来自桔青霉的阿魏酸酯酶的分离纯化、理化性质

Purification and characterization of ferulic acid esterase from Penicillium citrinum

摘要:

摘要：【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 Sepharose 6 Fast 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31 kDa，全酶分子量约为58 kDa。其最适pH为6.0，最适温度为45℃－65℃，在pH 5.0-6.0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5 U 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。

Abstract:

Abstract: [Objective] Extracellular feruloyl esterase (EC 3.1.1.73) 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°C. The enzyme was stable at pH from 5.0 to 6.0 and temperature from 25 to 55°C. 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.