枯草芽孢杆菌表面展示氨肽酶的构建及协同水解大豆蛋白

doi:10.16085/j.issn.1000-6613.2023-0368

化工进展 ›› 2024, Vol. 43 ›› Issue (3): 1466-1473.DOI: 10.16085/j.issn.1000-6613.2023-0368

• 生物与医药化工 • 上一篇

枯草芽孢杆菌表面展示氨肽酶的构建及协同水解大豆蛋白

袁晓黎¹(), 李青云¹^,², 刘幽燕¹^,², 李进¹, 白雪¹, 唐爱星¹^,²()

^1.广西大学化学化工学院，广西南宁 530004
^2.广西生物炼制重点实验室，广西南宁 530007

收稿日期:2023-03-10 修回日期:2023-05-06 出版日期:2024-03-10 发布日期:2024-04-11
通讯作者: 唐爱星
作者简介:袁晓黎（1998—），女，硕士研究生，研究方向为生物化工。E-mail：yxl@163.com。
基金资助:
国家自然科学基金(21966007);广西自然科学基金(2018GXNSFAA281278)

Construction of aminopeptidase on the surface of Bacillus subtilis and its synergic hydrolysis of soybean protein

YUAN Xiaoli¹(), LI Qingyun¹^,², LIU Youyan¹^,², LI Jin¹, BAI Xue¹, TANG Aixing¹^,²()

^1.College of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, Guangxi, China
^2.Guangxi Key Laboratory of Biorefinery, Nanning 530007, Guangxi, China

Received:2023-03-10 Revised:2023-05-06 Online:2024-03-10 Published:2024-04-11
Contact: TANG Aixing

摘要/Abstract

摘要：

氨肽酶是蛋白质深度水解的重要协同酶，但游离酶使用成本高且稳定性差，限制了其工业化应用。本研究利用表面展示技术将来源于铜绿假单胞菌GF31的氨肽酶基因Aps展示到Bacillus subtilis WB800N芽孢的表面，构建了一种易分离且稳定性好的新型全细胞生物催化剂。通过免疫荧光分析，证明氨肽酶在芽孢表面正确表达。在最适反应温度60℃、pH=9.0的条件下，氨肽酶活力最高可达75.61U/g芽孢。将表面展示氨肽酶协同碱性蛋白酶水解大豆蛋白，在60℃、碱性蛋白酶与表面展示氨肽酶加入比例为2∶1、水解pH先为10.0后为9.0的条件下，水解5.0%的大豆蛋白8h，水解度最高可达55.50%，分别是表面展示氨肽酶与碱性蛋白酶单独水解时的3.3倍、1.5倍。同时16种游离氨基酸含量大幅度提升，其中疏水性氨基酸亮氨酸、酪氨酸和苯丙氨酸含量分别增加19.21mg/L、8.59mg/L和16.77mg/L，鲜味氨基酸谷氨酸、天冬氨酸含量增加13.98mg/L和4.11mg/L，说明表面展示氨肽酶在蛋白质的深度水解及对蛋白水解液脱苦、增鲜方面具有显著的作用，具有良好的工业应用前景。

关键词: 氨肽酶, 表面展示, 枯草芽孢杆菌, 大豆蛋白, 协同水解

Abstract:

Aminopeptidase is an important synergistic enzyme for protein deep hydrolysis, however the high cost and poor stability of free enzyme remain the primary limiting factors for their industrial applications. In this study, we constructed a new easily isolated and well tolerated whole-cell biocatalyst which displayed the aminopeptidase derived from Pseudomonas Aeruginosa GF31 on the surface of Bacillus subtilis WB800N spores by surface-display technology. And immunofluorescence analysis indicated that the aminopeptidase was correctly expressed on the surface of spores. The enzyme activity of surface-displayed aminopeptidase reached 75.61U/g at 60℃ and pH=9.0. The aminopeptidase and alkaline protease were used in a synergistic hydrolysis of soybean protein, in which 5.0% of soybean protein was hydrolyzed for 8h at 60℃ with an enzyme ratio of 2∶1 between the alkaline protease and the aminopeptidase, with an initial hydrolysis pH of 10.0 followed by 9.0. The maximum hydrolysis degree was 55.50%, which was 3.3-fold and 1.5-fold times higher than that achieved by surface-displayed aminopeptidase or alkaline protease alone, respectively. Simultaneously, the content of 16 kinds of free amino acids was significantly increased. Specifically, the content of hydrophobic amino acids including leucine, tyrosine, and phenylalanine increased by 19.21mg/L,8.59mg/L and 16.77mg/L, respectively. While the content of umami amino acid including glutamic acid and aspartic acid increased by 13.98mg/L and 4.11mg/L, respectively. These results suggested that aminopeptidase in deep hydrolysis could significantly reduce the bitterness of protein hydrolysate and enhance its umami favor, and had excellent potential for practical use.

Key words: aminopeptidase, surface-display, Bacillus subtilis, soybean protein, synergistic hydrolysis

中图分类号:

Q556

袁晓黎, 李青云, 刘幽燕, 李进, 白雪, 唐爱星. 枯草芽孢杆菌表面展示氨肽酶的构建及协同水解大豆蛋白[J]. 化工进展, 2024, 43(3): 1466-1473.

YUAN Xiaoli, LI Qingyun, LIU Youyan, LI Jin, BAI Xue, TANG Aixing. Construction of aminopeptidase on the surface of Bacillus subtilis and its synergic hydrolysis of soybean protein[J]. Chemical Industry and Engineering Progress, 2024, 43(3): 1466-1473.

图/表 6

图1 芽孢表面展示氨肽酶Aps质粒构建图M—DNA maker；1—重组质粒PHY-P43-cotG-Aps双酶切后的质粒片段

图2 cotG-Aps融合蛋白的免疫荧光分析

图3 表面展示氨肽酶的最适反应pH及pH稳定性

图4 表面展示氨肽酶的最适反应温度、热稳定性及最适反应条件下的半衰期

图5 碱性蛋白酶与表面展示氨肽酶协同水解大豆蛋白的条件优化与水解效果分析

图6 大豆蛋白水解液中游离氨基酸的含量

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枯草芽孢杆菌表面展示氨肽酶的构建及协同水解大豆蛋白

Construction of aminopeptidase on the surface of Bacillus subtilis and its synergic hydrolysis of soybean protein

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