重组毕赤酵母发酵甘油制备α-葡聚糖酶的研究

摘要

【目的】考察甘油对组成型毕赤酵母（Pichiapastoris ）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。%Objective]The aim of this paper is mainly to investigate the effects of glycerol on Pichia pastoris growth and constitutive expression ofα-glucanase.[Methods]The initial concen-tration of glycerol in fermentation medium,glycerin concentration in fed-batch,ventilation and other effects on constitutive expression ofα-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 andα-glucanase expression were not affected,while the growth andα-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 andα-glucanase expres-sion.When glycerin concentration became higher,the growth of Pichia pastoris andα-glu-canase expression were restrained.Meanwhile,improving the ventilation was helpful to the ex-pression 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 forα-glucanase prepara-tion with glycerol as carbon source,which great-ly improvedα-glucanase activity and laid a solid foundation forα-glucanase large-scale produc-tion.