嗜热厌氧芽孢杆菌偶氮还原酶蛋白结构模拟分析及原核表达条件优化

Optimization of expression conditions and protein structure simulation analysis of azoreductase from Anoxybacillus sp.

嗜热厌氧芽孢杆菌PDR2(Anoxybacillus sp. PDR2)是一株极具潜力的偶氮染料降解菌,但目前对一种菌株中分离纯化两种具有不同催化活性的偶氮还原酶的相关研究还未见报道.从具有降解偶氮染料活性的嗜热厌氧芽孢杆菌的全基因组序列中,获得两种偶氮还原酶基因,将其命名为FMN和AZO,两种蛋白不具亲缘关系,但蛋白结构均属于α/β型结构.两种蛋白结构中的配体FMN可能为蛋白与底物结合提供电子和质子,且配体结构中异咯嗪环分子的关键原子N5分别与蛋白的Asn104和His75形成氢键,在稳定配体和蛋白活性可能起到重要作用,可作为研究蛋白与底物结合特异性的关键氨基酸.此外,采用原核表达技术成功将两种基因在大肠杆菌Escherichia coli BL21(DE3)中表达.优化两种重组菌的诱导表达条件,AZO重组菌最佳诱导条件为添加诱导剂时菌体量OD600为0.8、诱导剂IPTG浓度为0.2 mmol/L、诱导时间24 h及诱导温度16℃,此时胞内重组酶降解甲基红的比酶活达到15.65 U/mg;FMN重组菌最佳诱导条件为添加诱导剂时菌体量OD600为0.8、诱导剂IPTG浓度为0.4 mmol/L、诱导时间10 h及诱导温度20℃,此时胞内重组酶降解甲基红的比酶活达到18.66 U/mg.本研究表明优化两种偶氮还原酶原核表达条件能够显著提高酶活性,结合其蛋白结构特性可为后续蛋白纯化及分析蛋白质与底物结合特异性提供理论基础.(图11表1参33)

Anoxybacillus sp. PDR2 is a high potential azo-dye-degrading bacteria;however, the investigation of the separation and purification of two azoreductases with different catalytic activity in the same strain has never been reported. In this study, two azoreductase genes were obtained from the whole genome sequence of the Anoxybacillus sp. PDR2 and named FMN and AZO. Both proteins have α/β type structures, although there is no relationship between the two. Through analysis, it was found that the ligand FMN in the two protein structures may provide electrons and protons for protein-to-substrate binding, and the key atom N5 of the heteroquine ring molecule in the ligand FMN structure formed hydrogen bonds with the proteins Asn104 and His75, respectively. These proteins may play an important role in stabilizing ligand and protein activity and can be used as key amino acids to study the specificity of protein-to-substrate binding. Furthermore, the use of prokaryotic expression technology successfully put these two genes in E. coli E., and they were expressed in E·coli BL21(DE3). The optimum induction conditions for AZO recombinant bacteria are: the inducer isopropyl β-d-1-thiogalactopyranoside(IPTG) concentration is 0.2 mmol/L, the induction time is 24 h, and the induction temperature is 16 ℃, at which time the intracellular recombinant enzyme activity of intracellular recombinase reduced methyl red reaches 15.65 U/mg. The best induction conditions for FMN recombinant bacteria are: the amount of bacterium OD600 is 0.8 when the inducer is added, the inducer IPTG concentration is 0.4 mmol/L, the induction time and temperature are 10 h and 20 ℃, at which time the intracellular recombinant enzyme activity of intracellular recombinase reduced methyl red reaches 18.66 U/mg. This study shows that optimizing the expression conditions of the two azoreductases prokaryotic can significantly improve the enzyme activity, and the combination of the protein structure characteristics can provide a theoretical basis for subsequent protein purification and the analysis of protein-substrate binding specificity.