Released reducing sugar was determined using known amounts of xylose as a standard. All of the experiments were performed in triplicate. Specific AlX activity was expressed as

U mg−1 protein. Protein was determined by the Bradford assay (Bradford, 1976) using bovine serum albumin as a standard (Bio-Rad Laboratories, Hercules, CA). The effect of different seed media on AlX production was investigated by growing 10 representative transformants (A1–A10 containing Pcat300/xylanase/pAN56-1; K1–K10 containing Pcat924/xylanase/pAN56-1) of both the constructs in Sabouraud’s (glucose 40 g L−1, peptone 10 g L−1; pH 6.0)/wheat flour medium (Maida 55.2 g L−1, Soya Peptone 4.08 g L−1, Alpelisib solubility dmso Mono ammonium

phosphate 0.2 g L−1, copper sulphate 0.08 g L−1; pH 6.0). After 48 h, inoculums were transferred in production medium as described above. One selected transformant (K6) harboring Pcat924/xylanase/pAN56-1 was subjected to various inducing conditions and the expression pattern of AlX was analysed. H2O2, CaCO3 and a combination of both were used as inducers in the study. The inducers were added to the seed media in which K6 was grown. Different concentrations of the inducers were used to determine the optimum concentration required for the maximum reporter gene activity. The promoter-less xylanase/pAN56-1 vector was constructed using EVPAN7-1 and pAN56-1 alk-xylanase Rebamipide (truncated) (Fig. 1). Pcat300 and Pcat924 were amplified by using specific primers, cloned and sequenced (Fig. 2). Pcat300 and Pcat924 were cloned in promoter-less find more xylanase/pAN-56-1 to check the functional activity of Pcat300 and Pcat924 (Fig. 3a). Constructs (Pcat300/xylanase/pAN56-1 and Pcat924/xylanase/pAN56-1) were transformed

in A. niger. Genomes of putative transformants were initially screened for the presence of introduced construct using the E. coli ori primers, which amplified a 400-bp fragment from all the transformants, confirming that the construct was integrated successfully in the genome of the host, whereas from the host there was no amplification (data not shown; Fig. 3b). To study the regulation of catR promoter, the transformants were grown in two different seed media (Sabouraud’s and wheat flour media) to check the effect of seed media composition on the expression of AlX. In Sabouraud’s media, the AlX-specific activity profile of the transformants carrying Pcat(300) xylanase/pAN56-1, and Pcat924bp xylanase/pAN56-1 constructs are shown in Table 1. The activity was in the range of 41.91–91.4 U mg−1. Among the transformants carrying Pcat(300) xylanase/pAN56-1, A8 showed maximum 3.21-fold increase in specific activity compared to transformant containing promoter-less xylanase/pAN-56-1, whereas A5 showed the minimum change, with a 1.86-fold increase in specific activity.