产芳香腈水解酶的恶臭假单胞菌Pseudomonas putida CGMCC3830的筛选、鉴定及发酵优化

Screening, identification and culture optimization of a newly isolated aromatic nitrilase-producing bacterium– Pseudomonas putida CGMCC3830

摘要:

近年来微生物腈水解酶水解腈类化合物制备有机酸已逐步受到关注。本研究分离到一株表现出较高腈水解酶活力的细菌菌株，通过形态学、生理生化实验以及16S rRNA基因序列分析将其鉴定为恶臭假单胞菌Pseudomonas putida CGMCC3830。结合单因素及响应面法对该菌株产腈水解酶的发酵条件进行了优化，获得最适培养条件为：甘油13.54 g/L，胰蛋白胨11.59 g/L，酵母粉5.21 g/L，KH2PO4 1 g/L，NaCl 1 g/L，脲1 g/L，初始pH 6.0及培养温度30 ℃。通过优化，酶活由2.02 U/mL提升至36.12 U/mL。对该菌株底物特异性的考察结果表明，恶臭假单胞菌腈水解酶对芳香族腈类化合物具有较高的水解活力。将其应用于烟酸的生物合成中，2 mg/mL游离细胞能90 min内将20.8 g/L 3-氰基吡啶彻底转化，制备得到相应烟酸。这些结果表明恶臭假单胞菌P. putida CGMCC3830在烟酸的规模化生产中具有一定的应用潜力。

Abstract:

Microbial nitrilases have attracted increasing attention in nitrile hydrolysis for carboxylic acid production in recent years. A bacterium with nitrilase activity was isolated and identified as Pseudomonas putida CGMCC3830 based on its morphology, physiological and biochemical characteristics, as well as 16S rRNA gene sequence. The nitrilase production was optimized by varying culture conditions using the one-factor-at-a-time method and response surface methodology. Glycerol 13.54 g/L, tryptone 11.59 g/L, yeast extract 5.21 g/L, KH2PO4 1 g/L, NaCl 1 g/L, urea 1 g/L, initial pH 6.0 and culture temperature 30 ℃ were proved to be the optimal culture conditions. It resulted in the maximal nitrilase production of 36.12 U/mL from 2.02 U/mL. Investigations on substrate specificity demonstrate P. putida nitrilase preferentially hydrolyze aromatic nitriles. When applied in nicotinic acid synthesis, 2 mg/mL P. putida cells completely hydrolyzed 20.8 g/L 3-cyanopyridine into nicotinic acid in 90 min. The results indicated P. putida CGMCC3830 displayed potential for industrial production of nicotinic acid.