桔黄赛多孢霉外泌弹性蛋白酶的生化特性

Biochemical properties of Scedosporium aurantiacum extracellular elastase-like protease

摘要:

外泌弹性蛋白酶是桔黄赛多孢霉(Scedosporium aurantiacum)主要毒性蛋白酶之一，本文在前期研究的基础上，对这种蛋白酶的序列、结构和酶学特性进行了研究。首先通过柱层析技术将S. aurantiacum培养上清液中的蛋白进行了分离，然后通过酶谱电泳纯化得到了弹性蛋白酶条带。从凝胶中提取了弹性蛋白酶，通过质谱技术对其序列进行了检测，并对其反应特性、活力和反应动力学参数进行了研究。结果显示，S. aurantiacum外泌弹性蛋白酶对弹性蛋白和牛跟腱胶原蛋白(bovine achilles tendon collagen, BATC)具有较好的水解性能，对鱼皮胶原蛋白(fish skin collagen, FSC)的水解效率高于对鱼鳞明胶的水解效率，对酪蛋白的水解性最差。作用于弹性蛋白时，其催化效率小于猪胰腺弹性蛋白酶。Zn²⁺对酶活力有提升作用，而Ca²⁺、Mg²⁺、Na⁺、(2S)-2-[(4S)-2-氨基-1,4,5,6-四羟基4-嘧啶基]-N-[[[(1S)-1-羰基-3-甲基丁基]氨基]羰基]甘氨酰- N1-[(1S)-1-甲基-2-氧乙基]-l-谷氨酸甲酰胺(elastatinal)和苯甲基磺酰氟(phenylmethanesulfonyl fluoride, PMSF)均对酶活有抑制作用。该蛋白酶和青霉(Paecilomyces lilacinus)外泌丝氨酸蛋白酶(PDB Entry: c3f7oB_)的序列最相似，且有多段保守序列的氨基酸个数多于7个，可以作为PCR反应引物设计的模板。酶学特性实验表明，S. aurantiacum外泌弹性蛋白酶对肺组织中的弹性蛋白具有降解作用，其蛋白表达和毒性机制需要进一步的研究。

Abstract:

Extracellular elastase-like protease is one of the key virulence proteases of Scedosporium aurantiacum. To date, little is known about this enzyme in terms of genetic information, structure, properties and virulence mechanism due to the difficulties in purification caused by its low secretion amount, high specific activity, uncompleted genome sequencing and annotation. This work investigated the gene, structure and enzymatic properties of this enzyme. The S. aurantiacum elastase-like protease from the fungal culture supernatant was analyzed through tandem mass spectrometry (MS/MS) approach, illustrating its primary structure. Bioinformatics tools were employed to predict the conserved domain and tertiary structure, the enzymatic properties were also studied. It turned out that S. aurantiacum extracellular elastase-like protease demonstrated well hydrolysis towards elastin and bovine achilles tendon collagen, with V_max of 18.14 μg/s and 17.57 μg/s respectively, better than fish scale gelatin, with the lowest hydrolysis effect on casein. Its activity towards elastin was lower than that of the elastase from porcine pancreas, with values of K_cat/K_m of 3.541 (μg/s) and 4.091 (μg/s), respectively. It was an alkaline protease, with optimal pH 8.2 and temperature 37 ^oC. Zn²⁺ promoted the enzymatic activity while Ca²⁺, Mg²⁺, Na⁺, elastatinal and PMSF inhibited its activity. Its sequence was similar to Paecilomyces lilacinus secreted serine protease (PDB Entry: c3f7oB_) with multiple conserved fractions each containing more than 7 amino acids, thus suitable for design of PCR primer. This study increased our knowledge on S. aurantiacum extracellular elastase-like protease in terms of structure and enzymatic properties, and may facilitate later studies on protein expression and virulence mechanism.