Cloning, expression, characterization, and high cell-density production of recombinant endo-1,4-beta-xylanase from Aspergillus niger in Pichia pastoris
Ruanglek, V.; Sriprang, R.; Ratanaphan, N.; Tirawongsaroj, P.; Chantasigh, D.; Tanapongpipat, S.; Pootanakit, K.; Eurwilaichitr, L.
Enzyme and Microbial Technology 41(1-2): 19-25
A recombinant gene XylB (564 bp) encoding endo-1,4-beta-xylanase, obtained from Aspergillus niger BCC14405, was successfully cloned and secreted as a 21 kDa in Pichia pastoris under the control of AOX1 promoter. The activity of the recombinant xylanase was highest at 55 degrees C which was 5 degrees C higher than native xylanase. In addition, the recombinant xylanase was active over the range of pH 3.6-6.5 with maximal activity at pH 5 (8007 U/mg). When compared to a commercial enzyme, in vitro digestibility of the recombinant enzyme was 1.8- and 2.4-folds higher digesting rates of rice bran and soybean meal fibers, respectively.Two-liter production of xylanase was performed with BSM medium which increased cell concentration up to 84.5 g(dry-weight)/L via the 80% mu(max), exponential feed strategy. This process provided maximum xylanase production (3676 U/mL) with highest specific activity (7352 U/mg(protein)) and volumetric productivity (22,832 U/L/h) at 3.0% (v/v) methanol induction. By far, this was the highest xylanase expression in P pastoris host system being reported. Thus, this BCC14405 recombinant xylanase could be produced and used effectively as a feed additive for animals.