黑曲霉低聚葡萄糖氧化酶的分子克隆与生化特征

低聚葡萄糖氧化酶能够氧化低聚葡萄糖末端残基生成相应的低聚糖酸,在低聚糖酸的制备、食品、化工等方面具有潜在的应用价值。从黑曲霉基因组中发现1个疑似编码低聚葡萄糖氧化酶开放读框,共编码473个氨基酸序列,包含2个保守结构域(FAD结合域和黄素结构域)和4个活性位点(His80、Cys141、Asp377和Try428);在毕赤酵母中表达的重组酶蛋白大小为61 kDa,比酶活0.33 U/mg。重组酶的最适作用温度和pH值分别为75℃和9.0;在55～85℃和pH 5.0～9.0范围内孵育4 h,仍能保持70%以上的酶活;Mn²⁺和Fe³⁺分别对酶活具有最强的激活和抑制作用。该酶的K_m和V_max为0.48 mmol/L和2.22μmol/(L·min),对麦芽三糖、麦芽四糖和麦芽五糖具有相对较高酶活。

Molecular cloning and characterization of gluco-oligosaccharide oxidase from Aspergillus niger

The gluco-oligosaccharide oxidases oxidize the reducing end of glycosyl residues linked by alpha-or beta-1,4 bonds and glucose.Due to their significant application in aldonic acids production,these enzymes have high values in feed,food and chemical industries.A novel potential gluco-oligosaccharide oxidase gene from Aspergillus niger was successfully cloned into and heterologously expressed by Pichia pastoris GS115 followed by enzymatic properties analysis.The gene encoded 473 amino acids,containing 2 conserved domains(flavin adenine dinucleotide(FAD)binding domain and flavin structure domain)and 4 active sites(His80,Cys141,Asp377,and Try428).The mature protein revealed a molecular weight of 61 kDa with specific activity of 0.33 U/mg.The optimum temperature and pH of the recombinant enzyme was 75℃ and 9.0,respectively.After 4 hours of incubation at 55-85℃,pH 5.0-9.0,more than 70% of the enzyme activity was retained.The ion Mn^2+ revealed strong activation on the enzyme activity,and Fe^3+ exhibited inhibition.The Km and Vmax on lactose were 0.48μmol/L and 2.22μmol/(L·min),respectively.In addition,the enzyme has highest oxidation activity on maltotetraose followed by maltotriose and maltopentose.