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p ET-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 k Da,其最适作用温度为35°C,30–45°C的范围内稳定性较高,50°C则迅速失活,具有热不稳定的特征;最适p H为7.0,在p H 4.0–10.0的范围内仍能保持50%以上的活性;金属离子Fe^(3+)、Be^(2+)、Zn^(2+)和Ca^(2+)均能显著提高Aga3311的活性,特别是Ca^(2+)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。
[Objective] The complete genome of the agarolytic bacterium Pseudoalteromonas sp. NJ21 from Antarctic sample was analyzed by bioinformatics methods and putative agarase aga331/was 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.
出处
《微生物学报》
CAS
CSCD
北大核心
2016年第9期1468-1476,共9页
Acta Microbiologica Sinica
基金
南北极环境综合考察与评估专项(CHINARE2014-01-05)
国家海洋局海洋生物活性物质与现代分析技术重点实验室开放基金(MBSMAT-2015-06)
青岛市应用基础研究计划项目(14-2-4-14-jch)
关键词
琼胶酶
表达
酶学性质
新琼二糖
agarase, expression enzymatic characterization, neoagarobiose
