Sakamoto S, Terada I, Iijima M, Matsuzawa H, Ohta T
Life Science Research Laboratory, Japan Tobacco Inc., Yokohama.
Appl Microbiol Biotechnol. 1994 Dec;42(4):569-74. doi: 10.1007/BF00173922.
Escherichia coli TG1, transformed with an expression plasmid pAQN carrying the aqualysin I (AQI) gene derived from Thermus aquaticus YT-1 under the control of the tac promoter, was cultivated under various conditions in order to find fermentation conditions for the efficient production of the thermophilic protease, AQI. The amount of AQI produced was closely related to the growth phase at the time of isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, and the highest production was obtained when it was added during the exponential growth phase. The addition of yeast extract had a greater effect on AQI production than did Polypeptone or casamino acids, and AQI productivity increased from 1.1 x 10(3) kU/g to 2.7 x 10(3) kU/g cells when 2 g/l yeast extract was supplied. Furthermore, the specific growth rate improved from 0.35 h-1 to 0.89 h-1 when 5 g/l yeast extract was supplied. The culture temperature also affected AQI gene expression. When the temperature was shifted from 37 degrees C to 34 degrees C at the time of IPTG induction, 19 kU/ml enzymatically active AQI was obtained, corresponding to a 28% increase over the amount produced in a batch culture without a shift. This is about a 44-fold higher yield than was obtained from the original strain, T. aquaticus YT-1.
用携带源自嗜热水生栖热菌YT-1的嗜热栖热菌蛋白酶I（AQI）基因的表达质粒pAQN转化的大肠杆菌TG1，在各种条件下培养，以寻找高效生产嗜热蛋白酶AQI的发酵条件。AQI的产生量与异丙基-β-D-硫代半乳糖苷（IPTG）诱导时的生长阶段密切相关，在指数生长期添加IPTG时产量最高。添加酵母提取物对AQI产量的影响比添加蛋白胨或酪蛋白氨基酸的影响更大，当提供2 g/l酵母提取物时，AQI生产率从1.1×10³ kU/g细胞提高到2.7×10³ kU/g细胞。此外，当提供5 g/l酵母提取物时，比生长速率从0.35 h⁻¹提高到0.89 h⁻¹。培养温度也影响AQI基因表达。在IPTG诱导时将温度从37℃转变为34℃，可获得19 kU/ml的具有酶活性的AQI，比未转变的分批培养物中产生的量增加了28%。这比从原始菌株嗜热水生栖热菌YT-1获得的产量高约44倍。
