Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Callinstr. 5, D-30167 Hannover, Germany.
Bioresour Technol. 2012 Mar;108:231-9. doi: 10.1016/j.biortech.2011.12.097. Epub 2011 Dec 24.
Valencene dioxygenase (ValOx) from the edible basidiomycete Pleurotus sapidus converted the sesquiterpene (+)-valencene to the valuable grapefruit flavour (+)-nootkatone and to nootkatols through intermediate hydroperoxides. Expression of the enzyme was carried out in the cytosol and periplasm of Escherichia coli. The heterologous production led to high yields of inclusion bodies. The poor yield of soluble recombinant protein was improved by various strategies including cold shock expression, chaperone co-expression, and employment of mutant E. coli strains. Up to 60 mg of the biologically active, soluble ValOx was produced by cold shock under control of the cspA promoter at 8 °C in the BL21(DE3)Star strain and co-expression of the E. coli trigger factor. The recombinant enzyme, purified using the N-terminal His tag, showed the catalytic properties of the wild-type enzyme, as was confirmed by the LC-MS analysis of hydroperoxide intermediates and GC-MS analysis of the volatile products.
来自可食用担子菌美味侧耳（Pleurotus sapidus）的 Valencene 双加氧酶（ValOx）将倍半萜（+）-Valencene转化为有价值的葡萄柚味（+）-诺卡酮和诺卡醇中间体过氧化物。该酶的表达在大肠杆菌的细胞质和周质中进行。异源生产导致包涵体产量很高。通过各种策略提高了可溶性重组蛋白的低产量，包括冷休克表达、伴侣蛋白共表达和使用突变型大肠杆菌菌株。在 BL21(DE3)Star 菌株中，在 cspA 启动子的控制下，在 8°C 下进行冷休克，并共表达大肠杆菌触发因子，可生产出高达 60 毫克具有生物活性的可溶性 ValOx。使用 N 端 His 标签纯化的重组酶表现出野生型酶的催化特性，这通过对过氧化物中间体的 LC-MS 分析和对挥发性产物的 GC-MS 分析得到了证实。
