枇杷果肉漆酶酶学特性研究

Study on Characteristics of Laccase from Eriobotrya japonica Pulp

研究枇杷果肉漆酶(EC1.10.3.2)的提取及其理化性质,为探讨漆酶在冷藏枇杷果实木质化败坏中的作用机理提供参考。以‘解放钟’枇杷(Eriobotrya japonica Lind‘l.Jiefangzhong’)果实为试材,采用硫酸铵盐析、透析和DEAE-纤维柱层析等提取漆酶(EC1.10.3.2)研究其理化特性,包括温度、pH、抑制物、金属离子对漆酶活性的影响及其底物特性,测定果实贮藏期间pH变化。结果表明,漆酶的最适温度为45℃,最适pH为4.4,pH为3.2～4.4时酶的稳定性较好。金属离子Cu2+、K+对漆酶有激活作用,而Ag+、Fe2+、Fe3+起抑制作用。EDTA和L-半胱氨酸抑制物对漆酶均有抑制作用,以L-半胱氨酸抑制作用最强。枇杷果肉漆酶以2,2’-连氮-双(3-乙基苯并噻唑-6-磺酸)(简称ABTS)为底物的Km值为0.00512mol/L。

The aim of this paper was to study the extraction 1.10.3.2） from Eriobotrya japonica pulp, and to establish and physical-chemical properties of laecase （EC foundation for discussion on flesh woodiness mechanism of laccase in loquat fruits under low temperature storage. The laccase from an E. japonica cv. ＇Jiefangzhong＇ was extracted and purified by （NH4）~SO4 salting out, dialysis and DEAE-cellulose column chromatography. The influences were investigated of different temperature, pH, substrates, inhibitors and metal ions on the laecase activity. The pH change was determined in loquat fruits during storage. Optimal temperature of laccase was 45~C, and optimal pH was 4.4.. The laccase activity was stable in the pH range of 3.2-4.4, was increased by the addition of Cu^2＋ and K^＋, and was inhibited by the addition of Ag^＋, Fe^2＋ and Fe^3＋. Both EDTA and L-cysteine had the strongest inhibitory effect on the laccase activity. The Km constants based on the substrate of 2,2-azobis （3-ethylbenzothiazole-6-sulfonaic acid） （ABTS） was 0.00512 mol/L.