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

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

涂兰兰^1a,,
许璟珅^1a,
陈润莎²,
吴金鸿^1a, ,,
李向红²,
张勇^1b

1.
农业与生物学院上海交通大学
2.
生命科学技术学院，上海 200241 上海交通大学
2.
长沙理工大学食品与生物工程学院，湖南长沙 410114

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
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- 文章访问数: 94
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- 被引次数: 0
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

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

1.
Shanghai Jiao Tong University School of Agriculture and Biology
2.
Shanghai Jiao Tong University School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200241, China
2.
School of Food Science and Bioengineering, Changsha University of Science and Technology, Changsha 410114, China

摘要

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。
- Patatin /
- 脂肪酶 /
- 固定化 /
- 酶催化特性
Abstract: Patatin, an ester hydrolase with lipase catalytic activity, can be applied to the hydrolysis of fat. To solve the problems of poor stability of free patatin lipase and difficult reuse in industrial production, we immobilized patatin lipase by coupled nano (ConA) magnetic beads to improve its catalytic characteristics of its industrial application. The results show that the average adsorption rate of ConA magnetic beads was 24.50%, and PAA-Fe₃O₄ was determined as the most suitable immobilization material. The optimal immobilization conditions are as follows: immobilization time of 47.2 min, immobilization temperature of 25.3 ℃, magnetic bead addition of 3.0 mg·mL⁻¹. Immobilized patain had the highest hydrolytic substrate activity at 40.0 ℃ and pH 7.0. Compared with free patatin lipase, its temperature tolerance increased by about 123% and the pH tolerance increased by about 47%. After five consecutive reactions, 56.60% patatin activity was still retained, indicating that the immobilized nano magnetic beads improved the enzymatic catalytic characteristics of patatin, which provides a new enzyme system with stronger catalytic performance for its application in the processing of aquatic products such as oil fish.
- Patatin /
- Lipase /
- Immobilization /
- Enzyme catalytic properties

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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图 13 固定化Patatin酯酶重复利用率

Figure 13. Reuse ratio of immobilized patatin lipase

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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 buffer	2.5
PNP-acetate 缓冲液 PNP-acetate buffer	250
Patatin 酯酶溶液 Patatin lipase solution	100
氯化钙溶液 CaCl₂ solution	100

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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	A	B	C
水平 Level	t/min	温度 Temperature/℃	磁珠添加量 Magnetic bead addition/(mg·mL⁻¹)
−1	40	20	2.5
0	50	25	3.0
1	60	30	3.5

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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/%
1	33.17	24.07±0.75
2	33.92	24.61±0.22
3	34.21	24.82±0.66
平均值 Average	33.77	24.50

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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.	A	B	C	酶比活力 Enzyme specific vitality/ (10⁴ U·g⁻¹)
编号 No.	时间 Time	温度 Temperature	磁珠添加量 Magnetic bead addition	酶比活力 Enzyme specific vitality/ (10⁴ U·g⁻¹)
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

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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	F	P	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).

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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

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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

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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

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