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

作　　者：徐菁雯 乌日娜[1,2] 王伟明 阎亦然 向雪琴 赵玉莲 武俊瑞 王亚琦[5] 邓丽 童星 史海粟 XU Jingwen;WU Rina;WANG Weiming;YAN Yiran;XIANG Xueqin;ZHAO Yulian;WU Junrui;WANG Yaqi;DENG Li;TONG Xing;SHI Haisu(College of Food Science,Shenyang Agricultural University,Shenyang 110866,China;Engineering Research Center of Food Fermentation Technology,Shenyang 110866,China;Heilongjiang Academy of Chinese Medicine Sciences,Harbin 150001,China;Shenyang Key Laboratory of Microbial Fermentation Technology Innovation,Shenyang 110866,China;Foshan Haitian(Gaoming)Flavouring&Food Co.Ltd.,Foshan 528000,China)

机构地区：[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

出　　处：《食品科学》2025年第4期68-80,共13页Food Science

基　　金：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)。

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。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(3025.59±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(8698.24±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(17287.02±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.

关 键 词：豆豉纤溶酶 异源表达 毕赤酵母 淡豆豉 

分 类 号：TS264.2[轻工技术与工程—发酵工程]