文章摘要
皱纹盘鲍脯氨酰内肽酶的分离纯化及性质研究
Purification and characterization of a prolyl endopeptidase from Haliotis discus hannai
投稿时间:2020-12-27  修订日期:2021-03-24
DOI:
中文关键词: 皱纹盘鲍  脯氨酰内肽酶  纯化  性质分析  性腺发育
英文关键词: Haliotis discus hannai  Prolyl Endopeptidase  Purification  Property analysis  Gonads development
基金项目:国家重点研发计划项目(2018YFD0901004),国家现代农业产业技术体系(CARS-49)
作者单位邮编
陈守峰 集美大学食品与生物工程学院 361021
杨汝晴 集美大学食品与生物工程学院 
陈玉磊 集美大学食品与生物工程学院 
孙乐常 集美大学食品与生物工程学院 
张凌晶 集美大学食品与生物工程学院 
刘光明 集美大学食品与生物工程学院 
曹敏杰 集美大学食品与生物工程学院 
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中文摘要:
      脯氨酰内肽酶(Prolyl Endopeptidase,PEP)广泛分布在动植物和微生物体内,参与细胞生长和代谢调控。PEP在哺乳动物生殖器官的生长发育过程中起着重要作用,但贝类PEP与性腺发育是否有关尚未有相关报道。本研究以皱纹盘鲍为对象,采用荧光定量PCR技术(qRT-PCR)在性腺检测到有最高的基因表达量和较高的酶活性。通过硫酸铵分级沉淀和连续柱层析,从皱纹盘鲍性腺中分离纯化得到分子量约为82 ku,等电点约为5.5的PEP,其最适温度和最适pH分别为25 °C和6.0,在15~25 °C和pH 5~8能够保持较高的酶活性。利用LC/MS/MS分析纯化蛋白,得到182个氨基酸残基,与皱纹盘鲍脯氨酰内肽酶一致。圆二色谱分析显示,温度对PEP二级结构有较大的影响,拟合热变性温度为51.4±0.2 °C。为了进一步探究PEP在鲍鱼性腺发育过程中的作用,对不同发育时期(生长初期、前期、中期和后期)的雌雄鲍鱼,采用免疫印迹(Western Blot)和qRT-PCR分析性腺中PEP的蛋白和基因表达水平。结果表明,PEP在雄性和雌性性腺的各个时期均能被检测到,在雄性性腺的成熟中期和雌性性腺的成熟后期表达量最高。PEP在鲍鱼性腺不同发育时期时表达量的差异表明其可能参与到性腺发育的过程。
英文摘要:
      Prolyl Endopeptidase (PEP) is widely distributed in animals,plants and microorganisms, which is involved in the regulation of cell growth and metabolism. PEP plays an important role in the growth and development of reproductive organs, but whether PEP of shellfish is related to gonadal development has not been reported. In this study, Haliotis discus hannai was selected as the object, fluorescence quantitative PCR technology (qRT-PCR) found that PEP has the highest expression level and higher enzyme activity in the gonads The PEP with a molecular weight of about 82 ku and the isoelectric point of about 5.5 was purified by ammonium sulfate fractional precipitation and continuous column chromatography from the abalone gonad. The optimum temperature and pH were 25 °C and 6.0, respectively, the enzyme activity remained high at 15~25 °C and pH 5~8. The 182 amino acid residues was obtained by LC/MS/MS analysis, which were completely consistent with the abalone prolyl endopeptidase. The circular dichroism analysis showed that temperature had a great influence on the secondary structure of PEP, and the fitting thermal denaturation temperature was 51.4±0.2 °C. In order to further explore the PEP in the role of abalone gonad development, Western Blot and qRT-PCR were used to analyze the protein and gene expression levels of PEP in the gonad of male and female abalone at different developmental stages (early, early, middle and late growth stages). The results showed that PEP could be detected in all stages of male and female gonads, and the expression level was highest in the middle maturity of male gonads and the late maturity of female gonads. The difference in the expression level of PEP at different gonad development stages in abalone suggests that PEP may be involved in gonad development.
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