Appl Microbiol Biotechnol (2012) 93:1601–1608 1603 Table 1 Oligonucleotides for amplification of the individual cellulase genes and the cdna1 promoter (with restriction sites underlined) Target cel7a cel7b cel12a Pcdna1 Oligonucleotides Cbh1_ClaI_F Cbh1_SalI_R Egl1_ClaI_F Egl1_HindIII_R Egl3_ClaI_F/ Egl3_HindIII_R Cdna1_XhoI Cdna1_ClaI colonies which showed regular growth were transferred to selective media. To test stability, the transformants were passaged three to four times to nonselective media before they were retested on selective media for growth and expression of the individual cellulases. Molecular analysis of transformants Genomic DNA of the transformants was extracted using a phenol–chloroform method (Seiboth et al. 2002). Transformants were screened for the presence of the different cellulase expression cassettes by amplifying the expression cassette by PCR with one oligonucleotide specific for the promoter region of tef1 or cDNA1 and the second specific for the terminator region of the respective cellulase gene. Southern analysis was used to verify the integration of the construct and determine the approximate copy number of the integrated cellulase genes. For Southern blot analysis, genomic DNA (~15–20 μg) of QM9414, the Δxyr1 strain and the transformants were digested with BamHI or PstI for cel7b, and XhoI or PstI for cel12a copy number determination. Digested DNA was size fractionated by gel electrophoresis and transferred to a Biodyne B 0.45-μm membrane (Pall Corporation). DNA labeling, hybridization, and detection were performed according to the DIG High Primer DNA Labeling and Detection Starter Kit I protocols (Roche Applied Science). DIG-labeled probes were amplified with oligonucleotides Egl1_ClaI_F/Egl1_HindIII_R for cel7b and Egl3_ClaI_F/ Egl3_HindIII_R for cel12a. The above-mentioned restriction enzymes cut the respective cellulase gene approximately in the middle of the coding region which results upon hybridization with the probe in two hybridizing bands of similar intensity. The copy number of integrated cellulase genes was hence determined by the number of additional fragments (n−1) to the endogenous cellulase gene copy. SDS-PAGE Supernatants were loaded to SDS gel after EtOH precipitation as follows: 500 μL of culture supernatant was mixed with 1 mL 96% EtOH and stored o/n at −20°C. Following centrifugation at 13,000 rpm for 20 min at 4°C, the protein pellets were resuspended with 40 μL ddH2O. Samples 5′-GTTATCGATTCCGGACTGCGCATCATGTATC-3′ 5′-CATGTCGACTTGTCTCCCTATGGGTCATTAC-3′ 5′-GTTATCGATTCTTAGTCCTTCTTGTTGTCCC-3′ 5′-GATAAGCTTGGTGGGAGAAGACTTTGGAC-3′ 5′-GTTATCGATTAGCGTCGCAATGAAGTTCC-3′ 5′-GGAAAGCTTGCCGTGAGAATTGTAC-3′ 5′-TGACTCGAGCAGACAATGATGGTAGCAGC-3′ 5′-AGTATCGATGAGAGAAGTTGTTGGATTGATC-3′ were run with constant current (15 mA/gel) on 12% denaturing SDS gel. Cellulase activity assays Cel7b transformants were assayed using 4-methylumbelliferyl-β-D-cellobioside (MUC) and cel7a transformants using 4-methylumbelliferyl-β-D-lacto- side (MULAC) as substrate. Substrates (MUC/MUCLAC) were dissolved in DMSO, and the activities were measured with 50 nmol substrate, 50 mM NaAc buffer (pH 5.0) and 50 μL supernatant in a reaction volume of 200 μL/well. Fluorescence was measured with a Gemini XS spectroflu- orometer with 1 min intervals for 1 h at 40°C using ddH2O as blank, with the following parameters: excitation 330 nm, emission 456 nm, and cutoff 455 nm. Initial linear release rate of fluorescence was used to calculate activities. The carboxymethylcellulase assays for CEL12A were done in quadruplets. To remove interfering D-glucose in the culture filtrate, the extracellular protein was precipitated by the addition of 2 vol. of ethanol and then resuspended in ddH2O. Reagents were preheated to 50°C and 96-well plates were covered with a plastic mat during incubations to prevent evaporation. Thirty-microliter supernatants were added to 96-well PCR plates and heated to 50°C. Thirty microliters 2% carboxymethylcellulose (CMC) in NaOAc Buffer (pH 4.8) was added to wells simultaneously, mixed quickly, and incubated for 30 min at 50°C. Sixty microliters DNS reagent was added to wells, mixed shortly, and incubated at 95°C for 5 min for color development. PCR plate was then placed on ice, and absorbances of 100 μL of mixtures were read at 550 nm after transferring them to flat bottomed 96-well microplate. CMCase activity was calcu- lated as described previously (Ghose 1987). One unit catalyzes the liberation of 1 μmol glucose from sodium carboxymethyl cellulose per min. Specific activities are related to the total extracellular protein produced. Total protein concentration and dry biomass determination Total protein concentrations in the culture supernatants were measured by the Bradford assay (Bradford 1976). For dry biomass determination, 20 mL of culture were