意蜂工蜂酸性磷酸酶的纯化及其酶学特性

›› 2004, Vol. 47 ›› Issue (3): 310-315.

意蜂工蜂酸性磷酸酶的纯化及其酶学特性

江琰¹，刘克武^2*，雷远成²，宋贤丽²

出版日期:2004-06-20 发布日期:2004-06-20

Purification and characterization of acid phosphatase from the Italian honeybee, Apis mellifera L. (Hymenoptera: Apidae)

IANG Yan¹, LIU Ke-Wu^2*, LEI Yuan-Cheng², SONG Xian-Li²

Online:2004-06-20 Published:2004-06-20
Contact: LIU Ke-Wu

摘要/Abstract

摘要： 从意蜂Apis mellifera工蜂体内分离提纯酸性磷酸酶（ACPase, EC3.1.3.2）,并对其性质进行了研究。将工蜂酸性磷酸酶的初提物经分段盐析、DEAE-Sepharose FF离子交换层析及Sephadex G-200 凝胶过滤等纯化步骤，得到经聚丙烯酰胺凝胶电泳为单一蛋白区带的酶液。提纯倍数为77.24，酶液比活力为16.22 U/mg（对硝基苯磷酸二钠作底物）。利用凝胶过滤法测定酶的相对分子质量为135 kD，SDS-PAGE测定酶的亚基相对分子质量为63 .1 kD。酶的等电点为4.46和4.79。非还原/还原（NR/R）单向、双向SDS-PAGE显示酶分子含有链内二硫键。对二级结构圆二色谱分析显示，酶分子中α-螺旋占13.84%，β-折叠占25.68%，无规则卷曲占56.34%。氨基酸组成分析结果表明, 酸性磷酸酶约含有507个氨基酸残基，富含门冬氨酸残基。

关键词: 意蜂工蜂, 酸性磷酸酶, 分离纯化, 性质

Abstract: Acid phosphatase (ACPase, EC3.1.3.2) was isolated and purified from the Italian honeybee, Apis mellifera L., and properties of the enzyme had been studied. Acid phosphatase was partially obtained from A. mellifera by homogenate, a mmunium sulfate fractionation, chromatography with DEAE-sepharose FF and gel filtration with Sephadex G-200 The purified enzyme moved as a single electrophoretic band in PAGE. The purification multiple was 77.24, and the specific activity 16.22 U/mg with pNPP as its substrate. The molecular weight of ACPase was 135 kD determined with gel filtration and its subunit weight was 63.1 kD determined with SDS-PAGE. Isoelectric focusing study showed that pI values of the enzyme were 4.46 and 4.79. NR/R single and two dimensions SDS-PAGE indicated that the enzyme contained intrachain disulfide bond. Circular dichroism spectrum was investigated, and it was found that the proportion of α-helix, β-sheet and random coil in the enzyme was 13.84%, 2.68% and 6.34% respectively. Amino acid composition analysis showed that there were about 507 amino acids in the ACPase, with plenty of Asp.

Key words: Apis mellifera, acid phosphatase, isolation and purification, properties

江琰¹，刘克武^2*，雷远成²，宋贤丽². 意蜂工蜂酸性磷酸酶的纯化及其酶学特性[J]. , 2004, 47(3): 310-315.

IANG Yan¹, LIU Ke-Wu^2*, LEI Yuan-Cheng², SONG Xian-Li². Purification and characterization of acid phosphatase from the Italian honeybee, Apis mellifera L. (Hymenoptera: Apidae)[J]. , 2004, 47(3): 310-315.

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