Fraser P D, Shimada H, Misawa N
Central Laboratories for Key Technology, Kirin Brewery Co., Ltd, Kanagawa, Japan.
Eur J Biochem. 1998 Mar 1;252(2):229-36. doi: 10.1046/j.1432-1327.1998.2520229.x.
An in vitro assay procedure for the carotenoid (beta-ionone ring) 3,3'-hydroxylase and 4,4'-oxygenase has been developed that enables efficient conversion of non-radiolabeled carotenoid substrates added directly into aqueous solution. The following enzymic conversions were demonstrated and apparent kinetic constants (Vmax, Km, and specificity constants) obtained: (a) 3,3'-hydroxylase (from Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) converted phoenicoxanthin (adonirubin) to astaxanthin, 3-hydroxyechinenone to 4-ketozeaxanthin (adonixanthin), 3'-hydroxyechinenone to 4-ketozeaxanthin, as well as echinenone to 4-ketozeaxanthin via 3- and 3'-hydroxyechinenone; (b) 4,4'-Oxygenase (from A. aurantiacum, Alcaligenes sp. strain PC-1 and Haematococcus pluvialis) converted 4-ketozeaxanthin to astaxanthin, 3-hydroxyechinenone to phoenicoxanthin, 3'-hydroxyechinenone to phoenicoxanthin, and echinenone to canthaxanthin. Determination of substrate specifities allowed assessment of biosynthetic routes to astaxanthin formation and demonstrated that pathways via mono-hydroxylated and ketolated products are enzymically feasible.
已开发出一种用于类胡萝卜素（β-紫罗酮环）3,3'-羟化酶和4,4'-加氧酶的体外测定方法，该方法能够有效地将直接添加到水溶液中的非放射性标记类胡萝卜素底物进行转化。已证明了以下酶促转化反应并获得了表观动力学常数（Vmax、Km和特异性常数）：（a）3,3'-羟化酶（来自橙色土壤杆菌和产碱杆菌属PC-1菌株）将凤仙花黄素（虾红素）转化为虾青素，3-羟基海胆酮转化为4-酮玉米黄质（虾黄质），3'-羟基海胆酮转化为4-酮玉米黄质，以及海胆酮通过3-和3'-羟基海胆酮转化为4-酮玉米黄质；（b）4,4'-加氧酶（来自橙色土壤杆菌、产碱杆菌属PC-1菌株和雨生红球藻）将4-酮玉米黄质转化为虾青素，3-羟基海胆酮转化为凤仙花黄素，3'-羟基海胆酮转化为凤仙花黄素，以及海胆酮转化为角黄素。底物特异性的测定允许评估虾青素形成的生物合成途径，并证明通过单羟基化和酮化产物的途径在酶学上是可行的。
