表达牛肉风味肽的重组毕赤酵母部分生物学特性的研究

Biological characteristics of recombinant Pichia pastoris 16B2 for beefy meaty peptide

对分泌表达16拷贝牛肉风味肽毕赤酵母工程菌株)的生物表型、生长特性、表达特性及遗传稳定性等进行了研究。结果表明:该重组菌株的表型为Mut+;在5L发酵罐中,采用BSM培养基,设定适合的菌体培养温度和pH值,通过调节搅拌速度、通气量和罐压等措施使溶氧(DO)维持在20%～35%,当诱导90h后(发酵110h),目的产物BMP的表达量达到最高为54mg/L,是其摇瓶水平的5.4倍;遗传稳定性分析实验及PCR检测结果证明,重组菌株具有良好的遗传稳定性和重组蛋白表达的稳定性。

The characteristics of recombinant Pichia pastoris GS 115-16B2 secreting the fusion peptide containing 16 tandem repeated beefy meaty peptide （BMP）, including phenotype, growth rate, expression for BMP, and stability of heredity. The results obtained from growing on medium of MM and MD confirmed that phenotype of recombinant strain was identified as Mut＋, utilizing methanol to grow as wild type. In order to produce BMP in a 5 L fermentor, recombinant P. pastoris GS115-16B2 was inoculated to carry out a two-step fermentation process, i.e., glycerol batch phase and a methanol fed-batch induction phase. Dissolved oxygen （DO） was maintained above 20-35% saturation by adjusting agitation rate, aeration and fermentor pressure. Samples were analyzed to determine the cell density （OD600） and BMP production. It is found that, after fermetation for 110 h, the cell density （OD600） and the yield of target protein is 54 mg/L. The production of BMP was 4.4-fold higher than that of 500 ml shake-flask. In the genetic stability tests, the growth characteristics and BMP expression level of the recombinant strain P. pastoris GS115-16B2 did not show differences in five generations. It is indicated that recombinant stain has good genetic stability.