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首页> 外文期刊>Applied Microbiology and Biotechnology >Screening for and characterization of phospholipase A(1) hypersecretory mutants of Tetrahymena thermophila
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Screening for and characterization of phospholipase A(1) hypersecretory mutants of Tetrahymena thermophila

机译：嗜热四膜虫磷脂酶A（1）高分泌突变体的筛选和表征

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摘要

We have described a procedure for the isolation of mutants of Tetrahymena thermophila with hyperscretion of phospholipase A(1) (PLA(1)). Using random chemical mutagenesis,. uniparental cytogamy, genetic crossing and a new, fast and effective screening procedure, four PLA(1)-hypersecretory mutants were isolated. The screening procedure is based on the formation of a halo appearing around cylindrical holes in a lecithin-containing agar plate filled with cell-free supernatants. About 3,940 clones were tested with this procedure in primary screening for hypersecretory features, of which 60 putative hypersecretory mutants were isolated, subcloned and tested in a secondary screening. Of these, four selected mutants showed 1.8-2.2 more PLA(1) activity in the cell-free supernatants compared to the wild-type strain CU 438.1. Hypersecretion was only observable for PLA(1); no increased activity for two other lysosomal enzymes could be detected. These hypersecretory mutants of T. thermophila can be very useful for increasing the yield of PLA(1) in fermentation processes. This is particularly relevant because, in contrast to other phospholipases, PLA(1) is not available on the commercial market for fine chemicals and little is known about the role of PLA(1) in cell signaling and metabolism. [References: 26]
机译：我们已经描述了一种程序，用于分离嗜热四膜虫的突变体，并带有磷脂酶A（1）（PLA（1））的超沉淀。采用随机化学诱变。单亲细胞分裂，遗传杂交和一个新的，快速和有效的筛选程序，四个PLA（1）-分泌过多的突变体被分离。筛选程序基于在充满无细胞上清液的含卵磷脂的琼脂平板中圆柱孔周围出现光环的形成。用此程序测试了约3,940个克隆，以初步筛选出分泌过多的特征，其中60个假定的分泌过多的突变体被分离，亚克隆并在二次筛选中进行了测试。其中，与野生型菌株CU 438.1相比，四个选定的突变体在无细胞上清液中的PLA（1）活性更高1.8-2.2。分泌过多仅对PLA（1）可见;没有检测到其他两种溶酶体酶的活性增加。这些嗜热嗜热菌的高分泌突变体对于提高发酵过程中PLA（1）的产量非常有用。这一点特别相关，因为与其他磷脂酶相比，PLA（1）在精细化学品的商业市场上不可用，并且对PLA（1）在细胞信号传导和代谢中的作用了解甚少。 [参考：26]

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