南极菌产琼胶酶aga3311的表达、性质及其降解特性

Expression and characterization of the agarase gene aga3311 from an Antarctic bacterium

摘要:

[目的]本文通过对具有琼胶降解能力的南极菌Pseudoalteromonas sp. NJ21全基因组进行生物信息学分析，筛选获得琼胶酶疑似序列aga3311，采用基因工程手段对该基因的功能和性质进行了验证和分析。[方法]首先对aga3311进行克隆和表达；采用Ni-NTA对重组酶进行纯化； DNS-还原糖法测定重组酶的酶学性质；用薄层层析（TLC）和质谱（MS）技术对Aga3311的酶解产物进行分析。[结果]构建的重组表达质粒pET-30（a）+aga3311能够在工程菌E. coli BL21（DE3）中实现高效表达，其中可溶性表达为30%左右；纯化的重组酶Aga3311分子量为87 kDa，其最适作用温度为35℃，30-45℃的范围内稳定性较高，50℃则迅速失活，具有热不稳定的特征；最适pH为7.0，在pH 4.0-10.0的范围内仍能保持50%以上的活性；金属离子Fe³⁺、Be²⁺、Zn²⁺和Ca²⁺均能显著提高Aga3311的活性，特别是Ca²⁺使其酶活提高1倍。该酶的酶解终产物经TLC和质谱分析主要为新琼二糖。[结论]重组酶Aga3311为Glyco_hydro_42家族的外切型β-琼胶酶，能够特异性降解琼脂糖生成新琼二糖。

Abstract:

[Objective] The complete genome of the agarolytic bacterium Pseudoalteromonas sp. NJ21 from Antarctic sample was analyzed by bioinformatics methods and putative agarase aga3311was screened. Expression and characterization of the putative agarase aga3311 were studied.[Methods] Gene aga3311 was cloned and expressed by genetic engineering method firstly; then, the recombinant enzyme was purified by Ni-NTA chromatography and the characterization of recombinant enzyme was determined by dinitrosalicylic acid method; the hydrolysis product of recombinant enzyme Aga3311 was analyzed by thin-layer chromatography (TLC) and mass spectrometry (MS).[Results] The recombinant expression vectors (pET-30(a)+aga3311) was overexpressed in E. coli BL21(DE3) and 30% of the recombinant protein was soluble. The purified agarase (Aga3311) revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 87 kDa. The optimum temperature of the recombinant agarase was 35℃, and it maintained higher activity between 30 and 45℃, but the activity declined rapidly above 50℃, typical of thermal instability enzyme. The optimum pH was 7.0, and it maintained 50% of its maximum activity between pH 4 and 10. Aga3311 was significantly activated by Fe³⁺, Be²⁺, Zn²⁺ and Ca²⁺, especially Ca²⁺ doubled the enzyme activity. The pattern of agar hydrolysis of Aga3311 is an exo-β-agarase, producing neoagarobiose (NA2) as the final main product.[Conclusion] Aga3311 is an exo-β-agarase of Glyco_hydro_42 family, producing neoagarobiose (NA2) as the final main product.