深海太平洋火色杆菌琼胶降解基因分析和琼胶酶Aga0950的表达及酶学性质

Analysis of agar degradation gene and characterization of an agarase Aga0950 from a deep-sea bacterium Flammeovirga pacifica WPAGA1

摘要:

[目的]对深海太平洋火色杆菌（Flammeovirga pacifica WPAGA1）全基因组进行生物信息学分析，筛选获得琼胶酶基因aga0950，采用基因工程手段对该基因的功能和性质进行验证和分析。[方法]采用Illumina HiSeq2500测序技术进行基因组测序分析；采用克隆表达和镍柱纯化方法获得纯aga0950基因表达产物；采用薄层层析（TLC）和离子色谱（IC）法分析酶降解琼胶产物；采用二硝基水杨酸法（DNS）测定琼胶酶活性。[结果]基因组序列分析表明，菌株WPAGA1全基因组拥有13个β-琼胶酶相关基因；氨基酸序列比对显示，同源性为60%–85%，其中aga0950是具有GH16家族典型特征的基因，同源性为67%。纯化的重组酶Aga0950比活力达51770 U/mg，具有高效降解琼胶活性，降解终产物为新琼四糖和新琼六糖；最适温度为50℃，最适pH为4.0–10.0；Co²⁺、Mn²⁺和Fe³⁺促进酶活，Cu²⁺抑制酶活。[结论]深海菌株WPAGA1具有丰富的琼胶酶基因；属于GH16家族的琼胶酶基因aga0950表达产物具有高效降解胶琼活性和良好的热、酸、碱稳定性。

Abstract:

[Objective] An agarase gene, aga0590, was found in the fine genomics mapping of a deep-sea bacterium Flammeovirga pacifica WPAGA1 using bioinformatics analysis. Recombinant Aga0950 was characterized with heterologous expression in E. coli. [Methods] Genomic sequencing of Flammeovirga pacifica WPAGA1 was performed by Illumina HiSeq2500 and agarase genes were annotated with BLAST. Subsequently, Aga0950 was obtained with heterologous expression in E. coli BL21 (DE3) and the agar-degrading activity was determined by dinitrosalicylic acid method. Enzymatic products of the agarase were analyzed by thin-layer chromatography and ion chromatography. [Results] Thirteen agarase genes with amino acid homologies from 60% to 85% were detected in the genome of Flammeovirga pacifica WPAGA1. The elected gene aga0950, which was aligned with highest identities of 67% in NCBI-nr database, possessed typical sequence characteristics of glucoside hydrolase family 16 (GH16). The specific activity of Aga0950 was 51770 U/mg, and the end-products of agar degradation by Aga0950 were neoagarotetraose (NA4) and neoagarohexaose (NA6). The optimum temperature and pH of Aga0950 was 50℃ and a range of 4.0 to 10.0, respectively. Meanwhile, rAga0950 showed outstanding temperature and pH stability. Activities of Aga0950 was enhanced by Co²⁺ and Mn²⁺ (1 mmol/L), while that was strongly inhibited by Cu²⁺. [Conclusion] Flammeovirga pacifica WPAGA1 has abundant polysaccharide-degrading genes. The agarase Aga0950 was indentified with high agar-degrading activity and excellent stability against acid, alkali and thermo.