快原子轰击质谱技术结合酶学方法鉴定黑曲霉1,6-缩水-β-D-吡喃葡萄糖激酶的诱导合成

Characterization of Inductive Synthesis of Levoglucosan Kinase by a Combined Strategy of Enzymological and Fast Atom Bombardment Mass Spectrometric Analysis

1 ,6 缩水 β D 吡喃葡萄糖是纤维素类物质热解的主要产物 ,黑曲霉突变株CBX 2 0 9能较好地利用该糖作为唯一的碳源和能源生长并产生有用的代谢产物柠檬酸 ,其效率与利用葡萄糖大致相当。利用葡萄糖氧化酶和辣根过氧化物酶复合系统测定证明该菌株不存在 1,6 缩水 β D 吡喃葡萄糖水解酶。采用快原子轰击质谱技术结合6 磷酸葡萄糖脱氢酶系统进行测定 ,结果表明经 (NH4) 2 SO4沉淀或阴离子交换层析处理后的无细胞提取液在加入ATP和Mg2 +的条件下能直接催化 1,6 缩水 β D 吡喃葡萄糖合成 6 磷酸葡萄糖 ,证明黑曲霉突变株中存在一个新酶 ,即 1,6 缩水 β D 吡喃葡萄糖激酶。

Levoglucosan is the main product derived from pyrolysis of cellulose. A mutant Aspergillus niger CBX-209 could grow on levoglucosan well fermenting it into citric acid with a yield comparable to that on glucose. Levoglucosan hydrolase was absent by measuring glucose formation with the glucose oxidase and peroxidase coupling system. Cell extracts were partly purified by ammonium sulfate fractionation and ion-exchange chromatograph. Direct formation of glucose 6-phosphate from levoglucosan in the presence of ATP and MgCl 2 was observed when it was reacted with partly purified enzyme by a combined strategy of enzymological and fast atom bombardment mass spectrometric analysis. These data showed that the mutant used a novel enzyme, levoglucosan kinase, to convert levoglucosan into glucose 6-phosphate. Levoglucosan kinase was an inductive enzyme.