| 冯怡畅,孔心茹,江欣,李昱龙.地衣芽孢杆菌耐碱纤维素酶的挖掘及其蛋白结构分析[J].农业环境科学学报,2026,45(3):777-785. |
| 地衣芽孢杆菌耐碱纤维素酶的挖掘及其蛋白结构分析 |
| Identification and structural analysis of an alkali-tolerant cellulase from Bacillus licheniformis |
| 投稿时间：2025-04-02 |
| DOI：10.11654/jaes.2025-0321 |
| 中文关键词: 地衣芽孢杆菌 蛋白结构 纤维素酶 酶学性质 GH9家族 |
| 英文关键词: Bacillus licheniformis protein structure cellulase enzymatic property GH9 family |
| 基金项目:宁夏回族自治区重点研发计划项目（2023BCF01023）；宁夏大学研究生创新项目（CXXM2025057） |
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| 中文摘要: |
| 为寻找高效强力的木质纤维素降解酶，本研究从农业秸秆堆肥样本中分离获得一株产纤维素酶的地衣芽孢杆菌（Bacilluslicheniformis）YC36。通过同源克隆技术获得其纤维素酶基因，构建pET-32a（+）重组质粒，并在大肠杆菌BL21（DE3）中实现异源表达。采用亲和层析法纯化重组蛋白，并对其酶学特性进行系统分析，同时结合生物信息学方法探讨其耐碱机制。结果表明：YC36重组纤维素酶分子量为70.4 kDa。酶学性质分析显示，该酶的最适反应条件为pH 9.0和55℃，在pH 5.0~9.0和温度50~65℃范围内时，酶活性保持在80%以上。热稳定性测试表明，在55℃条件下孵育60 min，酶活性仍能保持85%。在离子效应方面，Mg2+可显著增强酶活性，而Cu2+表现出较强的抑制作用。生物信息学分析发现，YC36具有GH9-CBM3结构域、高疏水性内核和碱性活性位点，这些特点赋予其优异的耐热性和较广的pH适应性。本研究揭示了YC36的GH9纤维素酶在高温碱性环境中的功能特性，通过其GH9-CBM3结构域、高疏水性内核及碱性活性位点等关键结构特征，阐明了该酶耐热性和pH适应性的分子机制。 |
| 英文摘要: |
| In search of efficient and powerful lignocellulose-degrading enzymes, a cellulase-producing Bacillus licheniformis strain YC36 was isolated from agricultural straw compost samples. The cellulase gene was cloned via homologous cloning techniques, inserted into the pET-32a（+）expression vector, and heterologously expressed in Escherichia coli BL21（DE3）. The recombinant protein was purified using affinity chromatography, and its enzymatic properties were systematically characterized. Bioinformatic approaches were also employed to elucidate its alkali tolerance mechanism. The results revealed that the molecular weight of the recombinant cellulase from YC36 was 70.4 kDa. Enzymatic assays demonstrated that the optimal reaction conditions were pH 9.0 and 55 ℃. The enzyme retained over 80% of its activity within a pH range of 5.0-9.0 and a temperature range of 50-65 ℃. Thermostability analysis showed that the enzyme maintained 85% activity after incubation at 55 ℃ for 60 minutes. Regarding ionic effects, Mg2+ significantly enhanced enzymatic activity, while Cu2+ exerted a strong inhibitory effect. Bioinformatic analysis indicated that YC36 cellulase possesses a GH9-CBM3 domain structure, a highly hydrophobic core, and alkaline-active site residues, which collectively confer excellent thermostability and broad pH adaptability. The study investigated the functional characteristics of YC-36′s GH9 cellulase under thermophilic and alkaline conditions. Through analysis of its critical structural features including the GH9-CBM3 domain, highly hydrophobic core, and alkaline-active site, we elucidated the molecular mechanisms underlying its thermostability and pH adaptability. These findings establish a theoretical foundation for developing industrial enzyme preparations with superior environmental adaptability while providing valuable genetic resources for enzymatic engineering applications. |
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