碳酸钙促进丙酮酸发酵过程中α-酮戊二酸的形成

CaCO_3 Stimulates α-ketoglutarate Accumulation During Pyruvate Fermentation by Torulopsis glabrata

在多重维生素营养缺陷型菌株光滑球拟酵母CCTCCM2 0 2 0 19发酵生产丙酮酸的摇瓶和发酵罐实验中发现 ,CaCO3的添加对发酵液中α 酮戊二酸 (α KG)的积累有重要影响。在维生素浓度不变且供氧充分的前提下 ,延迟CaCO3添加时间可明显抑制α KG的产生 ,并提高丙酮酸与α KG的碳摩尔比 (CPYR Cα KG) ;而增加培养基中的CaCO3浓度会导致α KG积累的增加。用不同物质调节发酵液中pH的实验证实 :在丙酮酸发酵过程中 ,Ca2 + 对α KG的积累起主要作用 ,CO32 - 起辅助作用 ,两者对α KG的积累具有协同效应。维持培养基中CaCO3浓度不变 ,改变培养基中硫胺素的浓度 ,对α KG的积累 ,特别是对CPYR Cα KG值没有影响 ;而增加培养基中生物素的浓度 ,则导致α KG的浓度不断上升且CPYR Cα KG 值不断下降。当有Ca2 + 存在时 ,胞内丙酮酸羧化酶的活性最高可提高 4 0 % ,而丙酮酸脱氢酶系的活性没有明显变化。结果表明 ,丙酮酸发酵过程中α KG的形成是由于CaCO3促进了丙酮酸羧化反应 ,其中Ca2 + 可显著提高丙酮酸羧化酶的活性 ,而CO32 - 则有可能作为丙酮酸羧化反应的底物。

A large amount of α-ketoglutarate (α-KG) (6 8 g/L) was accumulated in flask culture when CaCO 3 was used as a buffering agent in the production of pyruvate by multi-vitamin auxotrophic yeast Torulopsis glabrata CCTCC M202019 In a 5 L jar-fermentor, less α-KG (1 3 g/L) was produced when NaOH was used to adjust the pH, while more α-KG (11 5 g/L) detected when CaCO 3 was used as the buffer. In the latter case, the molar carbon ratio of pyruvate to α-KG (C PYR/C α-KG) was similar to that obtained in flask culture, suggesting the accumulation of α-ketoglutarate was related to the addition of CaCO 3 Furthermore, it was found that: (1) delaying the addition time of CaCO 3 decreased the α-ketoglutarate formation but increased C PYR/C α-KG; and (2) under vitamin limitation conditions increasing the concentration of CaCO 3 led to an increased α-KG accumulation at the expenses of pyruvate. To study which ions in CaCO 3 was responsible for the accumulation of α-KG, the effects of different pH buffers on the α-KG accumulation were studied. The level of α-KG was found to correlate with the levels of both Ca 2+ and CO 3 2-, with Ca 2+ played a dominant role and CO 3 2- played a minor role. To find out which pathway was responsible for the accumulation of α-KG, the effects of biotin and thiamine on α-KG accumulation was investigated. The increase in biotin concentration led to an increase in α-KG accumulation and a decrease in C PYR/C α-KG, while the levels of α-KG and C PYR/C α-KG were not affected by thiamine concentration. The activity of pyruvate carboxylase was increased as much as 40% when the medium was supplemented with Ca 2+. On the other hand, the activity of the pyruvate dehydrogenase complex was unaffected by the presence of Ca 2+. To conclude, the higher level of α-KG was caused by higher activity of pyruvate carboxylase stimulated by Ca 2+, with CO 3 2- served as the substrate of the reaction.