毛木耳漆酶纯化及其部分漆酶特性的研究

对毛木耳AuriculariapolytrichaAP4的粗酶液进行PAGE电泳后发现含有三种漆酶同工酶,并且通过运用NativeSDS-PAGE获得三种漆酶的分子量大小分别约为:LacA(110kD);LacB(84kD);LacC(65kD)。对漆酶粗酶液通过硫酸铵分级沉淀和离子交换柱层析进行纯化,用SDS-PAGE证明获得纯化的单一漆酶LacB。LacB漆酶的反应的最适温度为30℃,最适pH为3.0。此酶氧化ABTS的Km值为6.64×10-mmol/L,金属离子对酶活的影响很大,其中5Ca2+,Mg2+,Zn2+,Na2+,Ag2+对漆酶LacB有明显的激活作用;Co2+,Hg2+,Fe3+,Fe2+,Ba2+等对酶活有明显的抑制作用。LacB和其它真菌漆酶一样具有底物专一性不强的特点,并且LacB对RB亮兰染料有很好的脱色作用。

PURIFICATION AND PROPERTIES OF LACCASE PRODUCED BY AURICULARIA POLYTRICHA

The result of PAGE for laccase crude extract showed that three kinds of isoenzymes exist in the Auricularia polytricha AP4. The molecular weight of the three isoenzymes was determined by Native SDS-PAGE: LacA (110kD), LacB (84kD), LacC (65kD). The LacB was purified from AP4 by ammonium sulphate fractionation, DEAE sephrase fast flow chromatography.The optimum pH and temperature for LacB activity were 4.0℃ and 30℃, respectively. The LacB catalyzed the oxidation of ABTS with a Michaelis constant of 6.64× 10-5mol/L.Various metal ions showed different effects on the LacB activity. The activity was enhanced by Ca2+, Mg2+, Zn2+, Na2+, Ag2+, and was strongly inhibited by Co2+,Hg2+,Fe3+,Fe2+,Ba2+. LacB also is able to decolorize remazol brilliant blue R efficiently.