{Reference Type}: Journal Article
{Author}: Pezzella, C.; Lettera, V.; Piscitelli, A.; Giardina, P.; Sannia, G.
{Year}: 2013
{Title}: Transcriptional analysis of Pleurotus ostreatus laccase genes
{URL}: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=22395908&query_hl=1
{Tag}: 0
{Star}: 0
{Journal}: Appl Microbiol Biotechnol
{Volume}: 97
{Issue}: 2
{Pages}: 705-17
{DOI}: 10.1007/s00253-012-3980-9
{Date Displayed}: 2013 Jan
{Date}: 2013-01-01
{Type of Work}: Journal Article; Research Support, Non-U.S. Gov't
{Accession Number}: 22395908
{Abstract}: Fungal laccases (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) are multi-copper-containing oxidases that catalyse the oxidation of a great variety of phenolic compounds and aromatic amines through simultaneous reduction of molecular oxygen to water. Fungi generally produce several laccase isoenzymes encoded by complex multi-gene families. The Pleurotus ostreatus genome encodes 11 putative laccase coding genes, and only six different laccase isoenzymes have been isolated and characterised so far. Laccase expression was found to be regulated by culture conditions and developmental stages even if the redundancy of these genes still raises the question about their respective functions in vivo. In this context, laccase transcript profiling analysis has been used to unravel the physiological role played by the different isoforms produced by P. ostreatus. Even if reported results depict a complex picture of the transcriptional responses exhibited by the analysed laccase genes, they were allowed to speculate on the isoform role in vivo. Among the produced laccases, LACC10 (POXC) seems to play a major role during vegetative growth, since its transcription is downregulated when the fungus starts the fructification process. Furthermore, a new tessera has been added to the puzzling mosaic of the heterodimeric laccase LACC2 (POXA3). LACC2 small subunit seems to play an additional physiological role during fructification, beside that of LACC2 complex activation/stabilisation.
{Author Address}: Department of Organic Chemistry and Biochemistry, University of Naples Federico II, via Cinthia 4, 80126 Naples, Italy.
{Language}: eng

{Reference Type}: Journal Article
{Author}: Xiong, C.; Xia, Y.; Zheng, P.; Wang, C.
{Year}: 2013
{Title}: Increasing oxidative stress tolerance and subculturing stability of Cordyceps militaris by overexpression of a glutathione peroxidase gene
{URL}: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=22828981&query_hl=1
{Tag}: 0
{Star}: 0
{Journal}: Appl Microbiol Biotechnol
{Volume}: 97
{Issue}: 5
{Pages}: 2009-15
{DOI}: 10.1007/s00253-012-4286-7
{Date Displayed}: 2013 Mar
{Date}: 2013-03-01
{Type of Work}: Journal Article; Research Support, Non-U.S. Gov't
{Accession Number}: 22828981
{Abstract}: Like other filamentous fungi, the medicinal ascomycete Cordyceps militaris frequently degenerates during continuous maintenance in culture by showing loss of the ability to reproduce sexually or asexually. Degeneration of fungal cultures has been related with cellular accumulation of reactive oxygen species (ROS). In this study, an antioxidant glutathione peroxidase (Gpx) gene from Aspergillus nidulans was engineered into two C. militaris strains, i.e., the Cm01 strain which can fruit normally and the Cm04 strain which has lost the ability to form fruiting bodies on different media through subculturing. The results showed   that the mitotically stable mutants had higher Gpx activities and stronger capacity to scavenge cellular ROS than their parental strains. Most significantly, the fruiting ability of Cm04 strain was restored by overexpression of the antioxidant enzyme. However, after being successively transferred for up to ten generations, two of three Cm04 mutants again lost the ability to fruit on   insect pupae while Cm01 transformants remained fertile. This study confirms the relationship between fungal culture degeneration and cellular ROS accumulation. Our results indicate that genetic engineering with an antioxidant gene can be an   effective way to reverse fungal degeneration during subculturing.
{Author Address}: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China.
{Language}: eng

{Reference Type}: Journal Article
{Author}: Amore, A.; Amoresano, A.; Birolo, L.; Henrissat, B.; Leo, G.; Palmese, A.; Faraco, V.
{Year}: 2012
{Title}: A family GH51 alpha-L-arabinofuranosidase from Pleurotus ostreatus: identification, recombinant expression and characterization
{URL}: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=22080345&query_hl=1
{Tag}: 0
{Star}: 0
{Journal}: Appl Microbiol Biotechnol
{Volume}: 94
{Issue}: 4
{Pages}: 995-1006
{DOI}: 10.1007/s00253-011-3678-4
{Date Displayed}: 2012 May
{Date}: 2012-05-01
{Type of Work}: Journal Article; Research Support, Non-U.S. Gov't
{Accession Number}: 22080345
{Keywords}: Cloning, Molecular; Enzyme Stability; Exons; Gene Expression; Glycoside Hydrolases/chemistry/*genetics/*metabolism; Hydrogen-Ion Concentration; Introns; Kinetics; Kluyveromyces/enzymology/genetics; Lycopersicon esculentum/microbiology; Molecular Weight; Open Reading Frames; Pichia/enzymology/genetics; Pleurotus/*enzymology/*genetics; Recombinant Proteins/chemistry/genetics/metabolism; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Temperature
{Abstract}: An alpha-L-arabinofuranosidase produced by Pleurotus ostreatus (PoAbf) during solid state fermentation on tomato pomace was identified and the corresponding gene and cDNA were cloned and sequenced. Molecular analysis showed that the poabf gene carries 26 exons interrupted by 25 introns and has an open reading frame encoding a protein of 646 amino acid residues, including a signal peptide of 20 amino acid residues. The amino acid sequence similar to the other alpha-L-arabinofuranosidases indicated that the enzyme encoded by poabf can be classified as a family 51 glycoside hydrolase. Heterologous recombinant expression of PoAbf was carried out in the yeasts Pichia pastoris and Kluyveromyces lactis achieving the highest production level of the secreted enzyme (180 mg L(-1)) in the former host. rPoAbf produced in P. pastoris was purified and characterized. It is a glycosylated monomer with a molecular weight   of 81,500 Da in denaturing conditions. Mass spectral analyses led to the localization of a single O-glycosylation site at the level of Ser160. The enzyme   is highly specific for alpha-L-arabinofuranosyl linkages and when assayed with p-nitrophenyl alpha-L-arabinofuranoside it follows Michaelis-Menten kinetics with a K (M) of 0.64 mM and a k (cat) of 3,010 min(-1). The optimum pH is 5 and the optimal temperature 40 degrees C. It is worth noting that the enzyme shows a very high stability in a broad range of pH. The more durable activity showed by rPoAbf in comparison to the other alpha-L-arabinofuranosidases enhances its potential for biotechnological applications and increases interest in elucidating the molecular bases of its peculiar properties.
{Author Address}: Department of Organic Chemistry and Biochemistry, University of Naples Federico II, Complesso Universitario Monte S. Angelo, via Cintia 4, Naples, Italy.
{Language}: eng

{Reference Type}: Journal Article
{Author}: Nakajima, M.; Yamashita, T.; Takahashi, M.; Nakano, Y.; Takeda, T.
{Year}: 2012
{Title}: Identification, cloning, and characterization of beta-glucosidase from Ustilago esculenta
{URL}: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=21850431&query_hl=1
{Tag}: 0
{Star}: 0
{Journal}: Appl Microbiol Biotechnol
{Volume}: 93
{Issue}: 5
{Pages}: 1989-98
{DOI}: 10.1007/s00253-011-3538-2
{Date Displayed}: 2012 Mar
{Date}: 2012-03-01
{Type of Work}: Journal Article; Research Support, Non-U.S. Gov't
{Accession Number}: 21850431
{Keywords}: Aspergillus oryzae/enzymology/genetics; Chromatography, Gel; Chromatography, Ion Exchange; Cloning, Molecular; Molecular Weight; Phylogeny; Polysaccharides/metabolism; Recombinant Proteins/chemistry/genetics/metabolism; Sequence Homology, Amino Acid; Tandem Mass Spectrometry; Ustilago/*enzymology/genetics; beta-Glucosidase/chemistry/genetics/*metabolism
{Abstract}: Hydrolytic enzymes responsible for laminarin degradation were found to be secreted during growth of Ustilago esculenta on laminarin. An enzyme involved in   laminarin degradation was purified by assaying release of glucose from laminaribiose. Ion-exchange chromatography of the culture filtrate followed by size-exclusion chromatography yielded a 110-kDa protein associated with laminaribiose hydrolysis. LC/MS/MS analysis of the 110-kDa protein identified three peptide sequences that shared significant similarity with a putative glucoside hydrolase family (GH) 3 beta-glucosidase in Ustilago maydis. Based on the DNA sequence of the U. maydis GH3 beta-glucosidase, a gene encoding a putative GH3 beta-glucosidase in U. esculenta (Uebgl3A) was cloned by PCR. Based   on the deduced amino acid sequence, the protein encoded by Uebgl3A has a molecular mass of 91 kDa and shares 90% identity with U. maydis GH3 beta-glucosidase. Recombinant UeBgl3A expressed in Aspergillus oryzae released glucose from beta-1,3-, beta-1,4-, and beta-1,6-linked oligosaccharides, and from 1,3-1,4-beta-glucan and laminarin polysaccharides, indicating that UeBgl3A is a beta-glucosidase. Kinetic analysis showed that UeBgl3A preferentially hydrolyzed   laminaritriose and laminaritetraose. These results suggest that UeBgl3A is a key   enzyme that produces glucose from laminarioligosaccharides during growth of U. esculenta on laminarin.
{Author Address}: Iwate Biotechnology Research Center, 22-174-4, Kitakami, Iwate, 024-0003, Japan.
{Language}: eng