灵芝漆酶在黑曲霉中的分泌表达及性质研究

刘雨; 于涛; 张宇鑫; 张会; 刘天奇; 李杰

doi:10.13386/j.issn1002-0306.2022080103

灵芝漆酶在黑曲霉中的分泌表达及性质研究

doi: 10.13386/j.issn1002-0306.2022080103

刘雨^1,,
于涛²,
张宇鑫¹,
张会¹,
刘天奇¹,
李杰^1, ,

1.
东北农业大学生命科学学院，黑龙江哈尔滨 150030
2.
内蒙古昆明卷烟有限责任公司，内蒙古呼和浩特 010000

基金项目: 国产雪茄烟生物发酵技术研究（202115010534-JS-178）。

详细信息

作者简介:
刘雨（1997−），女，硕士研究生，研究方向：丝状真菌表达系统，E-mail：s200901077@neau.edu.cn

通讯作者:
李杰（1972−），男，博士，教授，研究方向：丝状真菌表达系统，E-mail：lijie_neau@126.com

中图分类号: Q814.4
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- 文章访问数: 33
- HTML全文浏览量: 17
- PDF下载量: 4
- 被引次数: 0
出版历程
- 收稿日期: 2022-08-10
- 网络出版日期: 2023-05-21
- 刊出日期: 2023-07-01

Secretory Expression and Properties of Laccase from Ganoderma lucidum in Aspergillus niger

Yu LIU^1
,,
Tao YU²,
Yuxin ZHANG¹,
Hui ZHANG¹,
Tianqi LIU¹,
Jie LI^{1
, ,}

1.
College of Life Sciences, Northeast Agricultural University, Harbin 150030, China
2.
Inner Mongolia Kunming Cigarette Co., Ltd., Hohhot 010000, China

摘要

摘要: 前期通过分泌蛋白质谱分析和转录组测序，确定了一株高产漆酶的白腐菌为灵芝，其分泌漆酶的编码基因为lcc1。本研究PCR扩增lcc1基因的基因组序列（lcc1-D）和cDNA序列（lcc1-C），分别构建表达载体（pSZH6R-lcc1-D、pSZH6R-lcc1-C）。通过农杆菌介导法转化黑曲霉TH-2，筛选出在糖化酶基因glaA位点发生同源重组的纯合黑曲霉转化子。摇瓶发酵后取发酵液上清进行Native-PAGE检测和酶活测定，结果表明，重组菌株lcc1-C的漆酶表达量和酶活力高于lcc1-D，酶活力最高值分别为431.94 U/L和218.06 U/L。Real-time PCR结果表明，lcc1-C中的漆酶mRNA水平是lcc1-D的3.38倍。对重组漆酶进行酶学性质分析，发现其最适反应温度为60 ℃、最适pH为3.5。在染料脱色实验中发现重组漆酶对孔雀石绿、中性红、甲基橙具有明显脱色作用，介体存在时能增强漆酶对染料的脱色效果，对中性红脱色效率最强，48 h脱色率可达到91.21%。为改善重组漆酶的折叠，向重组菌株lcc1-C的发酵培养基中添加渗透调节剂，结果显示，在添加0.4 mol/L TMAO时酶活力达到1301.67 U/L，提高了1.97倍；在添加0.5 mol/L脯氨酸时酶活力达到2037.22 U/L，提高了3.64倍；在添加0.5 mol/L甘氨酸时酶活力达到1434.03 U/L，酶活力提高了2.27倍。
- 漆酶 /
- 黑曲霉 /
- 异源表达 /
- 酶学性质 /
- 染料脱色
Abstract: 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.
- laccase /
- Aspergillus niger /
- heterologous expression /
- enzymatic properties /
- dyes decolorizing

HTML全文

图 1 重组表达载体pSZH6R-lcc1-D、pSZH6R-lcc1-C构建

Figure 1. Construction of recombinant expression vectors pSZH6R-lcc1-D and pSZH6R-lcc1-C

下载: 全尺寸图片幻灯片

图 2 目的基因扩增结果

Figure 2. Cloning of target gene by PCR

注：M：Marker；1：lcc1-D目的条带；2：lcc1-C目的条带。

下载: 全尺寸图片幻灯片

图 3 重组转化子PCR酶切鉴定结果

Figure 3. Identification results of PCR digestion of recombinant transformants

注：M：Marker；0：水；0⁻：出发菌株TH-2；A：pSZH6R-lcc1-D质粒；B：pSZH6R-lcc1-C质粒；1~2：转化子PCR酶切结果。

下载: 全尺寸图片幻灯片

图 4 重组菌株中漆酶Native-PAGE电泳分析

Figure 4. Analysis of laccase in recombinant strains by Native-PAGE electrophoresis

注：M：Marker；0：TH-2；A：4~11 d重组菌株lcc1-D发酵液；B：4~11 d重组菌株lcc1-C发酵液。

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图 5 重组菌株中漆酶酶活力测定结果

Figure 5. Results of laccase activity in recombinant strains

下载: 全尺寸图片幻灯片

图 6 重组菌株中漆酶基因转录水平的荧光定量PCR检测结果

Figure 6. Real-time PCR results of laccase gene transcription level in recombinant strains

注：****表示在0.0001水平上菌株之间具有显著差异（n=3）。

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图 7 不同温度下重组漆酶的酶活性

Figure 7. Enzymatic activity of recombinant laccase at different temperatures

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图 8 不同pH下重组漆酶的酶活性

Figure 8. Enzymatic activity of recombinant laccase at different pH

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图 9 重组漆酶对4种染料的脱色效果

Figure 9. Decolorization of 4 dyes by laccase in recombinant strains

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图 10 固体发酵培养基平板上重组菌株的形态

Figure 10. Morphology of recombinant strains on solid fermentation medium plates

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图 11 3种渗透调节剂对重组菌株lcc1-C产漆酶的影响

Figure 11. Effects of three osmotic regulators on laccase production by recombinant strain lcc1-C

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表 1 PCR引物序列

Table 1. PCR primer sequence

引物名称	引物序列（5'-3'）
P1：lcc1-sense	TCTAGAATCGGGCCCACGACCGAC
P2：lcc1-antisense	AAGCTTACTGGTCATCGGGAGAGAG
P3：YXgla.3p	CGCTATAGGCTGGTTCTCC
P4：HYXgla.4p	TCGGCTATTATTGAACTGGG
P5：lcc1qPCR-sense	CAGCGTTACTCGTTCGTGCT
P6：lcc1qPCR-antisense	GAGAGAGCGCGTTGTACGTC
P7：actA-RT-sense	CCACGAGACCACCTTCAACTCCA
P8：actA-RT-antisense	CCACCGATCCAGACGGAGTACTTGC

下载: 导出CSV

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