褶牡蛎氨肽酶B的分离纯化和性质研究

Purification and Characterization of Aminopeptidase B from Oysters (Alectryonella plicatula)

氨肽酶是一类能特异水解蛋白质N端氨基酸残基的外切酶,在食品行业有着广阔的应用前景。以褶牡蛎(Alectryonella plicatula)为原料,通过硫酸铵盐析,DEAE-sepharose,phenyl-sepharose及羟基磷灰石柱层析,分离纯化得到了一种高效水解碱性氨基酸的氨肽酶,其活性能被氨肽酶特异性抑制剂bestatin有效抑制。双向电泳结果显示该酶的分子质量约为100 ku,pI约为5.8。通过肽质量指纹图谱对其进行鉴定得到12个肽段,共含138个氨基酸残基,这些氨基酸序列与太平洋牡蛎中嘌呤霉素敏感性氨肽酶一致,表明纯化的酶为氨肽酶B。褶牡蛎氨肽酶B的二级结构以无规卷曲与反向平行为主,其中无规卷曲占42.6%,反向平行占32.0%。动力学研究获得其K_m和k_(cat)值分别为1.5μmol/L和117.5 s^(-1),k_(cat)/K_m为78.3 L/(μmol·s)^(-1)。在35℃,pH值7.0的条件下,该酶具有最大催化活性,能高效水解氨肽酶底物Lys-MCA和Arg-MCA,释放出游离态的Lys和Arg,推测与牡蛎呈味相关。

Aminopeptidase is a kind of exopeptidases that specifically cleaves amino acids from the N- ter- minal of proteins, and thus has wild applications in food industry. In this study, an aminopeptidase was isolated to homogeneity from oysters （Alectryonella plicatula） by ammonium sulfate fractionation and a series of chromatographic steps including DEAE- sepharose, phenyl-sepharose and hydroxyapatite. The aminopeptidase was strongly suppressed by bestatin, a specific aminopeptidase inhibitor. The 2D-PAGE results showed that molecular weight and pI value was approximately 100 ku and 5. 8. By MALDI- TOF/ TOF mass spectrometry analysis, 12 peptide fragments with 138 amino acid residues in total were obtained and these peptide fragments were identical to a puromycin- sensitive aminopeptidase from Cras-sostrea gigas, which confirmed that the purified enzyme was an aminopeptidase B. Circular dichroism spectroscopy analysis indicated that the secondary structure of aminopeptidase B （ APB） was mainly ran-dom coil （42. 6%） and reverse parallel （32. 0%）. The Km and kCat was 1.5 μmol/L and 117. 5 s^ -1 and the kcat /Km was 78. 3 L/（μmol·s）^ -1. The optimum temperature and pH of APB was 35 ℃ and 7. 0. Under these conditions, APB could hydrolyze Lys-MCA and Arg-MCA to release Lys and Arg, which had the oys-ter＇ s flavor.