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过程工程学报 ›› 2022, Vol. 22 ›› Issue (4): 506-514.DOI: 10.12034/j.issn.1009-606X.221108

• 研究论文 • 上一篇    下一篇

聚合物接枝对脂肪酶活性及稳定性的影响

刘宗浩1,2,史清洪1,2*   

  1. 1. 天津大学化工学院，天津 300350 2. 系统生物工程教育部重点实验室，天津 300072
  • 收稿日期:2021-03-31 修回日期:2021-05-25 出版日期:2022-04-28 发布日期:2022-04-24
  • 通讯作者: 史清洪 qhshi@tju.edu.cn
  • 作者简介:刘宗浩(1995-)，男，天津市人，硕士研究生，生物工程专业，E-mail: liuzonghaoa@tju.edu.cn；史清洪，通讯联系人，E-mail: qhshi@tju.edu.cn.

Efficient improvement on activity and stability of lipase via hydrophobic polymer grafting

Zonghao LIU1,2,  Qinghong SHI1,2*   

  1. 1. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China 2. Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin 300072, China
  • Received:2021-03-31 Revised:2021-05-25 Online:2022-04-28 Published:2022-04-24

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摘要： 酶的稳定性和活性一直是困扰其工业应用的主要瓶颈，因此改造酶分子以提升其催化性能是当前研究的重要课题。本工作引入甲基丙烯酸异丁酯(IBMA)和甲基丙烯酸乙酯(EMA)两种疏水性不同的单体分子，通过原子转移自由基聚合分别接枝于褶皱假丝酵母脂肪酶(CRL)表面，合成聚合物接枝脂肪酶pIBMA-g-CRL和pEMA-g-CRL。酶学实验结果显示，聚合物接枝脂肪酶的催化活性和稳定性显著提升。pIBMA-g-CRL和pEMA-g-CRL的催化效率分别达到了野生型CRL的4.39倍和4.68倍。在50℃条件下孵育6 h后，pIBMA-g-CRL和pEMA-g-CRL仍分别保留51%和92%的活性，而野生型CRL的活性则仅剩14%。同样，在pH=9条件下孵育3 h后pIBMA-g-CRL和pEMA-g-CRL能够分别保留52%和83%的活性，并在pH=4~9范围内显示出更好的耐受性。光谱学实验进一步证实了聚合物接枝脂肪酶催化性能的提升与其二级结构和三级结构的变化密切相关。本研究表明，IBMA和EMA的接枝对脂肪酶的稳定性和活性的提升效果显著，是改造脂肪酶的优良材料。

关键词: 甲基丙烯酸异丁酯, 甲基丙烯酸乙酯, 疏水性, 褶皱假丝酵母脂肪酶, 聚合接枝, 稳定性

Abstract: The stability and activity of enzymes are the main bottlenecks in enzyme's industrial applications. Therefore, engineering enzymes for improved catalytic performance is an important topic for industrial biocatalysis in the current status. In this work, hydrophobic monomers, isobutyl methacrylate (IBMA) and ethyl methacrylate (EMA) were grafted onto surface of Candida rugosa lipase (CRL) via atom transfer radical polymerization (ATRP), respectively, to synthesize two hydrophobic polymer-grafted lipases, pIBMA-g-CRL and pEMA-g-CRL. Compared with wide-type CRL, the catalytic activity and stability of pIBMA-g-CRL and pEMA-g-CRL improved significantly. After grafting with hydrophobic polymers, Michaelis parameter of CRL decreased from 9.98 mmol/L to 6.91 mmol/L (for pIBMA-g-CRL) and 8.54 mmol/L (for pEMA-g-CRL), whereas turnover number increased from 5.63 to 16.97 (for pIBMA-g-CRL) and 22.36 (for pEMA-g-CRL). As a result, catalytic efficiency of pIBMA-g-CRL and pEMA-g-CRL was 4.39 times and 4.68 times as high as that of wide-type CRL, respectively. Moreover, pEMA-g-CRL retained 92% activity after incubating at 50℃ for 6 h. It reflected that hydrophobic polymer grafting induced the opening of lipase "lid" structure and led to exposure of catalytic active site. Meanwhile, lipase in lid opening conformation had better stability. Spectroscopy experiments further confirmed that the improvement of lipase catalytic performance was closely related to the change in secondary and tertiary structure of lipase. CD results showed a decreased content of α-helix and an increased content of β-sheet in hydrophobic polymer-grafted CRLs. Moreover, as shown by fluorescence emission spectra, a blue shift of hydrophobic polymer-grafted CRLs was observed, which indicated a more compact structure than wild-type lipase. The results in this research demonstrated that IBMA and EMA, as the monomers for grafting CRL, had a significant improvement to the stability and activity of CRL, and the resulting polymer was a potential material for lipase modification.

Key words: isobutyl methacrylate, ethyl methacrylate, hydrophobicity, Candida rugosa lipase, polymer grafting, stability