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

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ca tPL Cgl,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 kDo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lh后,酶活剩余90％以上;最适作用温度为40℃,在25℃和40℃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24.9μmol/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l,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.%[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 ℃ at pH 7.0.The enzyme was stable at 25 ℃ and 40 ℃,retaining nearly 100％ activity after pre-incubation for 210 h.The kinetic parameters Kim,Vmax and kcat values were 24.9 μmol/L,8.3 μmol/(min·g) and 13.7 s-1respectively.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.