高产靛蓝色素大肠杆菌工程菌构建及其发酵转化条件  

Construction of Gene Engineering Bacterium and Optimization of Fermentation Conditions for High Production of Indigo from Tryptophan

作　　者：杜灵燕[1] 岳建明[1] 马金旺 王成涛[1] 孙宝国[1] 王子豪[1] 隆歆然 赵吉兴 Du Lingyan Yue Jianming Ma Jinwang Wang Chengtao Sun Baoguo Wang Zihao Long Xinran Zhao Jixing(Bering Advanced Innovation Center for Food Nutrition and Human Health, Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University （BTBU）, Beijing 100048 Department of Rural Development, Beijing Municipal Science and Technology Commission, Beijing 100195 Shandong Zhonghui Biotechnology Co. Ltd., Binzhou 251706, Shandong)

机构地区：[1]北京食品营养与人类健康高精尖创新中心、北京市食品添加剂工程技术研究中心北京工商大学,北京100048 [2]北京市科委农村科技发展处,北京100195 [3]山东中惠生物科技股份有限公司,山东滨州251706

出　　处：《中国食品学报》2017年第3期64-72,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(31571801);十三五国家重点研发计划课题(2016YFD0400802);科技创新服务能力建设-科技成果转化-提升计划项目(PXM2016-014213-000034);北京市科技计划项目(Z151100001215008)

摘　　要：天然食用蓝色系色素是一类稀缺资源。本文克隆假单胞菌B3中苯乙烯单加氧酶基因sty AB,将其超量表达于大肠杆菌DH5α,构建高产靛蓝色素工程菌,研究色氨酸发酵转化生成靛蓝色素的影响因素,通过正交设计试验优化靛蓝色素发酵条件。结果表明,构建的sty AB基因过表达大肠杆菌DH5α-sty AB具有较高靛蓝色素合成能力,而野生菌DH5α中未检测到靛蓝色素生成;发酵液起始pH、温度、时间、底物色氨酸浓度等因素条件对DH5α-sty AB靛蓝色素合成代谢有一定影响。正交设计试验优化的发酵条件为:起始pH 6.8、色氨酸质量浓度1.2 mg/m L、发酵温度33℃、发酵时间28 h。在此条件下,靛蓝色素产量达到592.50 mg/L,较未优化前(414.61mg/L)提高了42.9%,接近理论值770.60 mg/L;工程菌DH5α-sty AB发酵底物吲哚的靛蓝色素产量为60.00 mg/L,表明色氨酸更适宜为靛蓝色素发酵底物。上述结果为天然靛蓝色素生物转化提供了理论依据和技术指导。Indigo is one of oldest nature dyestuffs. The Pseudomonas putida B3was used to clone genes styAB eneodingstyrene monooxygenase for the production of indigo. The expression of genes styAB was constructed in DH5α by the method of recombinant DNA technique for high production of indigo from tryptophan. The indigo fermentation conditions were optimized in combination with single factor experiment and orthogonal experiment. The results showed that theDH5α- styAB could synthesis indigo from tryptophan while DH5αnot; the fermentation factors influenced the synthesis of indigo inordinately andoptimized fermentation conditions were determined as follows： initial pH of 6.8, culture temperature of 33 ℃, the substrate concentration of 1.2 mg/mL, the inoeulum amount of 1.2%, fermentation time of 28 h. The bio-indigo production reached up to 592.50 mg/L enhanced by 42.7% than the production before optimizing of 414.61 mg/L under thatconditions and was more closer to theory value of 770.60 mg/L. The production of the DH5α-styAB from indolewas 60.00 mg/L which was lower than from tryptophan, therefore tryptophan was better as the substratefor indigo production. The research offered theoretical and technical guidance for the bioconversion of indigo.

关 键 词：靛蓝色素 苯乙烯单加氧酶 色氨酸 基因工程菌株 发酵 

分 类 号：TQ920.1[轻工技术与工程—发酵工程] TS264.4