Computational protocol: Trichoderma reesei xylanase 5 is defective in the reference strain QM6a but functional alleles are present in other wild-type strains

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[…] In silico analysis of the encoded proteins was performed using SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/), NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/), and NetOGlyc 4.0 Server (http://www.cbs.dtu.dk/services/NetOGlyc/). The KEX2 processed aa sequence was used to predict the protein structure using I-TASSER (Roy et al. ; Yang et al. ; Zhang ) and PyMOL (The PyMOL Molecular Graphics System, Version 1.3r1 Schrödinger, LLC. 2010). [...] For the enzymatic characterization of XYN5 the enzyme was purified as described above. Xylanase activity was measured with the DNS method with 1.5% (w/v) beechwood xylan (Sigma-Aldrich, St. Louis, MO, USA; Product number X4252) dissolved in 10 mM NaAc buffer pH 4 (Bailey et al. ; König et al. ). For all reactions, the reaction volume was adjusted according to König et al. () to fit 1.5-ml tubes. All reactions were done in triplicates, and d-xylose was used as standard to calculate the amount of released sugars. One unit was defined as the enzyme amount liberating 1 μmol of reducing sugars in 1 min under the given conditions. Non-linear fitting for determination of the enzyme activity was performed using SigmaPlot v13.0 (Systat Software Inc., San Jose, CA). For the determination of the optimal temperature, xylanase activity was determined in 10 mM NaAc buffer (pH 4) using different temperatures from 30 to 70 °C. For the determination of the pH optimum, McIlvaine buffer was used from pH 2.5 to 6.5 and reactions were performed at 50 °C.To determine the enzymatic activity in the xyn5 transformants +xyn5a and +xyn5b and the Δtku70 reference strain, strains were cultured on MA medium containing either 1% (w/v) lactose, beechwood xylan, or steam exploded wheat straw (kindly provided by Alexander Jäger of the University of Applied Sciences Upper Austria, FH Wels) in biological duplicates. Culture supernatants were filtered through Miracloth and stored at 4 °C until further use. Biomass was determined in technical triplicates by pelleting 1.5 ml of culture biomass which was subsequently washed with tap water, pelleted again, and dried to constant weight at 80 °C. For the determination of the endo-1,4-ß-d-xylanase activity, the chromogenic substrate S-AXBL (Megazyme International Ireland, Wicklow, Ireland) was used. Reactions were performed as described in the manufacturer’s manual but volumes were reduced 2.5 times to fit 1.5-ml reaction tubes. [...] Protein identification was carried out on an UltrafleXtreme (Bruker Daltonik, Bremen, Germany) after in-gel digestion, by MS and MS/MS analysis. In brief, after excising the gel lanes and removing the Coomassie staining by incubating the gel pieces in acetonitrile/100 mM NH4HCO3 (pH 8.5) (1/1, v/v), the samples were reduced with 10 mM dithiothreitol, alkylated with 50 mM iodoacetamide and trypsinized (20 ng trypsin, porcine, Roche, Basel, Switzerland). After overnight digestion, peptides were extracted with acetonitrile/water, dried in a vacuum centrifuge, and micropurified with C18 ZipTips (Merck Millipore, Billerica, MA, USA) and then eluted onto a stainless steel target together with α-cyano-4-hydroxy-cinnamic acid (CHCA, 3 mg/ml in 50% acetonitrile containing 0.1% trifluoroacetic acid). For all enzymatic digestion data, autolytic tryptic products, keratin and gel blank artifacts were assigned and removed before database search using an in-house Mascot server (Perkins et al. ). The database search was performed with the following parameters: taxonomy fungi, monoisotopic mass values, peptide mass tolerance of ±0.3 Da, two missed cleavages, carboxyamidomethylation as fixed modification, and methionine oxidations as variable modification. A protein was considered correctly identified if the search result was above the statistical threshold for the peptide mass fingerprint and all respective sequencing experiments. […]

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