关键词: 磷脂酶A1；磷脂酶A1辅助蛋白；锚蛋白重复序列；基因剪接；酶学性质；分子截短

Abstract: This study aimed to explore the key regulatory region of the gene encoding phospholipase A1 accessory protein (PlaS) on the activity of phospholipase A1 (PlaA1). According to the structural characteristics of PlaS, truncated mutants were designed, the gene encoding plaS in the PlaA1 and PlaS co-expressed gene (plaB) was spliced by PCR, and the PlaA1 activity and properties of the truncated strains were analyzed. The results showed that PlaS belonged to the Ankyrin family, which was mainly composed of three parts: N-terminal domain, ANK domain, and C-terminal domain. The ANK domain contained 4 typical anchoring protein repeats (ANK repeat). Out of the five truncated strains, AN-3 and AN-4 were found to have relatively high extracellular PlaA1 activity. In contrast to the untruncated strain BP28, the specific activities of AN-3 and AN-4 were increased by 73% and 78%, respectively, and the catalytic efficiencies (Kcat/Km) by 216% and 211%, respectively, while the optimum temperature (45 ℃) and pH (6.0) were unchanged. AN had the lowest catalytic efficiency among all the truncated strains. The results of plaS splicing showed that at least three ANK repeats in the ANK domain of PlaS were required to promote the extracellular activity of PlaA1, and the C-terminal domain of PlaS played an important role in maintaining the structural stability of PlaA1. This study provides a theoretical basis for revealing the regulatory mechanism of PlaS on PlaA1.

Key words: phospholipase A1; phospholipase A1 accessory protein; ankyrin repeat; gene splicing; enzymatic characteristics; truncated mutation