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Secretion of Pseudomonas Aeruginosa Lipoxygenase by Pichia Pastoris upon Glycerol Feed.
Biotechnology Journal ( IF 4.7 ) Pub Date : 2020-08-04 , DOI: 10.1002/biot.202000089
Chiam Hashem 1, 2 , Holly Stolterfoht 1 , Claudia Rinnofner 1, 3 , Stefan Steinberger 1 , Margit Winkler 1, 2 , Harald Pichler 1, 2
Affiliation  

Pseudomonas aeruginosa lipoxygenase (PaLOX) catalyzes the peroxidation of unsaturated fatty acids. Not only linoleic acid, but also linolenic acid and oleic acid are oxidized. The natural host secretes PaLOX into the periplasmic space. Herein, the aim is to secrete PaLOX to the culture supernatant of Pichia pastoris. Since protein background in the culture supernatant is typically rather low, this strategy allows for almost pure production of PaLOX applicable for the valorization of renewable fatty acids, for example for the production of green leaf volatiles. Using the CAT1 promoter system and the well‐established α‐factor signal sequence for secretion, methanol‐ and glycerol‐induced secretion are compared and the latter shows four times more LOX activity in the culture supernatant under methanol‐free conditions. In addition, secreted PaLOX is purified and the specific activity with enzyme in culture supernatant is compared. Notably, the predominant specific activity is achieved for enzyme in culture supernatant ‐ 11.6 U mg−1 ‐ reaching five times higher specific activity than purified PaLOX.

中文翻译:

甘油饲料中巴斯德毕赤酵母分泌铜绿假单胞菌脂氧合酶。

铜绿假单胞菌脂氧合酶(Pa LOX)催化不饱和脂肪酸的过氧化。不仅亚油酸,而且亚麻酸和油酸都被氧化。天然宿主将Pa LOX分泌到周质空间。在此,目的是将Pa LOX分泌至巴斯德毕赤酵母的培养上清液。由于培养上清液中的蛋白质背景通常很低,因此该策略可实现Pa LOX的几乎纯净的生产,可用于可再生脂肪酸的增值,例如绿叶挥发物的生产。使用CAT1比较了启动子系统和建立良好的分泌,甲醇和甘油诱导的分泌的α因子信号序列,后者在无甲醇条件下的培养上清液中显示出四倍的LOX活性。另外,纯化分泌的Pa LOX,并比较培养上清液中酶的比活性。值得注意的是,在培养上清液中酶的主要比活性达到11.6 U mg -1,比纯化的Pa LOX达到五倍高。
更新日期:2020-08-04
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