Marinomonas mediterranea MMB-1 transposon mutagenesis:: Isolation of a multipotent polyphenol oxidase mutant
Marinomonas mediterranea MMB-1 transposon mutagenesis:: Isolation of a multipotent polyphenol oxidase mutant
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DOI：
10.1128/jb.182.13.3754-3760.2000
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发表时间：
2000-07-01
影响因子：
3.2
通讯作者：
Sanchez-Amat, A
中科院分区：
文献类型：
--
作者：
Solano, F;Lucas-Elío, P;Sanchez-Amat, A
地中海海洋单胞菌是一种表达多功能多酚氧化酶（PPO）的产黑素海洋细菌，该多酚氧化酶能够氧化漆酶和酪氨酸酶的底物特征，以及产生经典的酪氨酸酶。本文提出了一种新的快速筛选漆酶活性的方法，以检测受此酶活性差异影响的突变体。首次将转座子诱变技术应用于M.通过使用负载在基于R6K的自杀递送载体中的不同的微型转座子，通过缀合可动员，来表达介体。通过使用编码卡那霉素或庆大霉素抗性的迷你Tn10衍生物获得了更高的插入频率。应用该方案后，获得了多功能PPO阴性突变体。通过使用抗生素抗性盒作为标记，克隆侧翼区。然后通过PCR扩增野生型基因，克隆并测序。这是第一次报道一个编码具有漆酶活性的原核酶的基因的克隆和测序。推导的氨基酸序列显示了其他蓝铜蛋白，包括真菌漆酶的特征铜结合位点。此外，它还显示了一些额外的铜结合位点，这可能与其多能酶促能力有关。
Marinomonas mediterranea is a melanogenic marine bacterium expressing a multifunctional polyphenol oxidase (PPO) able to oxidize substrates characteristic for laccases and tyrosinases, as well as produce a classical tyrosinase. A new and quick method has been developed for screening laccase activity in culture plates to detect mutants differentially affected in this PPO activity. Transposon mutagenesis has been applied for the first time to M. mediterranea by using different minitransposons loaded in R6K-based suicide delivery vectors mobilizable by conjugation. Higher frequencies of insertions were obtained by using mini-Tn10 derivatives encoding kanamycin or gentamycin resistance. After applying this protocol, a multifunctional PPO-negative mutant was obtained. By using the antibiotic resistance cassette as a marker, flanking regions were cloned. Then the wild-type gene was amplified by PCR and was cloned and sequenced. This is the first report on cloning and sequencing of a gene encoding a prokaryotic enzyme with laccase activity. The deduced amino acid sequence shows the characteristic copper binding sites of other blue copper proteins, including fungal laccases. In addition, it shows some extra copper-binding sites that might be related to its multipotent enzymatic capability.