{"title":"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","authors":"O. Pinar, Candan Tamerler Behar, A. Karataş","doi":"10.3906/BIY-1605-51","DOIUrl":null,"url":null,"abstract":"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.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":null,"pages":null},"PeriodicalIF":1.1000,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1605-51","citationCount":"11","resultStr":"{\"title\":\"Heterologous expression and characterization of a high redox potential laccase from Coriolopsis polyzona MUCL 38443\",\"authors\":\"O. Pinar, Candan Tamerler Behar, A. Karataş\",\"doi\":\"10.3906/BIY-1605-51\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"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.\",\"PeriodicalId\":23358,\"journal\":{\"name\":\"Turkish Journal of Biology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.1000,\"publicationDate\":\"2017-04-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.3906/BIY-1605-51\",\"citationCount\":\"11\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Turkish Journal of Biology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.3906/BIY-1605-51\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Turkish Journal of Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.3906/BIY-1605-51","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOLOGY","Score":null,"Total":0}
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。
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.
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.