Characterization and high-level expression of a metagenome-derived alkaline pectate lyase in recombinant Escherichia coli
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- 作者：Huilin Wang ; Xiaoman Li ; Yanhe Ma ; Jiangning Song
- 关键词：Metagenomic-derived ; Alkaline pectate lyase ; Recombinant Escherichia coli ; Characterization ; Overproduction
- 刊名：Process Biochemistry
- 出版年：January, 2014
- 年：2014
- 卷：49
- 期：1
- 页码：69-76
- 全文大小：2158 K
文摘
Alkaline pectate lyases (PLs) play an important role in mild and eco-friendly bioscouring pretreatment processes in the textile industry. However, to date, only a few PLs can be applied in industrial-scale production, and many of them exhibit high production cost, low activity, and/or do not meet the treatment requirements. In this study, an alkaline PL gene was cloned from the metagenomic DNA of alkaline environment soils. The gene pelB consisted of 1263 nucleotides and encoded a mature protein (PelB) of 399 amino acids, which was expressed in Escherichia coli. The maximum catalytic activity of the enzyme exhibited a bimodal distribution at pH 8.1 and 9.8 and an optimal temperature of 55 掳C. The Km and Vmax values of PelB were 1.78 g/L and 1084.8 渭mol/(L min) at 45 掳C, respectively. Substrate specificity analysis demonstrated the high cleavage capability of PelB on a broad range of substrates of natural methylated pectin. Based on the degradation products, PelB was considered to be an endo-acting lyase. Using high-cell-density cultivation in 7-L bioreactor, the highest PL activity (1816.2 U/mL) was achieved. Thus, the recombinant PelB, with promising properties for use in bioscouring in the textile pretreatment process, should be a potential enzyme for industrial applications.