The PCR product was analyzed in a 2% agarose Ixazomib concentration gel and purified from the gel using the gel extraction kit (Qiagen). The purified fragment was then inserted into the cloning vector (pGEMT; Promega) to confirm their identity. Plasmid isolation and purification were done using the Wizard plus SV Minipreps DNA purification

system (Promega). The presence of insert in the plasmid was checked by double digestion with restriction enzymes NotI plus NcoI. Plasmid containing the insert was sequenced using an automatic DNA Sequencer (310 Genetic Analyser; Applied Biosystems, Foster City, CA). The catR promoters (Pcat300, Pcat924) were then inserted into the promoter-less xylanase/pAN56-1 plasmid to check their functionality. Pcat300 and Pcat924 were re-amplified using the above-mentioned primers and Pfu DNA polymerase to get blunt-ended amplified products. Promoter-less xylanase/pAN56-1 vector was digested with EcoRV and de-phosphorylated. Digested and de-phosphorylated vector was ligated to Pfu-amplified Pcat300 and Pcat924 promoter fragments. Both ligated mixtures were click here electroporated in JM110-competent cells using gene pulser (Bio-Rad). The plasmids were isolated with Qiagen’s spin column according

to the instructions of the manufacturer. The presence of insert in the plasmids and orientation of the Pcat300 and Pcat924 in promoter-less xylanase/pAN-56-1 was checked by digestion with NcoI. Transformation of A. niger by constructs (Pcat300/xylanase/pAN56-1, Pcat924/xylanase/pAN56-1) was carried out by electroporation as described by Sanchez & Aguirre (1996). Transformed spores were spread on minimal medium agar plates containing 175 μg mL−1 hygromycin (Biogene; Imperial Biomedics) as the selective agent, and incubated at 37 °C (Tilburn et al., 1983; Malardier et al., 1989). Transformants were observed after 36–48 h at 37 °C. Individual clones were transferred to fresh Sabouraud’s/hygromycin plates. selleck Genomic DNA of putative transformants was extracted and amplified by the E. coli ori primers (Varadarajalu & Punekar, 2005) to confirm that each construct had

been integrated into the genome of A. niger. The transformants were further evaluated quantitatively for xylanase production by growing in seed medium under shaking conditions (200 rpm) for 48 h at 28 °C (inoculum size was 2 × 106 spores per flask) and then 10% inoculum was transferred in wet wheat bran (production medium pH 6.0) under static conditions for 96 h. The AlX enzyme from production medium was extracted by shaking at 30 °C for 2 h using 0.05 M phosphate buffer (pH 8.0) and filtered through a wet muslin cloth by squeezing. The extract was centrifuged at 6000 g for 5 min. Clear supernatant sample from each transformant was taken and used for the enzyme assay. Xylanase activity was estimated by quantifying the release of reducing sugar and expressed in terms of IU mL−1 (Gupta et al., 2000).