重组毕赤酵母发酵甘油制备α-葡聚糖酶的研究

Study on the Fermentation Conditions for α-Glucanase Production by Recombinant Pichia pastoris Using Glycerol as Carbon Source

关键词:  毕赤酵母  α-葡聚糖酶  甘油  发酵条件

Key words:  Pichia pastoris  α-glucanase  glycerol  fermentation conditions

摘要:

[目的]考察甘油对组成型毕赤酵母(Pichia pastoris)生长和α-葡聚糖酶表达量的影响。[方法]采用单因素实验法考察初始发酵培养基中甘油浓度、补料阶段甘油残留和通气量等对α-葡聚糖酶表达量的影响。[结果]发酵培养基中初始甘油浓度从50 g/L提高到150 g/L,菌体生长和α-葡聚糖酶表达量均未受到影响,继续提高到200 g/L时,α-葡聚糖酶表达量明显下降。补料过程甘油残留在0~5 g/L,菌体生长和α-葡聚糖酶表达量最佳,当甘油残留较多时,菌体生长和α-葡聚糖酶的表达量均受到影响。提高通气量有利于增加α-葡聚糖酶的表达量,发酵78 h为宜,在此条件下α-葡聚糖酶酶活力达1763 U。[结论]该工艺优化了以甘油作为碳源制备α-葡聚糖酶的发酵条件,提高了α-葡聚糖酶酶活力,为大规模生产α-葡聚糖酶奠定了基础。

Abstract:

[Objective] The aim of this paper is mainly to investigate the effects of glycerol on Pichia pastoris growth and constitutive expression of α-glucanase.[Methods] The initial concentration of glycerol in fermentation medium, glycerin concentration in fed-batch, ventilation and other effects on constitutive expression of α-glucanase were examined by means of single factor experiment.[Results] When the initial concentration of glycerol ranged from 50 g/L to 150 g/L, the growth and α-glucanase expression were not affected, while the growth and α-glucanase expression appeared to be markedly reduced when the glycerol increase to 200 g/L.Glycerol concentration between 0 to 5 g/L in fed-batch lead to the best growth and α-glucanase expression. When glycerin concentration became higher, the growth of Pichia pastoris and α-glucanase expression were restrained.Meanwhile, improving the ventilation was helpful to the expression of α-glucanase, and suitable fermentation time was 78 h.Under this condition, α-glucanase activity reached about 1763 U.[Conclusion] The process optimized the fermentation conditions for α-glucanase preparation with glycerol as carbon source, which greatly improved α-glucanase activity and laid a solid foundation for α-glucanase large-scale production.