Han Sang-Eun, Kwon Hawk-Bin, Lee Seung-Bum, Yi Bu-Young, Murayama Ikuo, Kitamoto Yutaka, Byun Myung-Ok
Molecular Physiology Division, National Institute of Agricultural Biotechnology, 441-707 Suwon, Republic of Korea.
Protein Expr Purif. 2003 Aug;30(2):194-202. doi: 10.1016/s1046-5928(03)00104-9.
Complementary DNA for a gene encoding trehalose phosphorylase (TP) that reversibly catalyzes trehalose synthesis and degradation from alpha-glucose-1-phosphate (alpha-Glc-1-P) and glucose was cloned from Pleurotus sajor-caju. The cDNA of P. sajor-caju TP (designated PsTP, GenBank Accession No. AF149777) encodes a polypeptide of 751 amino acids with a deduced molecular mass of 83.7 kDa. The PsTP gene is expressed in mycelia, pilei, and stipes of fruiting bodies. Trehalose phosphorylase PsTP was purified from PsTP-transformed Escherichia coli. The enzyme catalyzes both the phosphorolysis of trehalose to produce alpha-Glc-1-P and glucose, and the synthesis of trehalose. The apparent K(m) values for trehalose and Pi in phosphorolytic reaction at pH 7.0 were 74.8 and 5.4 mM, respectively. The PsTP gene complemented Saccharomyces cerevisiae Deltatps1, Deltatps2 double-mutant cells, allowing their growth on glucose medium. Furthermore, yeast transformed with PsTP produced 2-2.5-fold more trehalose than non-transformants or cells transformed with empty vector only.
从凤尾菇中克隆出了编码海藻糖磷酸化酶（TP）的互补DNA，该酶可催化α-葡萄糖-1-磷酸（α-Glc-1-P）和葡萄糖之间可逆的海藻糖合成与降解反应。凤尾菇TP的cDNA（命名为PsTP，GenBank登录号为AF149777）编码一个由751个氨基酸组成的多肽，推导分子量为83.7 kDa。PsTP基因在菌丝体、菌盖和子实体的菌柄中表达。从经PsTP转化的大肠杆菌中纯化出海藻糖磷酸化酶PsTP。该酶既能催化海藻糖磷酸解生成α-Glc-1-P和葡萄糖，也能催化海藻糖的合成。在pH 7.0的磷酸解反应中，海藻糖和磷酸的表观K(m)值分别为74.8和5.4 mM。PsTP基因互补了酿酒酵母Deltatps1、Deltatps2双突变细胞，使其能够在葡萄糖培养基上生长。此外，用PsTP转化的酵母产生的海藻糖比未转化的酵母或仅用空载体转化的细胞多2 - 2.5倍。
