中国食品学报

可溶性乳酸菌蛋白酶的克隆表达及酪蛋白源生物活性肽的制备

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武晓英 ¹ , 李博 ² , 燕平梅 ¹ , 刘地 ¹

中国食品学报 | 基础研究 2026,26(1): 24-36

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中国食品学报 | 基础研究 2026, 26(1): 24-36

可溶性乳酸菌蛋白酶的克隆表达及酪蛋白源生物活性肽的制备

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武晓英¹, 李博², 燕平梅¹, 刘地¹

作者信息

^1.太原师范学院生物科学与技术学院山西晋中 030619

^2.太原理工大学太原 030024

Cloning and Expression of Soluble Lactic Acid Bacterial Proteinase and Production of Casein-Derived Bioactive Peptides

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出版时间: 2026-01-31 doi: 10.16429/j.1009-7848.2026.01.003

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目的:通过可溶性乳酸菌蛋白酶高效制备牛乳蛋白生物活性肽。方法:筛选具备高蛋白水解活性的优势乳酸菌，通过PCR技术扩增其蛋白酶基因的Pre-H结构域，去除编码细胞壁连接区域的序列，使其转化为可溶性蛋白。构建食品级乳酸乳球菌NZ9000表达载体，诱导目的蛋白酶的分泌表达，并分析其酶学特性及酪蛋白水解产物的生物活性。结果:筛选获得的优势乳酸菌菌株被鉴定为德氏乳杆菌乳酸亚种，其蛋白酶基因经改造后得到prtB3基因，成功构建重组表达载体pNZ8048-prtB3，实现了蛋白酶PrtB3在乳酸乳球菌NZ9000中的高效分泌表达。与野生型蛋白酶相比，重组蛋白酶PrtB3的底物特异性显著拓宽，可有效降解α-、β-和κ-酪蛋白；PrtB3水解物对血管紧张素转化酶(ACE)抑制率最高可达78%，为野生型水解产物的2倍，半抑制浓度(IC50)为1.69 mg/mL，且ACE抑制活性在8周内保持稳定；同时，水解产物对金黄色葡萄球菌ATCC 25923和大肠杆菌K14 IBC 4004的生长有明显的抑制作用。结论:成功克隆、表达了可溶性的乳酸菌蛋白酶PrtB3，该酶可高效降解酪蛋白并显著提升水解产物的生物活性与稳定性，在降解酪蛋白制备生物活性肽方面具有潜力，为生物活性肽的高效制备提供了新的技术路径。

关键词

生物活性肽 / 酪蛋白 / 乳酸菌 / 可溶性蛋白酶 / 血管紧张素转化酶抑制肽 / 抑菌肽

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Objectives: The efficient release of bioactive peptides (BPs) from bovine milk proteins is of great significance. This study aimed to explore the potential of using the soluble proteinase of lactic acid bacteria (LAB) for production of BPs. Methods: A LAB strain exhibiting high proteolytic activity was screened and identified. The gene encoding the proteinase in the selected LAB was amplified via PCR and then modified by removing the sequence encoding the cell-wall-associated region， enabling it to be a soluble protein. Subsequently， the modified gene prtB3 was cloned and expressed in the food-grade expression strain Lactococcus lactis NZ9000. After purification， a comprehensive evaluation was conducted on the enzymatic properties of the recombinant proteinase PrtB3 and the biological activities of the hydrolysates resulting from casein degradation. Results: The screened LAB strain was identified as Lactobacillus delbrueckii subsp. lactis. A soluble， secreted， C-terminally Myc and His-tagged derivative PrtB3 was constructed and expressed in Lactococcus lactis via the NICE■ Expression System. Compared with the wild-type proteinase， PrtB3 demonstrated a significant expansion in substrate specificity. It was proteolytically active in the hydrolysis of α-， β-， and κ-casein. Regarding biological activity， the casein hydrolysate produced by PrtB3 showed remarkable angiotensin-converting enzyme (ACE) inhibitory activity. The maximum ACE inhibition rate reached 78%， which was twice that of the hydrolysate produced by the wild-type proteinase. The half-maximal inhibitory concentration (IC50) was 1.69 mg/mL， and the ACE inhibitory activity remained stable over an 8-week period. Furthermore， the enzymatically produced hydrolysate showed antimicrobial activity against Staphylococcus aureus and Escherichia coli. Conclusions: In this study， the soluble proteinase PrtB3 was successfully cloned and expressed. It effectively enhanced the degradation of casein and significantly improved the biological activity and stability of the casein hydrolysate. PrtB3 demonstrated great potential in producing BPs by degrading casein， providing a novel approach for the preparation of bioactive peptides.

Key words

bioactive peptides / casein / lactic acid bacteria / soluble proteinase / ACE inhibitory peptides / antibacterial peptides

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武晓英, 李博, 燕平梅, 刘地. 可溶性乳酸菌蛋白酶的克隆表达及酪蛋白源生物活性肽的制备. 中国食品学报, 2026 , 26 (1) : 24 -36 . DOI: 10.16429/j.1009-7848.2026.01.003

. Cloning and Expression of Soluble Lactic Acid Bacterial Proteinase and Production of Casein-Derived Bioactive Peptides[J]. Journal of Chinese Institute of Food Science and Technology, 2026 , 26 (1) : 24 -36 . DOI: 10.16429/j.1009-7848.2026.01.003

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山西省基础研究计划

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2026年第26卷第1期

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doi: 10.16429/j.1009-7848.2026.01.003

接收时间：2025-02-10
首发时间：2026-05-22
出版时间：2026-01-31

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收稿日期：2025-02-10

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山西省基础研究计划

作者信息

^1.太原师范学院生物科学与技术学院山西晋中 030619

^2.太原理工大学太原 030024

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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