| 安可,史晓翀,崔方元,等.噬琼胶卵链菌β-琼胶酶基因YM01-5的克隆表达及其酶学性质的研究[J].中国海洋药物,2018,37(1):31-39. |
| 噬琼胶卵链菌β-琼胶酶基因YM01-5的克隆表达及其酶学性质的研究 |
| Cloning, expression, and characterization of beta-agarase YM01-5 from Catenovulum agarivorans YM01T |
| 投稿时间：2017-04-25 修订日期：2017-06-02 |
| DOI： |
| 中文关键词: β-琼胶酶YM01-5 克隆表达 酶学性质 新琼二糖 |
| English Keywords:beta-agarase YM01-5 Cloning and expression characterization neoagarobiose |
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| 中文摘要: |
| 目的 从青岛近海海水中分离鉴定了1株能够降解琼胶的海洋新菌—嗜琼胶卵链菌（Catenovulumagarivoransgen. nov. sp. nov.）YM01T，并对其进行了全基因组测序。本文对该菌的1个β-琼胶酶基因YM01-5进行了克隆表达，并对重组琼胶酶的酶学性质进行了研究。方法 利用镍柱亲和层析对重组琼胶酶进行了分离纯化，采用DNS法测定重组酶的酶学性质，薄层层析(TLC)和质谱(MS)法对AgaYM01-5的酶解产物进行分析。结果 β-琼胶酶基因YM01-5全长2 412 bp，编码803个氨基酸，预测分子量为91.6 kDa，其催化模块属于糖苷水解酶GH50家族。重组琼胶酶YM01-5酶学性质的研究结果表明，该酶的最适温度为40 ℃，最适pH为9.0。在35 ℃以下具有良好的热稳定性，pH 6～10之间保持较高的稳定性，在该范围的pH缓冲液中放置12 h后，YM01-5仍能保持80%以上的酶活力。此外，该重组琼胶酶降解琼脂糖的Km、Vmax值分别为15.6 mg/mL和188 U/mg， 对降解产物的薄层层析及质谱分析结果表明，β-琼胶酶基因YM01-5以外切酶的形式作用于琼脂糖产生新琼二糖作为终产物。结论 该酶降解产物单一，有利于琼胶寡糖的制备，具有较高的工业应用潜力，它的发现也为研究菌株YM01中琼脂糖的代谢通路提供了参考。 |
| English Summary: |
| objective An agar-degrading bacterium, Catenovulum agarivorans YM01T, was isolated from the seawater of Qingdao, China and identified as a novel genus and species. The genome sequencing of this strain has already been completed. In this study, a new β-agarase gene YM01-5 was cloned and overexpressed in E.coli BL21 (DE3) system. Then the characterizations of the recombinantagaraseYM01-5 were analyzed. Methods The His-tagged recombinant proteins were purified using NTA-Ni2+agarose affinity chromatography from the crude extract. The characterizations of the recombinant agarase YM01-5 were analyzed by dinitro salicylic acid method. The hydrolysis products of AgaYM01-5 was analyzed by thin-layer chromatography (TLC) and mass spectrometry (MS). Results The gene agaYM01-5 consisted of 2,412 bp and encoded a protein of 803 amino acids with an apparent molecular mass of 91.6 kDa. YM01-5 belonged to glycoside hydrolase 50 family based on the amino acid sequence homology. The optimum pH and temperature of YM01-5 were 7.0 and 50 °C, respectively. It was stable at pH 6.0~10.0 and temperatures below 35 °C. It retained more than 80% activity after being preincubated at a wide range of pH 6.0～10.0 at 4 °C for 12 h. The Km and Vmax for agarose were 15.6 mg/mL and 188 U/mg，respectively. Thin layer chromatography (TLC) and ion trap mass spectrometer of the YM01-5 hydrolysis products displayed that YM01-5 was an exo-type β-agarase and degraded agarose into neoagarobiose directly. Conclusion The enzyme YM01-5 could digest agarose yielding neoagarobiose as the single end product, which had potential applications for the production of oligosaccharides with remarkable activities. While it also provided a better understanding of agarolytic metabolic pathway in YM01T. |
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