Combined coagulation/decantation-ozone/hydrogen peroxide-biological aerated filter process for advanced treatment of bio-treated leachate from Chinese herbal medicine residue.

To reach discharge limit, further treatment of bio-treated leachate from Chinese herbal medicine residue (BLCHMR) was very imperative. In this study, performance of combined coagulation/decantation-ozone/hydrogen peroxide (O3/H2O2)-biological aerated filter (BAF) technology used to further treat BLCHMR was investigated with pilot-scale experiment. Under optimal conditions, the COD (40-44 mg/L) and color (13-17 time) in treated BLCHMR indicated that combined process can efficiently treat BLCHMR. O3/H2O2 had good performance not only in mineralization and decomposing of organic matter but also in decolorization of BLCHMR. For dissolved organic matter (DOM), O3/H2O2 reacted with non-biodegradable fraction preferentially and oxidized different molecular weight (MW) fractions equally. O3/H2O2-BAF generated more higher MW DOM (namely F2 fraction) than BAF alone. Meanwhile, O3/H2O2 mainly influenced the amount and biodegradability on DOM but not the removal rate on DOM. Accordingly, the helpful influence on O3/H2O2 perhaps can be owed to the generalized influence on DOM but not the specific influence on improved biodegradability. Finally, inspiring (namely very low) cost (respectively, 0.3419 $/per ton bio-treated leachate and 0.5766 $/Kg COD removed) was achieved in the combined process.

One single basic amino acid at the ω-1 or ω-2 site is a signal that retains glycosylphosphatidylinositol-anchored protein in the plasma membrane of Aspergillus fumigatus.

Although the plasma membrane is the terminal destination for glycosylphosphatidylinositol (GPI) proteins in higher eukaryotes, cell wall-attached GPI proteins (GPI-CWPs) are found in many fungal species. In yeast, some of the cis-requirements directing localization of GPI proteins to the plasma membrane or cell wall are now understood. However, it remains to be determined how Aspergillus fumigatus, an opportunistic fungal pathogen, signals, and sorts GPI proteins to either the plasma membrane or the cell wall. In this study, chimeric green fluorescent proteins (GFPs) were constructed as fusions with putative C-terminal GPI signal sequences from A. fumigatus Mp1p, Gel1p, and Ecm33p, as well as site-directed mutations thereof. By analyzing cellular localization of chimeric GFPs using Western blotting, electron microscopy, and fluorescence microscopy, we showed that, in contrast to yeast, a single Lys residue at the ω-1 or ω-2 site alone could retain GPI-anchored GFP in the plasma membrane. Although the signal for cell wall distribution has not been identified yet, it appeared that the threonine/serine-rich region at the C-terminal half of AfMp1 was not required for cell wall distribution. Based on our results, the cis-requirements directing localization of GPI proteins in A. fumigatus are different from those in yeast.

Propyl 2-(1H-indol-3-yl)acetate.

In the title compound, C13H15NO2, the acetate group [C-C(=O)-O] makes a dihedral angle of 62.35 (13)° with the mean plane of the indole ring system [maximum deviation = 0.011 (3) Å]. In the crystal, mol-ecules are linked by N-H⋯O hydrogen bonds, forming helical chains propagating along [010].

Ku80 gene is related to non-homologous end-joining and genome stability in Aspergillus niger.

In this study, the ku70 and ku80 homologs from the Aspergillus niger genome were identified and their function was analyzed using targeted mutagenesis. The role of the ku80 gene in non-homologous end-joining (NHEJ) was investigated by calculating the frequency of homologous recombination. The transformation test verified that the frequency of homologous recombination significantly increased, from 1.78 to 65.6% in ku80 single deletion strains and to 100% in ku70/ku80 double deletion strains. These results suggest that the ku80 gene is important for non-homologous end-joining. Although the morphology of the ku deletion strains colonies was similar to that of the wildtype strain, mutants were more sensitive to the mutagen phleomycin. Furthermore, the purified ku80 deletion strain produced some sectored colonies on hygromycin B-containing plates. This result suggests that the ku80 gene deletion leads to genomic instability in A. niger.

[Analysis of acid-phosphatase localization in Aspergillus fumigatus by a secreted chimeric green fluorescent protein as reporter].

OBJECTIVE: In yeast glycosylphosphatidylinositol (GPI) anchoring is a signal directing localization of GPI proteins to the plasma membrane or cell wall. Some of the cis-requirements for the localization of GPI proteins are now understood, however, little is known the signals directing distribution of the GPI proteins in filamentous fungi. Previously, AfPhoA, a GPI-anchored acid phosphatase in filamentous fungus Aspergillus fumigatus, was first isolated from the cell membrane and latter found to be associated with the cell wall. The actual distribution of the AfPhoA remains unclear. Meanwhile, the signature amino acid motif that determines the distribution of GPI protein in yeast is not found in the C-terminal sequence of the AfPhoA. We aimed to elucidate the cell distribution of the AfPhoA. METHODS: The green fluorescent protein (GFP) was used as reporter to track the localization of the AfPhoA. The C-terminal sequence of the AfPhoA was fused to the C-terminus of the GFP. RESULTS: We first constructed the expression plasmid pchiGFP, in which the N-terminal signal sequence of the A. fumigatus AfChiB1 was fused to the N-terminus of the GFP. After transformation, a secreted expression of the GFP was achieved in A. fumigatus. Based on this construct, The C-terminal sequence of the AfPhoA was fused to the C-terminus of the GFP to construct a chimeric GFP. After the co-transformation of the fusion construct with plasmid pCDA14, a transformant was confirmed to harbor the chimeric GFP in its genome and could express the chimeric GFP. The transformant cultivated with or without chitin induction could express the chimeric GFP mainly attached to the cell membrane, a prolonged cultivation led to a minor distribution of the chimeric GFP in the cell wall. Although a 30KD of GFP fragment, instead of an intact 43.5KDa chimeric GFP, was also detected in the culture supernatant, which might be released by the cleavage between the fusion protein and its GPI anchor. CONCLUSION: Our results suggest that GPI anchoring determines the distribution of the AfPhoA in the cell membrane. In addition to our investigation of the GPI anchoring, an expression vector was also constructed, which would be useful for analyses of the function and regulation of the genes and proteins in A. fumigatus.

Isolation of four pepsin-like protease genes from Aspergillus niger and analysis of the effect of disruptions on heterologous laccase expression.

Four new aspartic protease genes pepAa, pepAb, pepAc and pepAd from Aspergillus niger were identified using a comparative genomic approach. All four gene products have highly conserved attributes that are characteristic of aspartic proteases; however, each one has novel sequence features. The PEPAa protease appears to represent an ortholog of a pepsin-type aspartic protease previously identified from Talaromyces emersonii and Scleotinia sclerotiorum. The PEPAb protease appears to be an ortholog of an aspartic protease previously identified from BcAP1 of Botryotinia fuckeliana. The PEPAc protease also appears to be an ortholog of BcAP5 from B. fuckeliana. These four genes appear to be conserved in many species of filamentous fungi, all except PEPAb contain a predicted signal peptide. Transcriptome analysis revealed that transcripts of the pepAa gene of Aspergillus nidulans were significantly up-regulated due to recombinant chymosin secretion, suggesting that silencing these genes may lead to improved yields of secreted proteins. To establish the effects of reduced protease activity on the stabilities of secreted proteins, three of the four genes were individually disrupted by double crossover, although we were unable to disrupt the pepAc gene. The secretion level of heterologous laccase in the pepAa, pepAb and pepAd disruption mutants were increased by about 21%, 42% and 30%, respectively. And their total glucogenic enzymes secretion were also increased by about 18.7%, 37.0% and 5.20%, respectively.

The synergetic effects of two CCAAT boxes in Aspergillus niger glaA gene promoter on activation of PglaA transcription.

EMSA and footprinting analyses have revealed that the -489-- -414 bp and the -390-- -345 bp (designated DC and PC respectively) upstream of the Aspergillus nigerT21 glaA gene were bound by one protein factor in the A. nigerT21 whole cell extract. Both DC and PC contained CCAAT pentanucleotides. The functions of DC and PC in regulation of expression of glucoamylase (GLA) were studied. CCAAT pentanucleotides were replaced with CGTAA and the mutated DNA fragments DCm and PCm lost the binding activities of protein factors in vitro. In vivo when either DC or PC was mutated or the relative orientations between them were changed on the PglaA, the transcriptional activity of PglaA decreased to a basal level. Introduction of multi-copies of DC into the original site at the PglaA in A. nigerT21 decreased the expression of endogenous GLA expression and the exogenous reporter E. coli uidA gene introduced under the PglaA promoter, while having no effect on the uidA gene under the control of PgpdA. EMSA revealed that the levels of the specific DNA-binding protein factors in the transformants maintained the same meaning that introduction of multi-copies of DC caused the titration effect. AnghapC gene was cloned from A. nigerT21 cDNA and introduced into the DC multi-copied strains. The expression of AnghapC improved the expression of the endogenous GLA and the exogenous gene controlled by PglaA. These results showed that both the CCAAT pentanucleotides were necessary for DC and PC binding to the protein factors, and the simultaneous binding of DC and PC to the protein was necessary for promoting the transcriptional activity of PglaA. AngHapC was the specific positive trans-acting protein factor binding to DC.

[Construction and functional analysis of the pepB gene disruptant in Aspergillus niger].

An integration plasmid pMW1-pepB for the pepB gene disruption in Aspergillus was constructed. The plasmid contained the pepB gene upstream (P) 1.4kb and downstream (T) 1.3kb homologous fragments with insertion of the expression unit of the hygromycin resistance gene (hph) between them. P and T DNA fragments were synthesized by PCR from Aspergillus niger chromosomal DNA. The integration plasmid was digested with the Hpa I restriction enzyme, the resultant 4.2kb linear fragment was introduced into the Aspergillus niger strain GICC2773 which expressing the glucoamylase/laccase fusion protein by PEG-mediated transformation. 62 Hygromycin resistance transformants were screened, and from them one strain named pepB29 was identified to be the pepB disruptant by PCR analysis. Data of functional assay of the pepB29 strain indicated that the disruption of the pepB gene secreted reduced acid proteolytic activity, and improved the heterologous protein laccase production.

[Integration and expression of beta-endoglucanase I from Trichoderma reesei in brewing yeast].

An integration plasmid pA15-PET for expression and secretion of beta-Endoglucanase I (EG I ) in yeast was constructed by insertion of EG I cDNA between yeast alcohol dehydrogenase promoter and terminator region. The plasmid contained part of yeast rDNA sequence, which was used as a homologous fragment for integration. The EG I cDNA was introduced into an engineered brewing yeast BE9711 containing alpha-acetolactate decarboxylase (alpha-ALDC) encoding gene and integrated onto its rDNA sequence of chromosomal DNA by co-transformation of pA15-PET and a YEP type plasmid pA15TXR carrying G418 resistance. The stable engineered brewing yeast expressing intracellulase alpha-ALDC and extracellular EG I simultaneously were obtained.

Detection of a protein, AngCP, which binds specifically to the three upstream regions of glaA gene in A. niger T21.

Electromobility shift assay (EMSA) was used to scan 600 bp of 5' cis regulatory sequence of Aspergillus niger (A. niger) T21 glucoamylase gene (glaA) for binding by partially fractionated T21 protein extracted from starchinduced mycelia. In this process, one protein, AngCP, was detected to bind specifically to three regions covering -374 to -344, -484 to -414 and -580 to -540 relative to the glaA translational start codon. UV-crosslinking of DNA-protein complex showed that MW of AngCP was 10 ku. DNase I footprinting analysis demonstrated that AngCP specifically binds to two CCAAT containing sequences within the regions between -374 and -344 and -484 and -414 bp. And the region between -580 and -540 bp contains CCAAT similar box, CCTAT. The results indicated that AngCP is probably one of the members of CCAAT-binding protein families, which are generally involved in enhancement of gene expression in filamentous fungi. These findings suggested that AngCP should be a transcription activator for high-level expression of glaA gene.

[Construction of recipient strain of expression-secretion system in filamentous fungi].

Glucoamylase overproducing A. niger T21 was mutated by UV mutagensis. An extracellular acid protease-deficient mutant, A. niger T21-201, which produced only 0.76% extracellular acid protease activity of the parent strain, was screened by casein-degradating plate and determination of protease activity. Moreover, the growth properties and the ability to secrete glucoamylase of A. niger T21-201 are identical to these of starting strain T21. The comparison of expression-secretion levels of heterologous gene in A. niger T21-201 and T21 was carried out with bacterial vhb as reporter, the level of expression-secretion of VHb in A. niger T21-201 was 6-7 times higher than that in T21, but the transcriptional levels of vhb gene in both strains were similar revealed by Northern blot. Therefore, it was demonstrated that the deficiency of acid protease of recipient T21-201 has significant effect on the protection of heterologous protein.