ-2, but in Trichoderma reesei RUT-C30, cellulose induces a more efficient response. To understand the reasons, we designed an artificially simulated plant biomass (cellulose plus xylan) to study the roles and relationships of each component in the production of lignocellulolytic enzymes by P. oxalicum GZ-2.ResultsThe changes in lignocellulolytic enzyme activity, gene expression involving (hemi)cellulolytic enzymes, and the secretome of cultures grown on Avicel (A), xylan (X), or a mixture of both (AX) were studied. The addition of xylan to the cellulose culture did not affect fungal growth but significantly increased the activity of cellulase and hemicellulase. In the AX treatment, the transcripts of cellulase genes (egl1, egl2, egl3, sow, and cbh2) and hemicellulase genes (xyl3 and xyl4) were significantly upregulated (P <0.05). The proportion of biomass-degrading proteins in the secretome was altered; in particular, the percentage of cellulases and hemicellulases was increased. The percentage of cellulases and hemicellulases in the AX secretome increased from 4.5% and 7.6% to 10.3% and 21.8%, respectively, compared to the secretome of the A treatment. Cellobiohydrolase II (encoded by cbh2) and xylanase II (encoded by xyl2) were the main proteins in the secretome, and their corresponding genes (cbh2 and xyl2) were transcripted at the highest levels among the cellulolytic and xylanolytic genes. Several important proteins such as swollenin, cellobiohydrolase, and endo-beta-1,4-xylanase were only induced by AX. Bray-Curtis similarity indices, a dendrogram analysis, and a diversity index all demonstrated that the secretome produced by P. oxalicum GZ-2 depended on the substrate and that strain GZ-2 directionally adjusted the compositions of lignocellulolytic enzymes in its secretome to preferably degrade a complex substrate.ConclusionThe addition of xylan to the cellulose medium not only induces more hemicellulases but also strongly activates cellulase production. The proportion of the biomass-degrading proteins in the secretome was altered significantly, with the proportion of cellulases and hemicellulases especially increased. Xylan and cellulose have positively synergistic effects, and they play a key role in the induction of highly efficient lignocellulolytic enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0162-2) contains supplementary material, which is available to authorized users.

BackgroundEndo-1,4-β-mannanase is an enzyme that can catalyze the random hydrolysis of β-1, 4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans and has a number of applications in different biotechnology industries. Penicillium oxalicum is a powerful hemicellulase-producing fungus (Bioresour Technol 123:117-124, 2012); however, few previous studies have focused on the cloning and expression of the endo-1,4-β-mannanase gene from Penicillium oxalicum.ResultsA gene encoding an acidophilic thermostable endo-1,4-β-mannanase (E.C. 3.2.1.78) from Penicillium oxalicum GZ-2, which belongs to glycoside hydrolase family 5, was cloned and successfully expressed in Pichia pastoris GS115. A high enzyme activity (84.4 U mL−1) was detected in the culture supernatant. The recombinant endo-1,4-β-mannanase (rPoMan5A) was tagged with 6 × His at its C-terminus and purified using a Ni-NTA Sepharose column to apparent homogeneity. The purified rPoMan5A showed a single band on SDS-PAGE with a molecular mass of approximately 61.6 kDa. The specific activity of the purified rPoMan5A was 420.9 U mg−1 using locust bean gum as substrate. The optimal catalytic temperature (10 min assay) and pH value for rPoMan5A are 80°C and pH 4.0, respectively. The rPoMan5A is highly thermostable with a half-life of approximately 58 h at 60°C at pH 4.0. The Km and Vmax values for locust bean gum, konjac mannan, and guar gum are 7.6 mg mL−1 and 1425.5 μmol min−1 mg−1, 2.1 mg mL−1 and 154.8 μmol min−1 mg−1, and 2.3 mg mL−1 and 18.9 μmol min−1 mg−1, respectively. The enzymatic activity of rPoMan5A was not significantly affected by an array of metal ions, but was inhibited by Fe3+ and Hg2+. Analytical results of hydrolytic products showed that rPoMan5A could hydrolyze various types of mannan polymers and released various mannose and manno-oligosaccharides, with the main products being mannobiose, mannotriose, and mannopentaose.ConclusionOur study demonstrated that the high-efficient expression and secretion of acid stable and thermostable recombinant endo-1, 4-β-mannanase in Pichia pastoris is suitable for various biotechnology applications.