Salinivibrio sp.YH4胞外丝氨酸蛋白酶EYHS耐盐性及生物信息学分析

Salt-tolerance and Bioinformatics Analysis on the Serine Protease EYHS Secreted by Salinivibrio sp.YH4

为探讨菌株Salinivibrio sp.YH4分泌的丝氨酸蛋白酶EYHS的耐盐性及结构特征,采用明胶底物酶谱法分析EYHS的耐盐性,并应用生物信息学手段对EYHS及6种耐盐的S8家族丝氨酸蛋白酶结构特征进行分析。结果表明EYHS在4 mol/L的NaCl溶液中仍具有活性,属于耐盐蛋白酶。EYHS及6种S8家族丝氨酸蛋白酶分子表面的loop区等无规则卷曲所占比例较高,α-螺旋与β-片层则主要位于酶分子内部。EYHS分子表面酸性氨基酸含量较高,且具有弱疏水内核。多序列比对发现蛋白酶的催化三联体两侧存在高度保守的基序和保守的极性氨基酸及芳香族氨基酸,并存在多个保守的Gly与Ala。同源模建和表面电荷分布显示,α螺旋和β片层围成了蛋白酶的催化腔,EYHS活性中心包含由Asp32、His65与Ser215组成的催化三联体,且催化位点区域表面静电势为负。上述结构特征可能有助于耐盐丝氨酸蛋白酶EYHS在高盐环境下维持其稳定性和适度柔性,并有助于其催化功能的发挥,为深入研究耐盐丝氨酸蛋白酶的高盐环境适应性提供了一定的理论依据。

To investigate the structural properties and salt tolerance of serine protease EYHS secreted by Salinivibrio sp.YH4,the gelatin immersing zymography was employed to analyze salt tolerance of EYHS.Bioinformatics tools were used to analyze structural properties of EYHS and six S8 family salt-tolerant serine proteases.The results showed that EYHS had high proteolytic activity at 4 mol/L NaCl.The proportion of irregular curls was high on the surface of EYHS and six kinds of S8 serine proteases,whileα-helix andβ-sheet were mainly located inside the enzyme.Furthermore,the content of acidic amino acids was high on the surface of serine protease and it has a weak hydrophobic core.Multiple sequence alignment showed that there were highly conserved motifs,polar amino acids and aromatic amino acids on both sides of the catalytic triad,in addition to high contents of Gly and Ala.Homologous modeling and surface charge distribution showed thatα-helix andβ-sheet formed the catalytic cavity of protease,the EYHS active center contained a catalytic triad composed of Asp32,His65 and Ser215,with the surface electrostatic potential being negative in the catalytic center area.The above structural characteristics of salt-tolerant serine protease may help maintain its stability and moderate flexibility in high salt environment,which was helpful for its catalytic function.This study provided a theoretical basis for studying the adaptability of salt tolerant serine protease to high salt environment.