从哈茨木霉中纯化和表征一种酸性磷酸酶。

Purification and characterization of an acid phosphatase from Trichoderma harzianum.

机构信息

Universidade Federal de Goiás, Campus Samambaia, ICB II, Cx. Postal 131, Goiânia, GO 74001-970, Brazil.

出版信息

Biotechnol Lett. 2010 Aug;32(8):1083-8. doi: 10.1007/s10529-010-0264-2. Epub 2010 Apr 3.

PMID:20364293

Abstract

An acid phosphatase from Trichoderma harzianum was purified in a single step using a phenyl-Sepharose chromatography column. A typical procedure showed 22-fold purification with 56% yield. The purified enzyme showed as a single band on SDS-PAGE with an apparent molecular weight of 57.8 kDa. The pH optimum was 4.8 and maximum activity was obtained at 55 degrees C. The enzyme retained 60% of its activity after incubation at 55 degrees C for 60 min. The K (m) and V (max) values for p-nitrophenyl phosphate (p-NPP) as a substrate were 165 nM and 237 nM min(-1), respectively. The enzyme was partially inhibited by inorganic phosphate and strongly inhibited by tungstate. Broad substrate specificity was observed with significant activities for p-NPP, ATP, ADP, AMP, fructose 6-phosphate, glucose 1-phosphate and phenyl phosphate.

摘要

哈茨木霉中的酸性磷酸酶可通过苯基-Sepharose 色层析柱一步法进行纯化。典型的步骤显示酶的纯化有 22 倍的提高，产率为 56%。SDS-PAGE 显示纯化的酶为单一条带，其表观分子量为 57.8 kDa。最适 pH 值为 4.8，在 55°C 时活性最大。该酶在 55°C 孵育 60 分钟后仍保留 60%的活性。以对硝基苯磷酸（p-NPP）为底物时，K(m)和 V(max)值分别为 165 nM 和 237 nM min(-1)。该酶被无机磷酸盐部分抑制，被钨酸盐强烈抑制。对 p-NPP、ATP、ADP、AMP、果糖 6-磷酸、葡萄糖 1-磷酸和苯磷酸有显著的活性，表现出广泛的底物特异性。