Secretory Expression and Properties of Laccase from Ganoderma lucidum in Aspergillus niger
In the early stages, a high laccase-producing white rot fungus was identified as Ganoderma lucidum and the gene coding laccase was identified as lcc1 by secretory proteomic analysis and transcriptome sequencing. In this study, the genomic sequence named lcc1-D and cDNA sequences named lcc1-C of lcc1...
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The editorial department of Science and Technology of Food Industry
2023-07-01
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| Online Access: | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2022080103 |
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| author | Yu LIU Tao YU Yuxin ZHANG Hui ZHANG Tianqi LIU Jie LI |
| author_facet | Yu LIU Tao YU Yuxin ZHANG Hui ZHANG Tianqi LIU Jie LI |
| author_sort | Yu LIU |
| collection | DOAJ |
| description | In the early stages, a high laccase-producing white rot fungus was identified as Ganoderma lucidum and the gene coding laccase was identified as lcc1 by secretory proteomic analysis and transcriptome sequencing. In this study, the genomic sequence named lcc1-D and cDNA sequences named lcc1-C of lcc1 gene were amplified by PCR, and their expression vectors pSZH6R-lcc1-D and pSZH6R-lcc1-C were constructed. By Agrobacterium tumefaciens-mediated transformation of Aspergillus niger TH-2, homozygous transformants of Aspergillus niger with homologous recombination at the glaA gene locus were screened. After shake flask fermentation, the laccase amount and enzyme activity of supernatant of recombinant strain lcc1-C and lcc1-D were detected by Native-PAGE and enzyme activity assay, the highest enzyme activity were 431.94 U/L and 218.06 U/L respectively. Real-time PCR results showed that the laccase mRNA level in lcc1-C was 3.38-fold higher than that in lcc1-D. The optimum reaction temperature and pH of recombinant laccase were 60 ℃ and 3.5 respectively. In dye decolorization experiments, it was found that the recombinant laccase had a significant decolorization effect on malachite green, neutral red and methyl orange, an anthraquinone dye. In the presence of laccase mediator, the decolorization effect of laccase on dyes was enhanced, and the decolorization efficiency of neutral red was the highest, reaching 91.21% after 48 hours. In order to improve the folding of recombinant laccase, osmoregulators were added into the fermentation medium of recombinant strain lcc1-C. The results showed that the enzyme activity reached 1301.67 U/L when 0.4 mol/L TMAO was added, which increased 1.97-fold. When 0.5 mol/L proline was added, the enzyme activity reached 2037.22 U/L, which increased 3.64-fold. When 0.5 mol/L glycine was added, the enzyme activity reached 1434.03 U/L, which increased 2.27-fold. |
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| language | zho |
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| spelling | doaj.art-9e781aacd4744e319cff0cf6a89a905d2023-08-24T06:00:43ZzhoThe editorial department of Science and Technology of Food IndustryShipin gongye ke-ji1002-03062023-07-01441311912610.13386/j.issn1002-0306.20220801032022080103-13Secretory Expression and Properties of Laccase from Ganoderma lucidum in Aspergillus nigerYu LIU0Tao YU1Yuxin ZHANG2Hui ZHANG3Tianqi LIU4Jie LI5College of Life Sciences, Northeast Agricultural University, Harbin 150030, ChinaInner Mongolia Kunming Cigarette Co., Ltd., Hohhot 010000, ChinaCollege of Life Sciences, Northeast Agricultural University, Harbin 150030, ChinaCollege of Life Sciences, Northeast Agricultural University, Harbin 150030, ChinaCollege of Life Sciences, Northeast Agricultural University, Harbin 150030, ChinaCollege of Life Sciences, Northeast Agricultural University, Harbin 150030, ChinaIn the early stages, a high laccase-producing white rot fungus was identified as Ganoderma lucidum and the gene coding laccase was identified as lcc1 by secretory proteomic analysis and transcriptome sequencing. In this study, the genomic sequence named lcc1-D and cDNA sequences named lcc1-C of lcc1 gene were amplified by PCR, and their expression vectors pSZH6R-lcc1-D and pSZH6R-lcc1-C were constructed. By Agrobacterium tumefaciens-mediated transformation of Aspergillus niger TH-2, homozygous transformants of Aspergillus niger with homologous recombination at the glaA gene locus were screened. After shake flask fermentation, the laccase amount and enzyme activity of supernatant of recombinant strain lcc1-C and lcc1-D were detected by Native-PAGE and enzyme activity assay, the highest enzyme activity were 431.94 U/L and 218.06 U/L respectively. Real-time PCR results showed that the laccase mRNA level in lcc1-C was 3.38-fold higher than that in lcc1-D. The optimum reaction temperature and pH of recombinant laccase were 60 ℃ and 3.5 respectively. In dye decolorization experiments, it was found that the recombinant laccase had a significant decolorization effect on malachite green, neutral red and methyl orange, an anthraquinone dye. In the presence of laccase mediator, the decolorization effect of laccase on dyes was enhanced, and the decolorization efficiency of neutral red was the highest, reaching 91.21% after 48 hours. In order to improve the folding of recombinant laccase, osmoregulators were added into the fermentation medium of recombinant strain lcc1-C. The results showed that the enzyme activity reached 1301.67 U/L when 0.4 mol/L TMAO was added, which increased 1.97-fold. When 0.5 mol/L proline was added, the enzyme activity reached 2037.22 U/L, which increased 3.64-fold. When 0.5 mol/L glycine was added, the enzyme activity reached 1434.03 U/L, which increased 2.27-fold.http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2022080103laccaseaspergillus nigerheterologous expressionenzymatic propertiesdyes decolorizing |
| spellingShingle | Yu LIU Tao YU Yuxin ZHANG Hui ZHANG Tianqi LIU Jie LI Secretory Expression and Properties of Laccase from Ganoderma lucidum in Aspergillus niger Shipin gongye ke-ji laccase aspergillus niger heterologous expression enzymatic properties dyes decolorizing |
| title | Secretory Expression and Properties of Laccase from Ganoderma lucidum in Aspergillus niger |
| title_full | Secretory Expression and Properties of Laccase from Ganoderma lucidum in Aspergillus niger |
| title_fullStr | Secretory Expression and Properties of Laccase from Ganoderma lucidum in Aspergillus niger |
| title_full_unstemmed | Secretory Expression and Properties of Laccase from Ganoderma lucidum in Aspergillus niger |
| title_short | Secretory Expression and Properties of Laccase from Ganoderma lucidum in Aspergillus niger |
| title_sort | secretory expression and properties of laccase from ganoderma lucidum in aspergillus niger |
| topic | laccase aspergillus niger heterologous expression enzymatic properties dyes decolorizing |
| url | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2022080103 |
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