摘要
目的:本研究构建一株重组枯草芽孢杆菌工程菌,实现以苯酚、丙酮酸和氨为底物全细胞生成L-酪氨酸(L-tyrosine,L-Tyr),并优化其细胞培养条件和催化反应条件,以期提高L-Tyr的产量。方法:将来源于Pantoea agglomerans的酪氨酸酚裂解酶(Tyrosine Phenol-Lyase,TPL)经密码子优化后在枯草芽孢杆菌中异源表达,通过单因素实验优化了重组菌诱导表达条件和全细胞转化条件,旨在提高L-Tyr产量。结果:重组枯草芽孢杆菌在20℃,2.0 g/L木糖诱导培养36 h时TPL活性最高达到4.65±0.15 U·g^(−1),在75 mmol/L苯酚、75 mmol/L丙酮酸钠、487 mmol/L氯化铵、2.0 g/L亚硫酸钠、2.0 g/L EDTA、0.08 g/L磷酸吡哆醛(Pyridoxal Phosphat,PLP)、湿菌体50 g/L、pH=8.0、35℃的全细胞转化条件下,L-Tyr达到9.38 g/L,转化率为73.24%。为了进一步改善高浓度苯酚导致TPL酶活力下降问题,在全细胞转化环节采用分批补料方式,20 h后得到15.12 g/L L-Tyr,转化率为75.51%。结论:研究结果表明重组枯草芽孢杆菌可以成功转化苯酚、丙酮酸钠合成L-酪氨酸,为全细胞生物制备食品级L-酪氨酸提供了理论和技术基础,具有良好的应用前景。
Objective:This study conducted a recombinant Bacillus subtilis strain capable of producing L-Tyr utilizing phenol,sodium pyruvate,and ammonia.Moreover,the conditions of cell culture and catalytic reaction were optimized to improve L-Tyr production.Methods:The tyrosine phenol-lyase(TPL)gene from Pantoea agglomerans was codonoptimized and successfully over-expressed in B.subtilis.The conditions of induction and whole-cell transformation were optimized using single-factor experiments for L-Tyr production.Results:The highest TPL enzyme activity was achieved 4.65±0.15 U·g^(−1) in the recombinant B.subtilis at 20℃with 2.0 g/L xylose after 36 hours.Under the whole-cell transformation conditions of 75 mmol/L phenol,75 mmol/L sodium pyruvate,487 mmol/L ammonium chloride,2.0 g/L sodium sulfite,2.0 g/L EDTA,0.08 g/L pyridoxal phosphate(PLP),wet cell mass of 50 g/L,pH8,and 35℃,the L-Tyr production increased to 9.38 g/L with a conversion rate of 73.24%.Coping with the issue of decreased TPL enzyme activity caused by phenol resistance,a batch feeding strategy was implemented during the whole-cell transformation,resulting in a final L-Tyr production of 15.12 g/L with a conversion rate of 75.51%after 20 hours.Conclusion:These findings demonstrate the successful conversion of phenol and sodium pyruvate into L-Tyr by the recombinant B.subtilis strain.The study would provide a theoretical and technical foundation for the production of food-grade L-Tyr using whole-cell biocatalysis,highlighting its potential for practical applications.
作者
林伟朝
孙雯
孙晓萱
朱显峰
张保国
LIN Weichao;SUN Wen;SUN Xiaoxuan;ZHU Xianfeng;ZHANG Baoguo(Institute of Microbial Engineering,School of Life Sciences,Henan University,Kaifeng 475000,China;Shanghai Advanced Research Institute,Chinese Academy of Sciences,Shanghai 201210,China)
出处
《食品工业科技》
CAS
北大核心
2024年第22期116-123,共8页
Science and Technology of Food Industry
基金
福建省中科院STS重大项目(2022T3004)。
