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适用鱼油加工的Patatin酯酶固定化及其特性研究

涂兰兰 许璟珅 陈润莎 吴金鸿 李向红 张勇

涂兰兰, 许璟珅, 陈润莎, 吴金鸿, 李向红, 张勇. 适用鱼油加工的Patatin酯酶固定化及其特性研究[J]. 南方水产科学, 2022, 18(2): 13-22. doi: 10.12131/20210291
引用本文: 涂兰兰, 许璟珅, 陈润莎, 吴金鸿, 李向红, 张勇. 适用鱼油加工的Patatin酯酶固定化及其特性研究[J]. 南方水产科学, 2022, 18(2): 13-22. doi: 10.12131/20210291
TU Lanlan, XU Jingshen, CHEN Runsha, WU Jinhong, LI Xianghong, ZHANG Yong. Preparation and characteristic research of immobilized patatin lipase suitable for fish oil processing[J]. South China Fisheries Science, 2022, 18(2): 13-22. doi: 10.12131/20210291
Citation: TU Lanlan, XU Jingshen, CHEN Runsha, WU Jinhong, LI Xianghong, ZHANG Yong. Preparation and characteristic research of immobilized patatin lipase suitable for fish oil processing[J]. South China Fisheries Science, 2022, 18(2): 13-22. doi: 10.12131/20210291

适用鱼油加工的Patatin酯酶固定化及其特性研究

doi: 10.12131/20210291
基金项目: 国家重点研发计划项目 (2019YFD0901902);国家自然科学基金项目 (31972017)
详细信息
    作者简介:

    涂兰兰 (1998—),女,硕士研究生,研究方向为功能性食品。E-mail: tulanlan@sjtu.edu.cn

    通讯作者:

    吴金鸿 (1978—),女,副研究员,博士,从事功能食品的开发与利用研究。E-mail: wujinhong@sjtu.edu.cn

  • 中图分类号: TQ 925+.6

Preparation and characteristic research of immobilized patatin lipase suitable for fish oil processing

  • 摘要: Patatin (马铃薯糖蛋白) 是一种酯水解酶,具有脂肪酶催化活性,可应用于脂肪的水解加工。针对游离Patatin酯酶稳定性差且工业生产中酶很难重复利用的缺点,利用ConA (刀豆蛋白A) 耦联的纳米磁珠材料固定化Patatin酯酶,以提高其工业化应用的催化特性。通过研究,发现ConA耦联的纳米磁珠的平均吸附率为24.50%。同时筛选出适宜的Patain酯酶固定化材料为PAA (聚丙烯酸)-Fe3O4,其最优固定化条件为固定化时间47.2 min,固定化温度25.3 ℃,磁珠添加量为3.0 mg·mL−1。Patain酯酶固定化纳米磁珠在40.0 ℃、pH 7.0时水解底物活性最高,且相比游离Patatin酯酶,其温度耐受性提高了123%左右,pH耐受性提高了47%左右,连续反应5次后仍保留56.60%酶活力,说明纳米磁珠固定化后提高了Patatin酯酶的酶催化特性,为其在水产品如鱼油中的加工应用提供了一种催化性能更强的新酶体系。
  • 图  1  亲和色谱分离组分SDS-PAGE分析结果

    Figure  1.  SDS-PAGE of fractiones separated by affinity chromatography

    图  2  BCA法测定蛋白质浓度标准曲线

    Figure  2.  Standard curve of protein concentration determined by BCA method

    图  3  固定化材料对Patatin酯酶固定化效果的影响

    Figure  3.  Effect of immobilized materials on immobilized patatin lipase

    图  4  固定化时间对Patatin酯酶固定化效果的影响

    Figure  4.  Effect of immobilized time on immobilized patatin lipase

    图  5  固定化温度对Patatin酯酶固定化效果的影响

    Figure  5.  Effect of immobilized temperature on immobilized patatin lipase

    图  6  磁珠添加量对Patatin酯酶固定化效果的影响

    Figure  6.  Effect of magnetic bead addition on immobilized patatin lipase

    图  7  各因素交互作用对Patatin酯酶固定化效果的响应曲面

    Figure  7.  Effects of various factors and their interactions on immobilized patatin lipase

    图  8  Patatin酯酶固定化前 (a, b) 与固定化后 (c, d) 表面特征

    Figure  8.  Surface characteristics of magnetic beads before (a, b) and after (c, d) patatin lipase immobilization

    图  9  pH对固定化Patatin酯酶酶解活性的影响

    Figure  9.  Effect of pH on enzymatic hydrolysis activity of immobilized patatin lipase

    图  10  温度对固定化Patatin酯酶酶解活性的影响

    Figure  10.  Effect of temperature on enzymatic hydrolysis activity of immobilized patatin lipase

    图  11  温度对固定化前后Patatin酯酶稳定性的影响

    Figure  11.  Effect of temperature on enzymatic hydrolysis activity before and after patatin lipase immobilization

    图  12  pH对固定化前后Patatin酯酶稳定性的影响

    Figure  12.  Effect of pH on enzymatic hydrolysis activity before and after patatin lipase immobilization

    图  13  固定化Patatin酯酶重复利用率

    Figure  13.  Reuse ratio of immobilized patatin lipase

    表  1  Patatin酯酶活性测定反应体系

    Table  1.   Reaction system for patatin lipase activity

    试剂    
    Reagent    
    体积
    Volume/μL
    Tris-HCl 缓冲液 Tris-HCl buffer2.5
    PNP-acetate 缓冲液 PNP-acetate buffer250
    Patatin 酯酶溶液 Patatin lipase solution100
    氯化钙溶液 CaCl2 solution100
    下载: 导出CSV

    表  2  固定条件Box-Benhnken响应面分析试验因素与水平

    Table  2.   Box-Benhnken response surface factors and levels of immobilization conditions

    水平
    Level
    ABC
    t/min温度
    Temperature/℃
    磁珠添加量
    Magnetic bead
    addition/(mg·mL−1)
    −140202.5
    050253.0
    160303.5
    下载: 导出CSV

    表  3  磁珠与ConA的偶联实验结果

    Table  3.   Results of coupling experiment between magnetic beads and ConA

    平行
    Parallel
    ConA吸附量 (磁珠)
    ConA adsorption capacity
    (magnetic beads)/(μg·mg−1)
    吸附率
    Adsorption rate/%
    133.1724.07±0.75
    233.9224.61±0.22
    334.2124.82±0.66
    平均值 Average33.7724.50
    下载: 导出CSV

    表  4  Box-Benhnken响应面分析试验设计及结果

    Table  4.   Box-Benhnken response surface design and experimental results

    编号
    No.
    ABC酶比活力
    Enzyme specific vitality/
    (104 U·g−1)
    时间
    Time
    温度
    Temperature
    磁珠添加量
    Magnetic bead
    addition
    1 −1 −1 0 2.440 0
    2 −1 0 −1 2.144 0
    3 −1 0 1 2.240 9
    4 −1 1 0 2.609 8
    5 0 −1 −1 2.009 6
    6 0 −1 1 2.047 0
    7 0 0 0 3.101 3
    8 0 0 0 3.150 0
    9 0 0 0 3.195 8
    10 0 0 0 3.200 3
    11 0 0 0 3.225 0
    12 0 1 −1 2.080 7
    13 0 1 1 2.082 0
    14 1 −1 0 1.729 6
    15 1 0 −1 1.549 3
    16 1 0 1 1.658 2
    17 1 1 0 1.874 6
    下载: 导出CSV

    表  5  Box-Benhnken响应面分析试验回归模型方差分析表

    Table  5.   Analysis of variance (ANOVA) and results of Box-Benhnken response surface

    方差来源  
    Variance source  
    平方和
    Sum of squares
    自由度
    Degree of freedom
    均方
    Mean square
    FP差异显著性
    Significance of difference
    模型 5.584 6 9 0.620 5 157.638 0 <0.000 1 ***
    A 0.860 0 1 0.860 0 218.482 3 <0.000 1 ***
    B 0.022 1 1 0.022 1 5.625 7 0.049 5 *
    C 0.007 5 1 0.007 5 1.898 4 0.210 7
    AB 0.000 2 1 0.000 2 0.039 1 0.848 9
    AC 0.000 0 1 0.000 1 0.009 1 0.926 5
    BC 0.000 3 1 0.000 3 0.082 8 0.781 9
    A2 1.435 3 1 1.435 3 364.630 0 <0.000 1 ***
    B2 0.768 2 1 0.768 2 195.146 7 <0.000 1 ***
    C2 2.019 3 1 2.019 3 513.003 2 <0.000 1 ***
    残差 Residual 0.027 6 7 0.003 9
    失拟项 Lack of fit 0.017 9 3 0.006 0 2.482 5 0.200 2
    误差 Error 0.009 6 4 0.002 4
    总离差 Total deviation 5.612 2 16
    $R^2 $=0.995 1; $R_{{\rm{Adj}}}^2$=0.988 8; $R_{{\rm{Pred}}}^2$=0.946 2; Adeq Precision=34.036 5
    注:*. 差异显著 (P<0.05);**. 差异高度显著 (P<0.01);***. 差异极其显著 (P<0.001)。 Note: *. Significant difference (P<0.05); **. Very significant difference (P<0.01); ***. Extremely significant difference (P<0.001).
    下载: 导出CSV

    表  6  磁珠固定Patatin酯酶前后ζ-电势

    Table  6.   ζ- potential of patatin lipase before and after magnetic beads immobilization

    组别   
    Group   
    平均ζ-电势
    Average ζ- potential/mV
    固定前 Before immobilization 86.673±4.233
    固定后 After immobilization 56.300±1.103
    下载: 导出CSV

    表  7  固定化前后Patatin酯酶温度耐受性的回归分析

    Table  7.   Regression analysis of temperature tolerance before and after patatin lipase immobilization

    酶种类   
    Enzyme   
    type   
    回归方程
    Regression
    equation
    相关系数
    Correlation
    coefficient
    IC50/℃
    游离Patatin酯酶
    Free patatin lipase
    y=181−2.65xy 0.974 18.87
    固定化Patatin酯酶
    Immobilized patatin lipase
    y=135−1.19xy 0.996 42.01
    下载: 导出CSV

    表  8  固定化前后Patatin酯酶pH耐受性的回归分析

    Table  8.   Regression analysis pH tolerance before and after patatin lipase immobilization

    酶种类
    Enzyme type
    回归方程
    Regression
    equation
    相关系数
    Correlation
    coefficient
    IC50
    游离Patatin酯酶
    Free patatin lipase
    y=−433+148x−10.5x2 0.886 1.915
    固定化Patatin酯酶
    Immobilized patatin lipase
    y=−225+84.06x−5.54x2 0.904 2.815
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-10-09
  • 修回日期:  2021-11-27
  • 录用日期:  2022-01-08
  • 网络出版日期:  2022-01-13
  • 刊出日期:  2022-04-01

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