翡翠贻贝副肌球蛋白的特性及在模拟胃肠液中的消化

为探究贝类副肌球蛋白(paramyosin,PM)的热稳定性、pH值稳定性及其在模拟胃肠液中的消化特性,以翡翠贻贝(Perna viridis)为对象,利用硫酸铵盐析和羟基磷灰石柱层析等方法,从肌肉中纯化PM,采用肽质量指纹图谱法对其进行鉴定,利用圆二色谱测定其二级结构及热变性温度。结果显示,翡翠贻贝PM分子质量为99.5 kDa;肽质量指纹图谱分析获得26个肽段,共330个氨基酸残基,与地中海贻贝(Mytilusgalloprovincialis)PM的序列一致性达到100%,表明纯化的蛋白为PM。圆二色谱结果显示,PM呈现典型的α-螺旋结构,其热变性温度为(56.3±0.2)℃。在30～100℃热处理30 min,PM未出现沉淀聚集现象,在pH 6～11范围内也较稳定,但当pH≤5时稳定性差,出现沉淀聚集现象。与胃蛋白酶、胰蛋白酶及胰凝乳蛋白酶对PM的单独消化作用相比,3种酶连续作用可使PM有效降解,但仍有分子质量30 kDa的片段未被完全消化。本研究表明,翡翠贻贝PM具有较好的耐热性及耐消化性,为贻贝深加工及PM的后续研究提供一定理论参考。

Characterization and Simulated Gastrointestinal Digestion of Paramyosin from Perna viridis

In order to investigate the thermal stability, pH stability and digestion characteristics of paramyosin (PM) in simulated gastrointestinal fluids, we purified PM to homogeneity from the muscle of Perna viridis by consecutive ammonium sulfate fractionation and hydroxyapatite chromatography, and we further identified it by peptide mass fingerprinting (PMF). Circular dichroism (CD) spectroscopy was employed to measure its secondary structure and thermal denaturation temperature. SDS-PAGE was carried out to explore its thermal stability, pH stability and digestion characteristics in simulated gastrointestinal fluids. PM showed a single band corresponding to 99.5 kDa on SDS-PAGE. Using peptide mass fingerprinting, a total of 26 peptide fragments, including 330 amino acid residues, were obtained from purified PM, which revealed 100% identity to PM from Mytilus galloprovincialis, indicating that the purified protein is PM. CD spectral analysis demonstrated that PM had a typical α-helix structure with thermal denaturation temperature (Td) of (56.3 ± 0.2) ℃. No obvious precipitate was observed when PM was heated in the temperature range from 30 to 100 ℃ for 30 min. PM was stable between pH 6.0–11.0. However, it was unstable below pH 5.0 and aggregated. PM could only be partially hydrolyzed by pepsin, trypsin or chymotrypsin individually, while it was effectively degraded by continuous hydrolysis with the three proteinases. However, protein bands with molecular mass of approximately 30 kDa remained undigested. In conclusion, PM from Perna viridis is thermally stable and resistant to gastrointestinal digestion. This work provides a valuable theoretical basis for mussel processing and further study on PM.