Pfeiffer Martin, Wildberger Patricia, Nidetzky Bernd
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, A-8010 Graz, Austria.
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, A-8010 Graz, Austria ; ACIB - Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria.
J Mol Catal B Enzym. 2014 Dec;110:39-46. doi: 10.1016/j.molcatb.2014.09.004.
Phosphomonoester hydrolases (phosphatases; EC 3.1.3.) often exhibit extremely relaxed substrate specificity which limits their application to substrate-selective biotransformations. In search of a phosphatase catalyst specific for hydrolyzing α-d-glucose 1-phosphate (αGlc 1-), we selected haloacid dehalogenase-like phosphatase 4 (HAD4) from and obtained highly active recombinant enzyme through a fusion protein (Z_HAD4) that contained Z, a strongly positively charged three α-helical bundle module, at its N-terminus. Highly pure Z_HAD4 was prepared directly from cell extract using capture and polishing combined in a single step of cation exchange chromatography. Kinetic studies showed Z_HAD4 to exhibit 565-fold preference for hydrolyzing αGlc 1- (/ = 1.87 ± 0.03 mM s; 37 °C, pH 7.0) as compared to d-glucose 6-phosphate (Glc 6-). Also among other sugar phosphates, αGlc 1- was clearly preferred. Using different mixtures of αGlc 1- and Glc 6- (e.g. 180 mM each) as the substrate, Z_HAD4 could be used to selectively convert the αGlc 1- present, leaving back all of the Glc 6- for recovery. Z_HAD4 was immobilized conveniently using direct loading of cell extract on sulfonic acid group-containing porous carriers, yielding a recyclable heterogeneous biocatalyst that was nearly as effective as the soluble enzyme, probably because protein attachment to the anionic surface occurred in a preferred orientation via the cationic Z module. Selective removal of αGlc 1- from sugar phosphate preparations could be an interesting application of Z_HAD4 for which readily available broad-spectrum phosphatases are unsuitable.
磷酸单酯水解酶（磷酸酶；EC 3.1.3.）通常表现出极其宽泛的底物特异性，这限制了它们在底物选择性生物转化中的应用。为了寻找一种特异性水解α-D-葡萄糖1-磷酸（αGlc 1-）的磷酸酶催化剂，我们从[具体来源未给出]中选择了卤代酸脱卤酶样磷酸酶4（HAD4），并通过在其N端包含带强正电荷的三α-螺旋束模块Z的融合蛋白（Z_HAD4）获得了高活性的重组酶。使用阳离子交换色谱一步结合捕获和纯化，直接从[具体来源未给出]细胞提取物中制备了高纯度的Z_HAD4。动力学研究表明，与6-磷酸-D-葡萄糖（Glc 6-）相比，Z_HAD4对水解αGlc 1-表现出565倍的偏好（/ = 1.87 ± 0.03 mM s；37°C，pH 7.0）。在其他糖磷酸酯中，αGlc 1-也明显更受青睐。使用αGlc 1-和Glc 6-的不同混合物（例如各180 mM）作为底物，Z_HAD4可用于选择性转化存在的αGlc 1-，而将所有的Glc 6-留作回收。通过将[具体来源未给出]细胞提取物直接负载到含磺酸基团的多孔载体上，方便地固定了Z_HAD4，得到了一种可循环使用的非均相生物催化剂，其效果几乎与可溶性酶相同，这可能是因为蛋白质通过阳离子Z模块以优选的方向附着在阴离子表面。从糖磷酸酯制剂中选择性去除αGlc 1-可能是Z_HAD4的一个有趣应用，而现有的广谱磷酸酶并不适合此应用。
