产阿魏酸酯酶菌株的筛选与产酶条件优化

Screening of feruloyl esterase-producing strains and optimization of its fermentation conditions

该研究从土壤中分离得到可利用阿魏酸乙酯为唯一碳源且产阿魏酸酯酶的菌株,经过摇瓶发酵和液相色谱定性定量分析,获得1株阿魏酸酯酶活力较高的菌株SK52.001,将发酵上清液的酶反应产物通过LC-MS分析产物分子质量,确定该上清液中含阿魏酸酯酶。对目标菌株进行分子生物学鉴定,并结合形态学特征和生理生化实验确认该菌株为短小芽孢杆菌,并命名为Bacillus pumilus SK52.001。通过对目标菌株进行发酵制备阿魏酸酯酶研究发现,在30℃,200 r/min,接种量5%的条件下,在45 g/L麦麸为碳源,5 g/L胰蛋白胨为氮源,初始pH值为6.0时,摇瓶培养26 h的酶活力可达到421 U/L,较优化前酶活力195 U/L提高115%。当菌株在以45 g/L葡萄糖为碳源的发酵培养基中培养8 h后,加入麸皮诱导产酶44 h,发酵液中阿魏酸酯酶活力达到808 U/L。

Feruloyl esterase can efficiently release ferulic acid by the deconstruction of plant biomass.However,the industrial application of feruloyl esterase is limited by the enzyme activity.Several strains producing feruloyl esterase were isolated from soil samples using ethyl ferulate as the sole carbon source of the screening medium.A strain named SK52.001 with high feruloyl esterase activity was obtained by shaking fermentation and HPLC analysis.It was confirmed that the strain secreted feruloyl esterase into the supernatant,which was analyzed by LC-MS to determine the molecular weight of the enzyme reaction mixture.Meanwhile,the strain was identified as Bacillus pumilus and named SK52.001 via the methods of morphology,molecular biology and physiological and biochemical characteristics.By one-factor-at-a-time method,the optimal fermentation conditions were determined as 45 g/L wheat bran,5 g/L tryptone and the initial pH value of 6.0.When the inoculation volume was controlled at 5%,the enzyme activity of the strain reached 421 U/L at 30°C and 200 r/min shaking for 26 h,enhanced by 115%compared to the initial strain.When the strains were preincubated in fermentation media with 45 g/L glucose as the carbon source for 8 h followed by supplementing 45 g/L wheat bran,the highest enzyme activity of 808 U/L was obtained after 44 h.