解淀粉芽孢杆菌Q-426酚酸脱羧酶的克隆表达及酶学性质鉴定*

目的:生物法脱羧制备4-乙烯基衍生物具有诸多优势和良好的发展前景,研究解淀粉芽孢杆菌Q-426酚酸脱羧酶(BaPAD-Q-426)的酶学性质,为其进一步应用提供理论基础。方法:从解淀粉芽孢杆菌中克隆酚酸脱羧酶基因;以pET-28a(+)为载体,将重组质粒转化至E. coli BL21(DE3)中,实现酚酸脱羧酶BaPAD-Q-426的高效表达,利用Ni-NTA亲和层析进行纯化,并进行酶学性质鉴定。结果:酚酸脱羧酶BaPAD-Q-426在pH 7.0~9.0范围内保持良好的pH稳定性,最适pH为8.0;在25~40℃范围内保持着较高的酶活性,最适温度为35℃,在4℃时保持30 min后该酶依然保持95%以上的酶活性;K⁺对BaPAD-Q-426的酶活具有明显促进作用,酶活力提高60%;该酶在石油醚中具有良好的耐受能力,在40%石油醚存在下,仍保留50%以上的酶活力;BaPAD-Q-426的最适底物为阿魏酸,酶活力达到19.5 IU/mL。结论:与其他来源的酚酸脱羧酶相比,BaPAD-Q-426在低温时具有更好的稳定性,在弱碱性环境下对阿魏酸的催化脱羧能力最强。

Cloning,Expression and Characterization of Phenolic Acid Decarboxylase from Bacillus amyloliquefaciens Q-426

Objective: The preparation of 4-vinyl derivatives by biological decarboxylation has many advantages and promising prospects. In this study, the enzymatic properties of Bacillus amyloliquefaciens Q-426 phenolic acid decarboxylase (BaPAD-Q-426) were studied in detail to provide theoretical basis for its future application. Methods: In this study, the phenolic acid decarboxylase gene was cloned from Bacillus amyloliquefaciens. Using pET-28a (+) as vector, the recombinant plasmid was transformed into E.coli BL21 (DE3), to achieve high expression of BaPAD-Q-426. It was purified by Ni-NTA affinity chromatography, and the enzymatic properties were identified. Results: BaPAD-Q-426 maintained good pH stability in the range of pH 7.0~9.0, and the optimum pH was 8.0. The enzyme maintained high enzyme activity in the range of 25~40℃, and the optimum temperature was 35℃. After holding at 4℃ for 30 minutes, the enzyme still maintained more than 95% enzyme activity. K⁺ significantly promoted the enzyme activity of BaPAD-Q-426 with an increase of 60%. This enzyme was well tolerated in petroleum ethers and retained more than 50% of the enzyme activity in the presence of 40% petroleum ethers. The optimum substrate of BaPAD-Q-426 was ferulic acid, and its enzyme activity reached 19.5 IU/mL. Conclusion: Compared with phenolic acid decarboxylases from other sources, BAPAD-Q-426 has better stability at low temperature and has the strongest catalytic decarboxylation of ferulic acid in weakly alkaline environment.