Paukner Regina, Staudigl Petra, Choosri Withu, Haltrich Dietmar, Leitner Christian
Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
Department of Food Technology, Ramkhamhaeng University, Bangkok, Thailand.
Protein Expr Purif. 2015 Apr;108:73-79. doi: 10.1016/j.pep.2014.12.010. Epub 2014 Dec 24.
A gene encoding a galactose oxidase (GalOx) was isolated from Fusarium sambucinum cultures and overexpressed in Escherichia coli yielding 4.4mg enzyme per L of growth culture with a specific activity of 159Umg(-1). By adding a C-terminal His-tag the enzyme could be easily purified with a single affinity chromatography step with high recovery rate (90%). The enzyme showed a single band on SDS-PAGE with an apparent molecular mass of 68.5kDa. The pH optimum for the oxidation of galactose was in the range of pH 6-7.5. Optimum temperature for the enzyme activity was 35°C, with a half-life of 11.2min, 5.3min, and 2.7min for incubation at 40°C, 50°C, and 60°C, respectively. From all tested substrates, the highest relative activity was found for 1-methyl-β-galactopyranoside (226Umg(-1)) and the highest catalytic efficiency (kcat/Km) for melibiose (2700mM(-1)s(-1)). The enzyme was highly specific for molecular oxygen as an electron acceptor, and showed no appreciable activity with a range of alternative acceptors investigated. Different chemicals were tested for their effect on GalOx activity. The activity was significantly reduced by EDTA, NaN3, and KCN.
从接骨木镰刀菌培养物中分离出一个编码半乳糖氧化酶（GalOx）的基因，并在大肠杆菌中进行过表达，每升生长培养物可产生4.4毫克该酶，比活性为159 U mg⁻¹。通过添加C末端His标签，该酶可通过一步亲和层析轻松纯化，回收率很高（90%）。该酶在SDS-PAGE上显示出一条带，表观分子量为68.5 kDa。半乳糖氧化的最适pH范围为6 - 7.5。酶活性的最适温度为35°C，在40°C、50°C和60°C孵育时的半衰期分别为11.2分钟、5.3分钟和2.7分钟。在所有测试的底物中，对1-甲基-β-吡喃半乳糖苷的相对活性最高（226 U mg⁻¹），对蜜二糖的催化效率（kcat/Km）最高（2700 mM⁻¹ s⁻¹）。该酶对作为电子受体的分子氧具有高度特异性，对一系列研究的替代受体没有明显活性。测试了不同化学物质对GalOx活性的影响。EDTA、NaN₃和KCN显著降低了该活性。
