豆豉纤溶酶基因的异源表达及其重组菌在淡豆豉中的应用

doi:10.7506/spkx1002-6630-20240628-211

食品科学 ›› 2025, Vol. 46 ›› Issue (4): 68-80.doi: 10.7506/spkx1002-6630-20240628-211

• 生物工程 • 上一篇

豆豉纤溶酶基因的异源表达及其重组菌在淡豆豉中的应用

徐菁雯，乌日娜，王伟明，阎亦然，向雪琴，赵玉莲，武俊瑞，王亚琦，邓丽，童星，史海粟

（1.沈阳农业大学食品学院，辽宁沈阳 110866；2.辽宁省食品发酵技术工程研究中心，辽宁沈阳 110866；3.黑龙江省中医药科学院，黑龙江哈尔滨 150001；4.沈阳市微生物发酵技术创新重点实验室，辽宁沈阳 110866；5.佛山市海天（高明）调味食品有限公司，广东佛山 528000）

发布日期:2025-02-07
基金资助:
国家自然科学区域创新联合发展基金项目（U20A20400）；沈阳农业大学科技创新平台项目（21-103-014；21-104-0-28）；沈阳农业大学人才引进（高层次）科研启动费项目（2023YJRC002）；广东省调味食品生物发酵先进技术企业重点实验室开放基金项目（2017B030302002）

Heterologous Expression of the Fibrinolytic Enzyme Gene from Bacillus velezensis 20-1, Isolated from Sojae Semen Praeparatum and Application of Recombinant Strain Containing It in the Production of Sojae Semen Praeparatum

XU Jingwen, WU Rina, WANG Weiming, YAN Yiran, XIANG Xueqin, ZHAO Yulian, WU Junrui, WANG Yaqi, DENG Li, TONG Xing, SHI Haisu

(1. College of Food Science, Shenyang Agricultural University, Shenyang 110866, China; 2. Engineering Research Center of Food Fermentation Technology, Shenyang 110866, China; 3. Heilongjiang Academy of Chinese Medicine Sciences, Harbin 150001, China; 4. Shenyang Key Laboratory of Microbial Fermentation Technology Innovation, Shenyang 110866, China; 5. Foshan Haitian (Gaoming) Flavouring & Food Co. Ltd., Foshan 528000, China)

Published:2025-02-07

摘要/Abstract

摘要： 从不同来源及不同发酵阶段的淡豆豉中分离筛选出65 株优势菌，通过酶联免疫吸附法测定纤溶酶活性，得到一株高产纤溶酶的贝莱斯芽孢杆菌（Bacillus velezensis）20-1，并扩增其纤溶酶基因，在毕赤酵母中异源表达，获得高产纤溶酶的重组菌株Pichia pastoris pPIC9K-his-aprEBS2，重组菌中该基因表达水平是出发菌株的5.8 倍，培养48 h后纤溶酶活性达到（3 025.59±201.27）U/mL，是出发菌株的8.9 倍；经优化，其活性可达（8 698.24±180.73）U/mL。通过急性毒性实验证明，该重组菌无毒。利用该重组菌纯种发酵淡豆豉，淡豆豉中纤溶酶活性达到（17 287.02±103.20）U/g，通过理化指标监测、感官特性分析及电子舌分析，重组菌可以提高淡豆豉的感官及风味。重组菌发酵的淡豆豉成品血栓溶解率可达到（60.55±1.58）%，高于市售淡豆豉。

关键词: 豆豉纤溶酶；异源表达；毕赤酵母；淡豆豉

Abstract: In this study, 65 strains of dominant bacteria were isolated from Sojae Semen Praeparatum (SSP) from different regions and screened for their fibrinolytic enzyme activity by enzyme-linked immunosorbent assay (ELISA). Bacillus velezensis 20-1 was selected from these strains for its high fibrinolytic enzyme activity, and its fibrinolytic enzyme gene was amplified and heterologously expressed in Pichia pastori. As a result, a recombinant strain with high fibrinolytic enzyme activity, P. pastoris pPIC9K-his-aprEBS2, was obtained. The results showed that the gene expression level of P. pastoris pPIC9K-his-aprEBS2 was 5.8 times as high as that of B. velezensis 20-1, and the enzyme activity reached (3 025.59 ± 201.27) U/mL after 48 h culture, which was 8.9 times as high as that of B. velezensis 20-1. The fibrinolytic enzyme activity increased to (8 698.24 ± 180.73) U/mL under optimized conditions. The acute toxicity test showed that the recombinant bacterium was non-toxic. SSP fermented by the recombinant bacterium was (17 287.02 ± 103.20) U/g. Physicochemical characterization, sensory evaluation and electronic tongue analysis demonstrated that the recombinant bacterium could improve the sensory quality and flavor of SSP. The thrombolysis rate of SSP fermented by it was (60.55 ± 1.58)%, which was higher than that of commercially available SSP.

Key words: Douchi fibrinolytic enzyme; heterologous expression; Pichia pastoris; Sojae Semen Praeparatum

中图分类号:

TS264.2

徐菁雯，乌日娜，王伟明，阎亦然，向雪琴，赵玉莲，武俊瑞，王亚琦，邓丽，童星，史海粟. 豆豉纤溶酶基因的异源表达及其重组菌在淡豆豉中的应用[J]. 食品科学, 2025, 46(4): 68-80.

XU Jingwen, WU Rina, WANG Weiming, YAN Yiran, XIANG Xueqin, ZHAO Yulian, WU Junrui, WANG Yaqi, DENG Li, TONG Xing, SHI Haisu. Heterologous Expression of the Fibrinolytic Enzyme Gene from Bacillus velezensis 20-1, Isolated from Sojae Semen Praeparatum and Application of Recombinant Strain Containing It in the Production of Sojae Semen Praeparatum[J]. FOOD SCIENCE, 2025, 46(4): 68-80.

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豆豉纤溶酶基因的异源表达及其重组菌在淡豆豉中的应用

Heterologous Expression of the Fibrinolytic Enzyme Gene from Bacillus velezensis 20-1, Isolated from Sojae Semen Praeparatum and Application of Recombinant Strain Containing It in the Production of Sojae Semen Praeparatum

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