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• 生物工程 •    下一篇

易错PCR技术定向进化褐藻胶裂解酶Alg-2的研究

李树1,张伟2,赵春梅2   

  1. 1. 山东大学(威海)海洋学院
    2. 江南大学生物工程学院
  • 收稿日期:2018-02-08 修回日期:2018-12-24 出版日期:2019-02-25 发布日期:2019-03-05
  • 通讯作者: 李树 E-mail:lishu2016@sdu.edu.cn
  • 基金资助:
    褐藻胶裂解酶Alg-2基因定向进化及其机制解析

Directed evolution research on alginate lyases Alg-2 based on error prone PCR

Shu LI 2, 2   

  • Received:2018-02-08 Revised:2018-12-24 Online:2019-02-25 Published:2019-03-05
  • Contact: Shu LI E-mail:lishu2016@sdu.edu.cn

摘要: 为提高褐藻胶裂解酶的酶活,采用易错PCR技术对海洋细菌Tamlana sp. S12中具有代表性的酶组分Alg-2进行了体外定向进化。经过2轮易错PCR及96孔板发酵筛选,分别获得了2株正突变和2株负突变菌株,其酶活分别是亲本(112.6 EU/ml)的155%、227%、51%、11%。酶的动力学分析显示,正突变株P2-81的Km值比亲本降低了50%,而负突变株N2-47的Km值比亲本提高了106%,表明突变株对底物的亲和力有极大的上升和下降。基因测序及氨基酸序列分析结果表明,Glu、Thr、Ser、Asp和Tyr对褐藻胶裂解酶的酶活提高起到了正面的积极作用,而Lys的突变起到了负面的消极作用,可能是Alg-2的保守区域。本研究有助于深入理解褐藻胶裂解酶的催化机制,更为后续利用理性设计手段改造褐藻胶裂解酶提供了理论参考。

关键词: 易错PCR, 定向进化, 褐藻胶裂解酶, 氨基酸序列

Abstract: In order to improve the enzymatic activity of alginate lyases Alg-2, a directed evolution research based on error prone PCR was developed for marine bacteria Tamlana sp. S12. After two rounds of error prone PCR and 96-well plate fermentation, two positive and two negative mutants were obtained, 155%、227%、51%、11% over the control, respectively. According to dynamics analysis, Km values of positive strain P2-81 was reduced by 50% while negative strain N2-47 was 106% higher than that of the parent, showing that the substrate affinity with enzyme in mutant strains was with great rising and falling. Results of gene sequencing and amino acid sequence analysis showed that Glu, Thr, Ser, Asp and Tyr played a positive role on increased enzyme activity of alginate lyase, while the mutation of Lys in conservative area had the negative effect. This study is helpful to understand the catalytic mechanism of alginate lyases, and to provide a theoretical reference for alginate lyases reconstruct using rational design methods.

Key words: error prone PCR, directed evolution, alginate lyases, amino acid sequence

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