Enhanced production of a recombinant xylanase (XT6): optimization of production and purification, and scaled-up batch fermentation in a stirred tank bioreactor

Abstract

Abstract The endoxylanase XT6 produced by Geobacillus stearothermophilus is a desirable candidate for industrial applications. In this study, the gene encoding XT6 was cloned using the pET-28a expression vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant XT6 production was improved by optimizing cell lysis (sonication, chemical, and enzymatic lysis) and expression conditions. Sonication in a 0.05 M sodium phosphate (pH 6.0) buffer resulted in the highest xylanase activity (16.48 U/ml). Screening and optimization of induction conditions using the Plackett–Burman Design and Box-Behnken Design (BBD) approaches revealed that cell density pre-induction (OD600 nm), post-induction incubation time, and IPTG concentration significantly (p < 0.05) influenced the expression levels of XT6 (16.48 U/ml to 40.06 U/ml) representing a 3.60-fold increase. BBD resulted in a further 8.74-fold increase in activity to 144.02 U/ml. Batch fermentation in a 5-l stirred tank bioreactor at 1 vvm aeration boosted recombinant xylanase production levels to 165 U/ml suggesting that heterologous expression of the XT6 enzyme is suitable for scaled-up production. The pure enzyme with a molecular weight of 43 kDa and a 15.69-fold increase in purity was obtained using affinity chromatography and a cobalt column. Future studies will include application of the purified recombinant xylanase to animal feed.