基于易错PCR的<i>β</i>-甘露聚糖酶体外分子定向进化研究

胡海艳; 甘祥武; 黄秀敏; 叶俊豪; 谢万勇

doi:10.12187/2020.04.002

CN 41-1437/TS ISSN 2096-1553

基于易错PCR的β-甘露聚糖酶体外分子定向进化研究

胡海艳 , 甘祥武 , 黄秀敏 , 叶俊豪 , 谢万勇

胡海艳, 甘祥武, 黄秀敏, 等. 基于易错PCR的β-甘露聚糖酶体外分子定向进化研究[J]. 轻工学报, 2020, 35(4): 8-15. doi: 10.12187/2020.04.002

引用本文: 胡海艳, 甘祥武, 黄秀敏, 等. 基于易错PCR的β-甘露聚糖酶体外分子定向进化研究[J]. 轻工学报, 2020, 35(4): 8-15. doi: 10.12187/2020.04.002

HU Haiyan, GAN Xiangwu, HUANG Xiumin, et al. Study on directed evolution of β-mannanase in vitro by error-prone PCR[J]. Journal of Light Industry, 2020, 35(4): 8-15. doi: 10.12187/2020.04.002

Citation: HU Haiyan, GAN Xiangwu, HUANG Xiumin, et al. Study on directed evolution of β-mannanase in vitro by error-prone PCR[J]. Journal of Light Industry, 2020, 35(4): 8-15. doi: 10.12187/2020.04.002

基于易错PCR的β-甘露聚糖酶体外分子定向进化研究

广州市微生物研究所, 广东广州 510663

作者简介: 胡海艳(1985-),女,湖南省株洲市人,广州市微生物研究所工程师,主要研究方向为微生物代谢产品利用与微生物防控.;

基金项目: 广州市科技计划项目（201710010154）
中图分类号: TS201.3;Q789

Study on directed evolution of β-mannanase in vitro by error-prone PCR

Guangzhou Institute of Microbiology, Guangzhou, 510663, China
Received Date: 2020-05-21

CLC number: TS201.3;Q789

摘要: 采用易错PCR方法，对β-甘露聚糖酶进行体外分子定向进化，通过调整dNTP比例和加入不同浓度的Mn²⁺，向β-甘露聚糖酶中随机引入突变，构建突变文库，并对筛选得到的突变菌株的突变酶酶学性质进行研究.结果表明：在突变文库的构建过程中，当Mn²⁺浓度为0.01 mmol/L时，阳性转化率最高，达到80%，氨基酸的突变个数为2.0个，符合易错PCR突变文库的构建原则；经过两轮易错PCR构建的突变文库库容量约为4000，从中筛选出的突变毕赤酵母菌X-33/pGAPZαA-GwMan26A-27的突变酶GwMan26A-27具有耐酸、耐高温且酶活力较高的特性；与突变前相比，GwMan26A-27具有较宽的酶促反应温度范围和适宜pH值范围，热稳定性和pH稳定性均有较大提升；在胃蛋白酶液和胰蛋白酶液中水浴2 h，GwMan26A-27的消化酶稳定性较突变前也均有所提升.
- β-甘露聚糖酶 /
- 易错PCR /
- 定向进化
Abstract: Error-prone PCR was used to directed evolution β-mannanase in vitro, random introduction of mutations into β-mannanase by adjusting dNTP ratio and setting Mn²⁺ of different concentrations, a mutant library was constructed, and the enzymatic properties of the mutant enzymes were studied. The results showed that during the construction of the mutation library, the positive conversion rate could reach the highest 80% and the number of amino acid mutations was 2.0 with the concentration of Mn²⁺ 0.01 mmol/L, which was in accordance with the general principles of error-prone PCR mutation libraries; after two rounds of error-prone PCR, a storage capacity of 4000 was constructed, the mutant enzymes GwMan26A-27 of mutant Pichia pastoris X-33/pGAPZαA-GwMan26A-27 with acid resistance, high temperature resistance and high enzyme activity was finally selected; compared with before mutation, GwMan26A-27 has a wider enzymatic reaction temperature before the mutation and a suitable pH range, the thermal stability and pH stability also had been greatly improved; in the pepsin solution and trypsin solution for water bath 2 h, the digestive enzymes stability of GwMan26A-27 had also been improved.
- β-mannanase /
- error-prone PCR /
- directed evolution
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沈阳化工大学材料科学与工程学院沈阳 110142

胡海艳, 甘祥武, 黄秀敏, 等. 基于易错PCR的β-甘露聚糖酶体外分子定向进化研究[J]. 轻工学报, 2020, 35(4): 8-15. doi: 10.12187/2020.04.002

引用本文: 胡海艳, 甘祥武, 黄秀敏, 等. 基于易错PCR的β-甘露聚糖酶体外分子定向进化研究[J]. 轻工学报, 2020, 35(4): 8-15. doi: 10.12187/2020.04.002

HU Haiyan, GAN Xiangwu, HUANG Xiumin, et al. Study on directed evolution of β-mannanase in vitro by error-prone PCR[J]. Journal of Light Industry, 2020, 35(4): 8-15. doi: 10.12187/2020.04.002

基于易错PCR的β-甘露聚糖酶体外分子定向进化研究

作者简介:胡海艳(1985-),女,湖南省株洲市人,广州市微生物研究所工程师,主要研究方向为微生物代谢产品利用与微生物防控.

广州市微生物研究所, 广东广州 510663

收稿日期: 2020-05-21

基金项目: 广州市科技计划项目（201710010154）

关键词:

摘要: 采用易错PCR方法，对β-甘露聚糖酶进行体外分子定向进化，通过调整dNTP比例和加入不同浓度的Mn²⁺，向β-甘露聚糖酶中随机引入突变，构建突变文库，并对筛选得到的突变菌株的突变酶酶学性质进行研究.结果表明：在突变文库的构建过程中，当Mn²⁺浓度为0.01 mmol/L时，阳性转化率最高，达到80%，氨基酸的突变个数为2.0个，符合易错PCR突变文库的构建原则；经过两轮易错PCR构建的突变文库库容量约为4000，从中筛选出的突变毕赤酵母菌X-33/pGAPZαA-GwMan26A-27的突变酶GwMan26A-27具有耐酸、耐高温且酶活力较高的特性；与突变前相比，GwMan26A-27具有较宽的酶促反应温度范围和适宜pH值范围，热稳定性和pH稳定性均有较大提升；在胃蛋白酶液和胰蛋白酶液中水浴2 h，GwMan26A-27的消化酶稳定性较突变前也均有所提升.