来源于芽孢杆菌HJ14耐热酯酶的克隆表达、酶学性质及降解邻苯二甲酸二乙酯研究

Cloning, heterologous expression and characterization of a thermostable esterase from Bacillus sp. HJ14 for diethyl-phthalate degradation

摘要:

[目的] 克隆芽孢杆菌HJ14的酯酶基因EstZ1并利用大肠杆菌表达得到相应的酯酶，分析重组酯酶的酶学性质和对邻苯二甲酸二乙酯（Diethyl phthalate，DEP）的降解。[方法] 特异性扩增酯酶基因EstZ1并对其全长测序，分析其氨基酸序列。利用pEASY-E2表达系统将EstZ1转化到Escherichia coli BL21（DE3）中完成异源表达。根据组氨酸标签纯化EstZ1，研究其酶学性质并利用HPLC和LC/MS检测系统定性分析其对DEP的降解。[结果] EstZ1全长903 bp，编码300个氨基酸残基，蛋白分子量33.84 kDa。EstZ1氨基酸序列分析结果显示，与NCBI数据库收录的HSL-like家族酯酶相似度最高可达到98%。酶学性质分析结果显示，EstZ1可水解碳链长度较短的p-NP底物，最适底物为p-NPC4（p-NP butyrate）。EstZ1的最适pH和最适温度分别为9.0和50℃，并且在pH 7.0–9.5和40–70℃范围内保持50%以上的酶活，为耐热碱性酯酶。EstZ1对多数金属离子和化学试剂保有良好的抗性。EstZ1可将DEP水解生成相应的单酯和醇。[结论] 本文报道了Bacillus sp.HJ14来源的酯酶基因并对其在大肠杆菌中表达获得的重组酶的酶学性质进行研究，EstZ1具有良好的碱性pH耐受性和热稳定性，能够部分降解DEP，本研究对邻苯二甲酸酯类的生物降解有一定的参考意义。

关键词:耐热酯酶;异源表达;酶学性质;DEP降解

Abstract:

[Objective] A thermostable esterase EstZ1 from Bacillus sp. HJ14 able to degrade diethyl-phthalate (DEP) was heterologously expressed in Escherichia coli BL21(DE3) and characterized.[Methods] Full-length EstZ1 was obtained based on specific amplification and genome sequencing, and amino acid sequence of EstZ1 was analyzed. EstZ1 was expressed in Escherichia coli BL21(DE3) using the pEASY-E2 expression system. EstZ1 was purified to electrophoretic homogeneity by Ni²⁺-NTA metal chelating affinity chromatography, and the enzyme was characterized. The degradation products from DEP were detected by high-pressure liquid chromatography and electrospray ionization mass spectrometry.[Results] The 903 bp full-length EstZ1 encoded 300 amino acid residues (EstZ1:33.84 kDa). EstZ1 showed the highest identity of 98% with hormone-sensitive lipase (HSL)-like family in NCBI databases. The optimal temperature and pH was 50℃ and 9.0, respectively, with p-NP butyrate as the best substrate. Meanwhile, it was stable between 40 and 70℃, pH 7.0 to 9.5. Most of metal ions, chemical agents had little impact. DEP could partially be degraded by EstZ1 to its corresponding monoalkyl and alcohol.[Conclusion] Our findings may serve as reference for phthalate esters degradation.

Key words:thermostable esterase;heterologous expression;enzyme characterizations;DEP degradation