Schwarz Alexandra, Goedl Christiane, Minani Alphonse, Nidetzky Bernd
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
J Biotechnol. 2007 Mar 30;129(1):140-50. doi: 10.1016/j.jbiotec.2006.11.022. Epub 2006 Dec 2.
Trehalose phosphorylase from the basidiomycete Pleurotus ostreatus (PoTPase) was isolated from fungal fruit bodies through approximately 500-fold purification with a yield of 44%. Combined analyses by SDS-PAGE and gelfiltration show that PoTPase is a functional monomer of approximately 55 kDa molecular mass. PoTPase catalyzes the phosphorolysis of alpha,alpha-trehalose, yielding alpha-d-glucose 1-phosphate (alphaGlc 1-P) and alpha-d-glucose as the products. The optimum pH of PoTPase for alpha,alpha-trehalose phosphorolysis and synthesis is 6.8 and 6.2, respectively. Apparent substrate binding affinities (K(m)) were determined at pH 6.8 and 30 degrees C: alpha,alpha-trehalose (79 mM); phosphate (3.5 mM); d-glucose (40 mM); alphaGlc 1-P (4.1mM). A series of structural analogues of d-glucose were tested as glucosyl acceptors for the enzymatic reaction with alphaGlc 1-P, and robust activity with d-mannose (3%), 2-deoxy d-glucose (8%), 2-fluoro d-glucose (15%) and 2-keto-d-glucose (50%) was detected. Arsenate replaces, with 30% relative activity, phosphate in the conversion of alpha,alpha-trehalose, and vanadate strongly inhibits the enzyme activity (K(i) approximately 4 microM). PoTPase has a half-life (t(0.5)) of approximately 1 h at 30 degrees C in the absence of stabilizing compounds such as alpha,alpha-trehalose (300 mM; t(0.5)=11.5 h), glycerol (20%, w/v; t(0.5)=6.5h) or polyethylenglycol (PEG) 4000 (26%, w/v; t(0.5)=70 h). Covalent modification of PoTPase with activated derivatives of PEG 5000 increases the stability by up to 600-fold. Sucrose was converted to alpha,alpha-trehalose in approximately 60% yield using a coupled enzyme system composed of sucrose phosphorylase from Leuconostoc mesenteroides, glucose isomerase from Streptomyces murinus and the appropriately stabilized PoTPase.
从担子菌平菇中分离得到海藻糖磷酸化酶（PoTPase），通过约500倍的纯化，得率为44%。SDS-PAGE和凝胶过滤联合分析表明，PoTPase是一种分子量约为55 kDa的功能性单体。PoTPase催化α,α-海藻糖的磷酸解，生成α-D-葡萄糖1-磷酸（αGlc 1-P）和α-D-葡萄糖作为产物。PoTPase催化α,α-海藻糖磷酸解和合成的最适pH分别为6.8和6.2。在pH 6.8和30℃下测定了表观底物结合亲和力（K(m)）：α,α-海藻糖（79 mM）；磷酸盐（3.5 mM）；D-葡萄糖（40 mM）；αGlc 1-P（4.1 mM）。测试了一系列D-葡萄糖的结构类似物作为与αGlc 1-P进行酶促反应的葡萄糖基受体，检测到其对D-甘露糖（3%）、2-脱氧-D-葡萄糖（8%）、2-氟-D-葡萄糖（15%）和2-酮-D-葡萄糖（50%）具有较强的活性。在α,α-海藻糖转化过程中，砷酸盐以30%的相对活性替代磷酸盐，钒酸盐强烈抑制酶活性（K(i)约为4 μM）。在30℃下，在没有稳定化合物如α,α-海藻糖（300 mM；t(0.5)=11.5 h）、甘油（20%，w/v；t(0.5)=6.5 h）或聚乙二醇（PEG）4000（2,6%，w/v；t(0.5)=70 h）的情况下，PoTPase的半衰期（t(0.5)）约为1 h。用PEG 5000的活化衍生物对PoTPase进行共价修饰可使稳定性提高多达600倍。使用由肠系膜明串珠菌的蔗糖磷酸化酶、鼠链霉菌的葡萄糖异构酶和适当稳定化的PoTPase组成的偶联酶系统，蔗糖转化为α,α-海藻糖的产率约为60%。
