Jacobsen, J., Lydolph, M., Lange, L.
"Culture independent PCR: An alternative enzymes discovery strategy"
(2005) Journal of Microbiological Methods, 60 (1), pp. 63-71.

Degenerate primers were designed for use in a culture-independent PCR screening of DNA from composite fungall communities, inhabiting residues of corn stovers and leaves. According to similarity searches and alignments amplified clone sequences affiliated with glycosyl hydrolase family 7 and glycosyl hydrolase family 45 though significant sequence divergence was observed. Glycosyl hydrolases from families 7 and 45 play a crucial role in biomass conversion to fuel ethanol. Research in this renewable energy source has two objectives: (i) To contribute to development of a renewable alternative to world's limited crude fossil oil reserves and (ii) to reduce air pollution. Amplification with 18S rDNA-specific primers revealed species within the ascomycetous orders Sordariales and Hypocreales as well as basidiomycetous order Agaricales to be present in these communities. Our study documents the value of culture-independent PCR in microbial diversity studies and could add to development of a new enzyme screening technology. © 2004 Elsevier B.V. All rights reserved. 

Becker, F., Schnorr, K., Wilting, R., Tolstrup, N., Bendtsen, J.D., Olsen, P.B.
"Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries"
(2004) Journal of Microbiological Methods, 57 (1), pp. 123-133.

To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less β-lactamase (′bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The ′bla gene was cloned into a bacteriophage Mu minitransposon enabling translational fusions between ′bla and target genes. Fusion of TnSig in the correct reading frame to a protein carrying transmembrane domains or signal peptides resulted in ampicillin resistance of the corresponding clone. Prokaryotic gene libraries from the alkaliphilic bacterium Bacillus halodurans C125 and the hyperthermophilic archaeon Sulfolobus solfataricus P2 were tagged with TnSig. The genomic sequences, which are publicly available (EMBL BA000004 and EMBL AE006641), were used for rapid open reading frame (ORF) identification and prediction of protein localisation in the cell. Genes for secreted proteins, transmembrane proteins and lipoproteins were successfully identified by this method. In contrast to previous transposon based identification strategies, the method described here is fast and versatile and essentially enables any selectable marker compatible library to be tagged. It is suited for identifying genes encoding extracytosolic proteins in gene libraries of a wide range of prokaryotic organisms. © 2004 Elsevier B.V. All rights reserved.

W. Helbert; H. Chanzy; T.L. Husum; M. Schülein; S. Ernst.
"Fluorescent cellulose microfibrils as substrate for the detection of cellulase activity".
Biomacromolecules, 4, 481-487 (2003)

In order to devise a sensitive cellulase assay based on substrates having most of the physical characteristics of native cellulose, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was used as a grafting agent to prepare suspensions of fluorescent microfibrils from bacterial cellulose. These suspensions were digested by a series of commercially relevant cellulases from Humicola insolens origin: cloned Cel6B and Cel 45A as well as crude H. insolens complex. The digestion induced the release of fluorescent cellodextrins as well as that of reducing sugars. After adequate centrifugation, these soluble products were analyzed as a function of grafting content, digestion time and cellulase characteristics. The resulting data allowed to optimize the grafting conditions in order to maximize the quantity of soluble products and therefore to increase the sensitivity of the detection. A comparison between the amount of released birefringence and that of released reducing sugar allowed to differentiate between processive exo and endo cellulase activities.  The casting of films of DTAF grafted microfibrils at the bottom of micro-well titer plates also led to sensitive cellulase detection. As these films kept their integrity and remained firmly glued to the well bottom during the digestion time, they are well tailored-made for a full automation of the cellulases testing.

V. Boyer; S. Fort; T.P. Frandsen; M. Schulein; S. Cottaz; H. Driguez.
"Chemoenzymatic synthesis of a bifunctionalized cellohexaoside as a specific substrate for the sensitive assay of cellulase by fluorescence quenching"
Chemistry-a European Journal, 8 (6),1389-1394 (2002)

A new bifunctionalized cellohexaose derivative was synthesized as a specific substrate for continuous assay of cellulases by resonance energy transfer. This cellohexaoside has a naphthalene moiety (EDANS) as a fluorescent energy donor at the reducing end and a 4-(4'- dimethylaminobenzeneazo)-benzene derivative as an acceptor chromophore at the non-reducing end. The key steps for the preparation of the target molecule involved trans glycosylation reactions of cellobiosyl and cellotetraosyl fluoride donors onto cellobiosyl acceptors catalysed by the E197A mutant of cellulase Cel7B from Humicola insolens. Upon digestion with various cellulases, the energy transfer was disrupted and an increase of fluorescence was observed.

S.F. Lassen; J. Breinholt; P.R. Østergaard; R. Brugger; A. Bischoff; M. Wyss; C.C. Fuglsang.
"Expression, Gene Cloning, and Characterization of Five Novel Phytases from Four Basidiomycete Fungi: Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens."
Appl. Environ. Microbiol., 67, 4701-4707 (2001)

P hytases catalyze the hydrolysis of phosphomonoester bonds of phytate (myo-inositol hexakisphosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi (Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens) and screened for phytase activity in yeast. One full-length phytase-encoding cDNA was isolated from each library, except for the Ceriporia sp. library where two different phytase- encoding cDNAs were found. All five phytases were expressed in Aspergillus oryzae, purified, and characterized. The phytases revealed temperature optima between 40 and 60 degreesC and pH optima at 5.0 to 6.0, except for the P. lycii phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1, 200 U . mg, of protein(-1) and were capable of hydrolyzing phytate down to myo-inositol monophosphate. Surprisingly, H-1 nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC

M. Skjøt; S. Kauppinen; L.V. Kofod; C.C.Fuglsang, M. Pauly; H. Dalbøge; L.N. Andersen.
"Functional cloning of an endo-arabinanase from Aspergillus aculeatus and its heterologous expression in A. oryzae and tobacco"
Mol. Genet. Genomics, 265, 913-921 (2001)

Functional cloning in yeast has been used to isolate full- length cDNAs encoding an endo-alpha -1, 5-L-arabinanase from the filamentous fungus Aspergillus aculeatus. Screening of a cDNA library constructed in a yeast expression vector for transformants that hydrolysed AZCL-arabinan identified 44 Saccharomyces cerevisiae clones all harbouring the same arabinanase-encoding cDNA. The cloned cDNA was expressed in A. oryzae and the recombinant enzyme was purified and characterized. The mode of action of the enzyme was studied by analysis of the digestion pattern towards debranched arabinan. The digestion profile obtained strongly suggests that the enzyme is an endo-arabinanase. In addition, the feasibility using Nicotiana tabacum as an alternative host for arabinanase expression was investigated.

M. Pauly; L.N. Andersen; S. Kauppinen; L.V. Kofod; W.S. York; P. Albersheim; A. Darvill.
"A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme".
Glycobiology, 9 (1), 93-100 (1999)

A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4-glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion-exc hange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 oC. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.