上海科技大学知识管理系统

α-L-arabinofuranosidases (EC 3.2.1.55; AFs) cause the release of arabinosyl residues from hemicellulose polymers such as xylans, and are receiving increased levels of research attention as they could be applied in a range of processes that involve the enzymatic degradation of xylans. The secretory production of bacterial AFs has not been attempted previously. In this study, we designed a unique induction system for the production of a recombinant AF in Bacillus subtilis in order to exploit its enzymic degradation of wheat bran. We found that non-starch phytochemicals were more efficient than D-xylose when inducing the expression of T7 RNA polymerase and driving the transcription of AF by the T7 promoter. The host cell, B. subtilis (ATCC 6051a-derived strain 164T7P) was engineered to incorporate a DNA cassette that expressed T7 RNA polymerase under the control of a D-xylose inducible promoter (PxylA). The T7 promoter engineered into 164T7P was initially tested and compared with P43 in terms of GFP expression; we found that the expression level of GFP by the T7 promoter was ten-fold higher than that achieved by P43. When cultured in a flask with gentle shaking, and with D-xylose as an inducer, the recombinant strain successfully expressed arbf, a family 51 (GH 51) glycoside hydrolase from Bacillus licheniformis, and secreted 141.4 ± 4.8 U/mL of enzyme, with a Km of 1.4 ± 0.1 mM and a kcat of 139.4 s−1. However, the protein was devoid of a secretary signal peptide. When cultures were supplemented with wheat bran, the maximal yield of the secreted AF reached 194.8 ± 4.1 U/mL. The results provide a foundation for the high level production of heterologous proteins using wheat bran as the inducer in B. subtilis. © 2020 Elsevier Inc.