多带毛桑MUCL 38443高氧化还原电位漆酶的异源表达及特性研究

IF 1.1 4区 生物学 Q3 BIOLOGY Turkish Journal of Biology Pub Date : 2017-04-01 DOI:10.3906/BIY-1605-51
O. Pinar, Candan Tamerler Behar, A. Karataş
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引用次数: 11

摘要

本研究从多带Coriolopsis polyzona MUCL 38443菌株中分离到一种新的漆酶基因Cplcc1及其cDNA,并对其进行了鉴定。Cplcc1基因由一个1563 bp的开放阅读框组成,编码520个氨基酸的蛋白质和20个残基的信号肽。Cplcc1基因全长2106 bp,包含10个内含子和5个潜在的n -糖基化位点。此外,分离的Cplcc1全长cDNA在毕赤酵母中成功表达。在1.5%甲醇、0.8 mM CuSO4和0.6% l -丙氨酸的条件下,该菌株的最高表达活性为800 U L-1。重组漆酶经部分纯化,分子量约为54 kDa。2,2-氮唑-(3-乙基苯并噻唑-6-磺酸)(ABTS)的氧化活性在pH 3.0时达到最大。最佳温度为70℃。在30℃条件下,酶稳定时间超过1周,半衰期大于8 h。重组漆酶的Km、Vmax、kcat和kcat Km-1分别为0.137 mM、288.6 μmol min-1 L-1、5.73 × 105 min-1和4.18 × 106 min-1 mM-1。叠氮化钠、l -半胱氨酸和SDS是常见的抑制剂。
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Heterologous expression and characterization of a high redox potential laccase from Coriolopsis polyzona MUCL 38443
In this study, a novel laccase gene, named as Cplcc1, and its corresponding cDNA were isolated and characterized from the Coriolopsis polyzona MUCL 38443 strain. The Cplcc1 gene consists of a 1563-bp open reading frame encoding a protein of 520 amino acids with a 20-residue putative signal peptide. The size of the Cplcc1 gene is 2106 bp and it contains ten introns and five potential N-glycosylation sites. Additionally, the isolated full-length Cplcc1 cDNA was successfully expressed in Pichia pastoris. The heterologous expression conditions were also optimized and the highest activity value increased to 800 U L-1 with 1.5% methanol, 0.8 mM CuSO4, and 0.6% L-alanine supplementation. The recombinant laccase was partially purified and the molecular weight was found as approximately 54 kDa. The maximum oxidation activity was observed for 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at pH 3.0. The optimal temperature was found as 70 °C. On the other hand, at 30 °C, the enzyme was stable for more than a week and its half-life was longer than 8 h. The Km, Vmax, kcat, and kcat Km-1 values of the recombinant laccase were identified as 0.137 mM, 288.6 μmol min-1 L-1, 5.73 x 105 min-1, and 4.18 x 106 min-1 mM-1, respectively. Sodium azide, L-cysteine, and SDS were found as usual inhibitors.
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来源期刊
CiteScore
4.60
自引率
0.00%
发文量
20
审稿时长
6-12 weeks
期刊介绍: The Turkish Journal of Biology is published electronically 6 times a year by the Scientific and Technological Research Council of Turkey (TÜBİTAK) and accepts English-language manuscripts concerning all kinds of biological processes including biochemistry and biosynthesis, physiology and metabolism, molecular genetics, molecular biology, genomics, proteomics, molecular farming, biotechnology/genetic transformation, nanobiotechnology, bioinformatics and systems biology, cell and developmental biology, stem cell biology, and reproductive biology. Contribution is open to researchers of all nationalities.
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