一种海洋琼胶酶的分离纯化、酶学性质研究及降解产物分析

Isolation and characterization of a marine agarase

【目的】对海洋Agarivorans albusQM38菌株所产琼胶酶的纯化工艺和酶学性质进行了研究。【方法】发酵液通过离心、(NH4)2SO4盐析、DEAE-Sepharose Fast Flow阴离子交换层析、Sephacryl S-100凝胶过滤等纯化步骤得到SDS-PAGE电泳级纯酶,并用质谱对酶的降解产物进行分析。【结果】得到琼胶酶A,纯化倍数为17.6倍,收率为15.21%,SDS-PAGE测定其分子量为127.8 kDa。对琼胶酶A进行了进一步的性质分析,其最适反应温度为35℃,最适反应pH为7.6,最适底物浓度为0.9%,多数金属离子为其活性抑制剂。琼胶酶A的降解产物经质谱分析主要为四糖和六糖。【结论】从菌株QM38的发酵液中纯化得到的琼胶酶A具有降解凝胶态琼胶的能力,其分子量与以往报道过的琼胶酶不同。

[Objective] This study was carried out to isolate and characterize an agarase from a marine bacterium Agarivorans albus QM38. [Methods] SDS-PAGE grade agarase was obtained from the fermentation broth after removing the bacteria by centrifugation, ammonium sulfate precipitation, DEAE-sepharose fast flow anion exchange chromatography and Sephacryl S-100 gel filtration. Enzyme＇s molecular weight was determined with SDS-PAGE. The catalysates of the isolated enzyme were determined with mass spectrography. [Results] Agarase A was isolated. The molecular weight of agarase A was 127.80 kDa. More characterizations of agarase A were studied and the results showed that the optimal reaction condition for agarase A was at 35℃, pH 7.6, and agar concentration of 0.9% （w/v）, while most of the metal ions inhibited the activity of it. The catalysates of agarase A were mainly tetrose and hexose. [ Conclusion] Agarase A was purified from the medium. It could hydrolyze jellied agar and yield simple catalysates. Its molecular weight is different from all the agarases reported so far.