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脂肪酶Sv-lip5的异源表达及其在虾青素酯水解中的应用

闫娇 高坤鹏 余可欣 孙建安 毛相朝

闫娇, 高坤鹏, 余可欣, 孙建安, 毛相朝. 脂肪酶Sv-lip5的异源表达及其在虾青素酯水解中的应用[J]. 南方水产科学. doi: 10.12131/20210293
引用本文: 闫娇, 高坤鹏, 余可欣, 孙建安, 毛相朝. 脂肪酶Sv-lip5的异源表达及其在虾青素酯水解中的应用[J]. 南方水产科学. doi: 10.12131/20210293
YAN Jiao, GAO Kunpeng, YU Kexin, SUN Jianan, MAO Xiangzhao. Heterologous expression of lipase Sv-lip5 and its application in hydrolysis of astaxanthin ester[J]. South China Fisheries Science. doi: 10.12131/20210293
Citation: YAN Jiao, GAO Kunpeng, YU Kexin, SUN Jianan, MAO Xiangzhao. Heterologous expression of lipase Sv-lip5 and its application in hydrolysis of astaxanthin ester[J]. South China Fisheries Science. doi: 10.12131/20210293

脂肪酶Sv-lip5的异源表达及其在虾青素酯水解中的应用

doi: 10.12131/20210293
基金项目: 国家重点研发计划“蓝色粮仓科技创新”重点专项 (编号?):水产品加工专用酶的创制关键技术 (2019YFD0901901)
详细信息
    作者简介:

    闫娇:闫 娇 (1998—),女,硕士研究生,研究方向为水产脂质加工专用酶的发掘与应用。E-mail: yanjiao19981031@163.com

    通讯作者:

    孙建安 (1984—),男,副教授,博士,从事食品生物技术研究。E-mail: Sunjianan@ouc.edu.cn

  • 中图分类号: Q 814.9

Heterologous expression of lipase Sv-lip5 and its application in hydrolysis of astaxanthin ester

  • 摘要: 虾青素具有多种生理活性,但在自然界中通常以不同脂肪酸结合的虾青素酯形式存在,将虾青素酯水解为游离虾青素是提高其吸收利用度和制备特定构型虾青素酯的重要研究方向。以杆菌状链霉菌 (Streptomyces violascens ATCC 27968) 来源的脂肪酶Sv-lip5为研究对象,将其在枯草芽孢杆菌 (Bacillus subtilis) 中进行异源表达,分析其酶学性质并探究其在虾青素酯水解中的应用。结果显示,Sv-lip5的最适温度为45 ℃,最适pH为10.0 (Gly-NaOH缓冲液),在35~55 ℃条件下孵育21 h后仍有高于55.7%的酶活。Sv-lip5为耐碱性酶,在碱性条件下孵育48 h后相对酶活仍可保持在70%以上。Sv-lip5可有效水解雨生红球藻 (Haematococcus pluvialis) 油中的虾青素酯,以对硝基苯酚棕榈酸酯作为底物测定该酶的比活力为12.46 U·g−1,水解虾青素酯的最佳反应条件为pH 8.0,乙醇与缓冲液比为1∶12,最佳加酶量和时间分别为900 mg和12 h。在5.5 mL反应体系中添加900 mg的酶粉后,1 h有明显水解效果,12 h时可达最高水解率98.27%,200 μg虾青素酯中游离虾青素产量为147.48 μg,实现了游离虾青素的高效生物水解制备。
  • 图  1  Sv-lip5进化树分析

    Figure  1.  Phylogenetic analysis of Sv-lip5

    图  2  Sv-lip5的核酸电泳结果和SDS-PAGE蛋白纯化结果

    (a) M. Maker;1~10. 转化子PCR原液;(b) M. Maker;1. 纯化后的Sv-lip5蛋白。

    Figure  2.  Nucleic acid electrophoresis results and SDS-PAGE protein purification results of Sv-lip5

    (a) M. Maker; 1−10. PCR stock of inverters; (b) M. Maker; 1. Purified Sv-lip5 protein.

    图  3  Sv-lip5的酶学性质分析

    Figure  3.  Analysis of enzymatic properties of Sv-lip5

    图  4  Sv-lip5在虾青素酯水解反应中的条件优化

    Figure  4.  Optimization of hydrolysis reaction conditions for Sv-lip5

    表  1  引物设计

    Table  1.   Sequences of primers

    引物名称
    Primer name
    碱基序列 (5'→3')
    Primer sequence (5'→3')
    Sv-lip5-Rcagtggtggtggtggtggtgccaggccagttgggc
    Sv-lip5-Ftaacacatgcctcagctgcagtgcacggccgggca
    Bone-Rtgcagctgaggcatgtgttac
    Bone-Fcaccaccaccaccaccactgatgaaagcttggcgtaatc
    Tong-Rcacacaggaaacagctatgacc
    Tong-Fgagttgctagtaacatctgaccg
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-10-10
  • 修回日期:  2021-12-08
  • 录用日期:  2022-01-07
  • 网络出版日期:  2022-01-13

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