粪便微生物宏基因组来源的热稳定性邻苯二酚1,2-双加氧酶异源表达及酶学性质

Expression and characterization of thermostable catechol 1,2-dioxygenase from a fecal microbial metagenome of Nycticebus pygmaeus

摘要:

【目的】克隆倭蜂猴粪便微生物宏基因组的邻苯二酚1,2-双加氧酶基因catPLCgl，并对该酶进行异源表达及酶学特性研究。【方法】利用宏基因组高通量测序技术获得catPLCgl，并对其氨基酸序列进行分析。将catPLCgl重组到载体pEASY-E2中并转化到大肠杆菌BL21(DE3)中异源表达，研究其酶学性质。【结果】catPLCgl全长852 bp，G+C含量48%，编码283个氨基酸，理论分子量为33.56 kD。重组CatPLCgl酶学性质分析显示最适作用pH为7.0，其中在pH 7.0–10.0范围内处理1 h后，酶活剩余90%以上；最适作用温度为40 °C，在25 °C和40 °C条件下稳定性较好，耐受210 h酶活性几乎不变。重组酶在最适条件下的动力学参数Km、Vmax和kcat分别为24.9 μmol/L、8.3 mmol/(min·g)和13.7 s–1；Fe2+、Hg2+、Cu2+、Triton X-100、SDS、Ag+强烈抑制该酶活性，而其它金属离子及有机试剂影响较小。【结论】从倭蜂猴粪便微生物宏基因组中克隆得到邻苯二酚1,2-双加氧酶基因catPLCgl，并对重组CatPLCgl酶学性质进行研究，该酶具有较好的热稳定性和耐碱性，在降解环境中的邻苯二酚和生产顺,顺-己二烯二酸方面具有应用潜力。

Abstract:

[Objective] The gene (catPLCgl) coding for catechol 1,2-dioxygenase from a fecal microbial metagenome of Nycticebus pygmaeus was cloned and expressed in Escherichia coli BL21(DE3), and the purified recombinant CatPLCgl was characterized. [Methods] The full-length catPLCgl was obtained based on the metagenomic high-throughput sequencing technology, and its amino acid sequence was analyzed. Then catPLCgl was cloned into the pEASY-E2 vector, and characterized by the heterologous expression in Escherichia coli BL21(DE3). [Results] The gene full-length of catPLCgl is 852 bp with a G+C content of 48% and encodes 283 amino acids, with a theoretical molecular weight of 33.56 kD. Characterization shows that the optimal pH was 7.0, and the enzyme activity remained more than 90% after being processed for 1 h at pH between 7.0 and 10.0. The thermal activity of purified recombinant CatPLCgl was optimal at 40 °C at pH 7.0. The enzyme was stable at 25 °C and 40 °C, retaining nearly 100% activity after pre-incubation for 210 h. The kinetic parameters Km, Vmax and kcat values were 24.9 μmol/L, 8.3 μmol/(min·g) and 13.7 s–1 respectively. Fe2+, Hg2+, Cu2+, Triton X-100, SDS and Ag+ dramatically reduced the enzymatic activity, and other metal ions or organic reagents have less effect. [Conclusion] Recombinant CatPLCgl has good temperature stability and alkali resistance.