Purification and characterization of ferulic acid esterase from Penicillium citrinum
- 化学化工－已发表论文 
【目的】从桔青霉的发酵液中分离纯化了胞外阿魏酸酯酶(PCfAE)并进行了酶学性质的研究,初步探讨了PCfAE对麦糟的酶解作用。【方法】利用(nH4)2SO4沉淀、dEAE-SEPHArOSE fAST flOW离子交换层析、PHEnyl SEPHArOSE6fAST flOW疏水层析纯化得到电泳纯的阿魏酸酯酶。【结果】从该菌株的发酵液中获得一阿魏酸酯酶,该酶亚基分子量约为31kdA,全酶分子量约为58kdA。其最适PH为6.0,最适温度为45℃-65℃,在PH5.0-6.0及25℃-55℃之间,酶保持了较好的稳定性。Mg2+、fE2+、Mn2+、CA2+和nA+对酶活有一定的促进作用,zn2+对PCfAE酶活有一定的抑制作用,而Cu2+、亮抑肽素、抑肽酶有显著的抑制作用,Hg2+、苯甲基磺酰氟几乎完全抑制了酶活。EdTA对PCfAE活性无明显影响。PCfAE的kCAT/kM对香豆酸甲酯、芥子酸甲酯、阿魏酸甲酯、咖啡酸甲酯的值分别为823、416、103、0,PCfAE对MPCA的催化效率最高。PCfAE作用于麦糟,当5u PCfAE/g麦糟时,其阿魏酸的释放量为7.2%。【结论】获得了一阿魏酸酯酶,其理化性质与至今报道的阿魏酸酯酶有所不同,为阿魏酸酯酶的开发提供了重要的实验依据。[Objective] Extracellular feruloyl esterase ( EC 126.96.36.199 ) from Penicillium citrinum culture filtrates was studied.The effect of feruloyl esterases on the enzymatic hydrolysis of brewer′ s spent grain was also investigated.[Methods ] Feruloyl esterase was purified to homogeneity by ammonium sulfate precipitation,ion-exchange chromatography with a DEAE-Sepharose Fast Flow column,and column chromatography with a Phenyl-Sepharose 6 Fast Flow column.[Results]The purified homogeneous preparation of native feruloyl esterase had a molecular mass of 58 kDa by native-PAGE and a subunit molecular mass of 31 kDa by SDS-PAGE.The optimum enzymatic activity was achieved at pH 6.0 and 45-65℃ .The enzyme was stable at pH from 5.0 to 6.0 and temperature from 25 to 55℃ .The enzymatic activity was slightly enhanced by Mg2 + ,Fe2 + ,Mn2 + ,Ca2 + ,and Na + ,whereas it was slightly inhibited by Zn2 + ,strongly inhibited by Cu2 + ,and completely inhibited by Hg2 + and phenylmethanesulfonyl fluoride.EDTA had a slight influence on the enzymatic activity.The determination of kcat/ Km revealed that the enzyme hydrolyzed methyl p-coumarate,methyl sinapate,methyl ferulate,methyl caffeate,and the values were 823,416,103,and 0,respectively.The kcat /Km values showed that the enzyme hydrolyzed MpCA faster and more efficiently than all the other substrates.When the crude feruloyl esterase was used to hydrolyze the brewer’s spent grain,about 7.2% of the alkaline-extractable ferulic acid could be released,with the concentration of 5 u feruloyl esterase /g.[Conclusion ] A feruloyl esterase was discovered.Its biochemical characteristics were different from what has been reported in literature.This provided an important basis for the exploitation of a feruloyl esterase.