碱性纤维素酶基因在巴氏毕赤酵母中的表达及重组酵母菌发酵工艺的优化

目的克隆碱性纤维素酶基因,构建酵母整合型表达质粒,在巴氏毕赤酵母中表达,并对重组菌的发酵工艺进行优化。方法应用PCR技术从嗜碱性芽孢杆菌ATCC21833中扩增碱性纤维素酶基因,克隆至酵母整合型表达载体pGAPZαA中,构建重组表达质粒pGAPZαA-ATCC21833,并转化至巴氏毕赤酵母GS115。通过单因素实验及正交实验,确定重组酵母的最佳发酵培养基。在20L发酵罐中进行高密度发酵,观察碳源对批式发酵的影响,并检测在4种流加方式(连续恒速流加、间歇匀速流加、间歇递减流加、维持底物浓度流加)下的菌体干重及发酵液中的酶活性。结果重组表达质粒pGAPZαA-ATCC21833经酶切及DNA测序证明构建正确,其基因序列与嗜碱性芽孢杆菌KSM-635的碱性纤维素酶基因序列一致。最佳发酵培养基组成为6%葡萄糖、2%硫酸铵、12g/L磷酸二氢钾。碳源浓度对于重组酵母菌体生长及产酶至关重要。SDS-PAGE表明表达产物的相对分子质量约为103000。维持底物浓度的流加方式可获得最高的菌体干重(29.8g/L)及酶活力(24U/ml)。结论已成功构建了表达碱性纤维素酶的巴氏毕赤酵母工程菌,并确定了维持底物浓度的流加方式为最佳发酵方式。

Expression of Gene Encoding Alkaline Cellulose in Pichia pastoris and Fermentation of Recombinant Pichia pastoris

Objective To clone the gene encoding alkaline cellulose, construct a yeast integrative expression plasmid and express in Pichia pastoris, and optimize the procedure for fermentation of recombinant P. pastoris. Methods The gene encoding alkaline cellulose was amplified from Bacillus sp. ATCC21833 by PCR and cloned into yeast integrative expression vector pGAPZαA. The constructed recombinant plasmid pGAPZαA-ATCC21833 was transformed to P. pastoris strain GS115. The medium for fermentation of recombinant P. pastoris was optimized by single factor test and orthogonal test. The high density fermentation of recombinant P. pastoris was performed in 20 L fermentor, and the effect of carbon source on batch fermentation was observed. The dry weights of P. pastoris and enzyme activity in fermentation liquid by 4 methods for fed-batch, i.e. continuous fed-batch at a constant speed, intermittent fed-batch at a constant speed, intermittent fed-batch at a degressive speed and fed-batch by maintaining a constant substrate concentration, were analyzed and compared. Results Both restriction analysis and DNA sequencing proved that recombinant plasmid pGAPZαA-ATCC21833 was constructed correctly. The gene sequence of recombinant plasmid pGAPZαA-ATCC21833 was consistent with that of alkaline cellulose gene of Bacillus sp. KSM-635. The optimal fermentation medium consisted of 6% glucose, 2% ammonium sulfate and 12 g / L postassium dihydrogen phosphate. The concentration of carbon source showed significant effect on the growth and enzyme production of recombinant P. pastoris. SDS-PAGE showed that the relative molecular mass of expressed product was about 103 000. The dry weight of P. pastoris and enzyme activity of fermentation liquid with fed-batch by maintaining a constant substrate concentration were 29. 8 g / L and 24 U / ml respectively, which were significantly higher than those by the other three methods for fed-batch. Conclusion The recombinant P. pastoris for expression of alkaline cellulose gene was successfully established, and the fed-batch by maintaining a constant substrate concentration was screened as optimal for fermentation.