0% microcrystalline cellulose and 01% yeast extract in the basal

0% microcrystalline cellulose and 0.1% yeast extract in the basal medium with 1% agar after step dilutions. Individual colonies were collected from the plate and inoculated in the basal medium containing 0.5% cellobiose and 0.1% yeast

extract. After five consecutive transfers, a fermentation experiment was carried out using the 200 mL basal medium containing 2 g of FP as the carbon source. The concentrations of fermentation products were analyzed by high-performance liquid CDK phosphorylation chromatography (HPLC) using an Aminex HPX-87H column (Bio-Rad, Hercules, CA). The detected fermentation products included acetate, ethanol, butyric acid, butanol, cellobiose and glucose. The fermentation broth was taken at 72 h to determine the crude enzyme activities. The sample was centrifuged at 10 000 g for 5 min, and the supernatant was used as crude extract. Clostridium thermocellum LQR1 was used as control, which was cultivated in CM3 medium with 1% FP as the carbon source (Weimer & Zeikus, 1977), and the crude enzyme was taken after 72 h cultivation. All assays were performed at 60 °C in 20 mM PIPES [piperazine-N,N-bis (2-ethanesulfonic acid)] buffer (pH 7) under static conditions for 60 min. FP hydrolysis activity (FPase) was determined using Whatman No. 1 FP and was expressed in filter paper units (FPU) (Wood & Kellogg, 1988). One FPU was defined as the

amount of enzyme capable of producing 1 μmol of reducing sugars in 1 min. Endoglucanase and xylanase activities were measured using carboxymethylcellulose (CMC) and birch wood xylan (Sigma-Aldrich), respectively, with learn more a 1% solution of CMC or xylan as the substrate. The β-glucosidase, β-xylosidase and pNPCase activities were determined using p-nitrophenyl-β-d-glucoside, p-nitrophenyl-β-d-xyloside and p-nitrophenyl-cellobioside (Sigma-Aldrich) as the substrate, respectively (Wood & Kellogg, 1988). One unit of enzyme releases 1 μmol equivalent of glucose, xylose or p-nitrophenol per minute. The release of reducing sugars was measured by the dinitrosalicylic colorimetric

(DNS) Methisazone method (Miller, 1959). Protein concentrations were determined with the Bradford assay kit (Biomed, Bejing, China) with bovine serum albumin as the standard. After five consecutive transfers using basal medium with Avicel as the growth substrate, total DNA of enrichment culture was extracted using the E.Z.N.A.™ Soil DNA Kit (Omega Bio-Tek). The DNA obtained from each set of triplicate extractions was pooled. Using the universal oligonucleotide primers 27F and 1492R, the extracted DNA was used in triplicate PCR amplifications targeting the 16S rRNA gene. PCR amplifications were prepared with 25 μL 2× Taq PCR Master Mix (Biomed), and 1 μM of each primer for a final volume of 50 μL, using 30 cycles of 94 °C (30 s), 50 °C (45 s), and 72 °C (90 s), with an initial denaturation at 95 °C (5 min) and a final extension at 72 °C (5 min).

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