Pseudomonas sp. F-12发酵优化及转化合成半胱氨酸的研究

摘要/Abstract

摘要： 通过不同发酵策略对Pseudomonas sp. F-12以DL-2-氨基-△-噻唑啉-4-羧酸（DL-ATC）为底物生产L-半胱氨酸的过程进行优化,以分批发酵和指数流加发酵的形式进行生产,将发酵过程中的DL-ATC浓度分别控制在5 g/L和15 g/L。结果表明15 g/L DL-ATC分批发酵生产L-半胱氨酸得到最高酶活为149.1 U/mL,比活为70.9 U/mg DCW; 采用15 g/L DL-ATC两阶段指数流加发酵,可使最高酶活达到313.3 U/mL,提高为分批发酵的2.1倍。同时在两阶段发酵过程中,能减少DL-ATC用量为5 g/L。两阶段发酵过程采用不同流加策略,证明菌体得率对酶活水平有显著影响。首次考察了Pseudomonas sp. F-12全静息细胞转化合成L-半胱氨酸的主要影响因素。结果表明Pseudomonas sp. F-12的最适转化pH为8.0,最适转化温度为30℃。加入甲苯等有机溶剂改变细胞膜通透性,使得半胱氨酸产量比对照组提高7.16倍,最高摩尔转化率达到93.3 %。不同金属离子对转化影响不同,0.1 mmol/L Fe对转化反应有促进作用,0.1 mmol/L Co、Zn、Ni等重金属离子有抑制作用。研究结果为微生物法发酵及全细胞转化合成半胱氨酸的后续工业化奠定了基础。

关键词: 假单胞菌, L-半胱氨酸, DL-2-氨基-&, #x025B3, 2-噻唑啉-4-羧酸（DL-ATC）, 发酵, 生物转化

Abstract: L-cysteine has been widely applied in food and pharmaceuticals. In this study, different fermentation strategies of Pseudomonas sp. F-12 transforming DL-ATC to L-cysteine were investigated. The results showed that the highest enzyme activity of 15 g/L DL-ATC batch fermentation was 149.1 U/mL, specific activity was 70.9 U/mg DCW. The highest enzyme activity of two phase fermentation using 15 g/L DL-ATC was 313.3 U/mL, 2.1 times higher than the batch fermentation. The amount of DL-ATC was reduced to 5 g/L. In this study, the major factors on the biotransformation of L-cysteine by Pseudomonas sp. F-12 were investigated. The optimal reaction conditions for cells were 30℃, pH 8.0. The addition of organic solvents could change cellular permeability and enhance the production of L-cysteine by 7.16 times. The maximum conversion of L-cysteine was 93.3%. 0.1 mmol/L Fe²⁺ benefitted the reaction, while 0.1 mmol/L Co²⁺, Zn²⁺, Ni²⁺ and other heavy metal ions inhibited transformation.

Key words: Pseudomonas, sp. F-12 L-cysteine, DL-ATC, Fermentation, Bioconversion

赵婧楠, 李志敏, 叶勤. Pseudomonas sp. F-12发酵优化及转化合成半胱氨酸的研究[J]. 生物技术通报, 2014, 0(10): 207-214.

Zhao Jingnan, Li Zhimin, Ye Qin. Study on Fermentation and Conversion of L-cysteine by Pseudomonas sp. F-12[J]. Biotechnology Bulletin, 2014, 0(10): 207-214.

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