초록
<P>A series of strategies were applied to improve expression level of recombinant endo-<I>β</I>-1,4-xylanase from<I> Aspergillus usamii</I> (<I>A. usamii</I>) in<I> Pichia pastoris</I> (<I>P. pastoris</I>). Firstly, the endo-<I>β</I>-1,4-xylanase (<I>xynB</I>) gene from<I> A. usamii</I> was optimized for<I> P. pastoris</I> and expressed in<I> P. pastoris</I>. The maximum xylanase activity of optimized (<I>xynB-opt</I>) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (<I>xynB</I>) gene. To further increase the expression of<I> xynB-opt</I>, the<I> Vitreoscilla hemoglobin</I> (<I>VHb</I>) gene was transformed to the recombinant strain containing<I> xynB-opt</I>. The results showed that recombinant strain harboring the<I> xynB-opt</I> and<I> VHb</I> (named X33/<I>xynB-opt-VHb</I>) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/<I>xynB-opt-VHb</I> in 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (<I>xynB-opt</I>) gene and wild-type (<I>xynB</I>) gene. Finally, the induction temperature of X33/<I>xynB-opt-VHb</I> was optimized in 50 L bioreactor. The maximum xylanase activity of X33/<I>xynB-opt-VHb</I> reached 58792 U/mL when the induction temperature was 22°C. The results presented here will greatly contribute to improving the production of recombinant proteins in<I> P. pastoris</I>.</P>