{"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}
引用次数: 11
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.
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.