Cloning of a phenol oxidase gene from Acremonium murorum and its expression in Aspergillus awamori
Cloning of a phenol oxidase gene from Acremonium murorum and its expression in Aspergillus awamori
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DOI：
10.1128/aem.67.6.2610-2616.2001
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发表时间：
2001-06-01
影响因子：
4.4
通讯作者：
Verrips, CT
中科院分区：
文献类型：
--
作者：
Gouka, RJ;van der Heiden, M;Verrips, CT
真菌多铜氧化酶在温和的条件下进行反应，具有潜在的工业应用前景。我们从真菌小顶藻变种中分离到一种酚氧化酶。这种酶能够使植物生色团(如花青素)脱色，这种酶对洗衣产品很有兴趣，因为它对生色团具有广泛的专一性。我们在酿酒酵母中表达了一个A，Murorum cDNA文库，随后根据它们对植物生色团脱色的能力鉴定了产酶酵母菌落。该序列含有1,806个核苷酸的开放阅读框，编码602个氨基酸的酶。通过疏水作用层析从发酵液中分离纯化得到的酚氧化酶在碱性pH(对丁香肼的最适作用pH为9)和高温(最适温度为60℃)下具有较强的活性，在碱性条件下至少在60℃下保持稳定。这些特性以及摇瓶中每升0.6克酚氧化酶的高产量水平(与糖淀粉酶蛋白的摩尔水平相同)，使该酶适合在碱性条件下发生的过程中使用，如洗衣。
Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins), This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A, murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography, The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60 degreesC) and is fully stable for at least I h at 60 degreesC under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning.