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Highly Active Modified Variants of Recombinant Phospholipase А2 from Streptomyces violaceoruber for Effective Expression in Yeasts
Applied Biochemistry and Microbiology ( IF 0.8 ) Pub Date : 2020-12-01 , DOI: 10.1134/s0003683820070029
S. E. Cheperegin , E. P. Sannikova , A. V. Malysheva , F. A. Klebanov , D. G. Kozlov

Abstract

The ability of yeast to produce highly active variants of the recombinant phospholipase PLA2 is confirmed. Highly active variants were based on an original enzyme from the Streptomyces violaceoruber А-2688 strain. Various approaches to reduce enzyme toxicity and increase its expression, including point mutations, the construction of artificial N- and/or C-end proregions, and the inactivation of glycosylation sites, were tested. The main result was the obtainment of modified PLA2 enzymes with the same secretion level as their weakly active prototype but a specific activity that was at least ten times higher. As the main feature, the selected mutants were characterized by a lower affinity for Ca2+, which probably accounts for their low toxicity at the stage of biosynthesis and the possibility of their activation under special conditions, e.g. by the addition of calcium during incubation with egg yolk. It appears that the obtained variants can significantly reduce the cost of the use of PLA2 enzyme preparations in industries that allow the use of high calcium concentrations.



中文翻译:

酵母链霉菌重组磷脂酶А2的高活性修饰变体,可在酵母中有效表达

摘要

证实了酵母产生重组磷脂酶PLA2的高活性变体的能力。高活性变体基于紫链霉菌А-2688菌株的原始酶。测试了多种降低酶毒性并增加其表达的方法,包括点突变,人工N和/或C端前区的构建以及糖基化位点的失活。主要结果是获得了修饰的PLA2酶,其分泌水平与其弱活性原型相同,但比活性至少高出十倍。作为主要特征,所选突变体的特征在于对Ca 2+的亲和力较低,这可能是由于它们在生物合成阶段的低毒性以及在特殊条件下被激活的可能性,例如在与蛋黄孵育期间添加钙。看来,所获得的变体可以显着降低在允许使用高钙浓度的工业中使用PLA2酶制剂的成本。

更新日期:2020-12-01
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