关键词: 右旋糖酐酶, 基因优化, 毕赤酵母, 表达

Abstract: The dextranase-encoding gene (dex) was amplified by reverse transcription PCR from the genome of Penicillium aculeatum F1001. The gene consisted of 1 866 base pairs and encoded a protein of 622 amino acid residues. According to the codon usage bias of Pichia pastoris, optimized gene (opt-dex) was obtained. The expression recombinant plasmids dex-pPICZαA and opt-dex-pPICZαA were constructed and then separately electro-transformed into P. pastoris X33 to form transformants. The dextranase-producing transformants were selected out using blue-dextran T-2000 specific plates and shake flask expression. The purified recombinant dextranase showed only one band about 65 kDa, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant dextranase reacted optimally at pH 5 and 35 ℃ and specifically cleaved α-1,6 glycosidic bonds. The shake-flask fermentation conditions of the recombinant strain were optimized as follows: temperature 25 ℃, initial pH 5.0, addition of 1% (V/V) methanol, 4 g/L Tween-80 and 5 g/L sorbitol at 24 h intervals, and 50 mL of medium contained in a 500-mL shake flask. Under the optimized conditions, the activity of dextranase was as high as 240.74 U/mL. In conclusion, this study indicates that P. pastoris X33 is suitable for heterologous expression of P. aculeatum dextranase and that the recombinant dextranase can be used as an alternative to the native dextranase in producing dextran for industrial application.

Key words: dextranase, optimization, Pichia pastoris, expression