Microbulbifer sp. FG5琼胶酶的分离纯化及酶学性质研究

doi:10.3969/j.issn.1001-1803.2022.08.003

摘要/Abstract

摘要：

琼胶酶是一种糖苷水解酶,能够特异性降解琼胶为琼胶寡糖。本研究从一株琼胶降解菌FG5的培养液中分离纯化得到纯净的琼胶酶并研究其酶学特性。将FG5菌株的发酵液离心后得到粗酶液,再通过超滤浓缩、透析、葡聚糖凝胶柱层析进行酶的分离纯化。收集得到葡聚糖凝胶柱层析紫外检测主峰组份,经SDS-PAGE电泳检测为单一条带蛋白质,分子量约为54 kD。酶活检测证明该蛋白具有琼脂酶活性,命名为琼胶酶FG5-A。采用单因素试验和正交试验设计对FG5-A的酶解反应条件进行优化分析。FG5-A琼胶酶活性最适条件为：pH 7.3,反应温度51 ℃,反应底物含量0.351%。探索了FG5-A的纯化流程,经葡聚糖凝胶柱纯化后琼胶酶活性相比发酵液提高了6.95倍,并初步优化了FG5-A琼胶酶的最适酶解条件,为高纯度琼胶酶的工业化生产和应用奠定理论与实践基础。

关键词: 琼胶降解菌, 琼胶酶, 葡聚糖凝胶分离

Abstract:

Through the separation and purification of the agar-degrading bacteria FG5 fermentation broth, agarase was obtained to study its enzymatic characterization, and whole genome sequencing was used to obtain high-potential agar-degrading engineering bacteria. The crude enzyme of FG5 agarase was obtained after centrifugation of the fermentation broth of FG5 strain followed by separation and purification via ultrafiltration concentration, dialysis, and dextran gel column chromatography. The DNS method was used to determine the reducing sugar content in the enzymatic hydrolysate, and then the enzyme activity was calculated. Then, a single factor experiment, and an orthogonal experiment design were utilized to optimize the enzymatic hydrolysis conditions for a single agarase component. A peak product with agarase activity is obtained after separation, purification, and UV detection. The molecular weight of the purified agarase protein is about 54 kD. The optimal conditions of FG5 agarase are as follows: pH 7.3, reaction temperature 51 ℃, and substrate concentration 0.351%. In this study, the FG5 crude enzyme is purified by 6.95 times using the dextran gel column, and the conditions for enzymatic hydrolysis of FG5 agarase are optimized. It provides a theoretical and practical foundation for the industrial production of high-purity agarase.

Key words: agar-degrading strain, agarase, dextran gel separation

中图分类号:

TQ658

刘明明,张力雄,龙昊. Microbulbifer sp. FG5琼胶酶的分离纯化及酶学性质研究[J]. 日用化学工业, 2022, 52(8): 812-818.

Liu Mingming,Zhang Lixiong,Long Hao. Isolation, purification and characterization of the agarase from Microbulbifer sp. FG5[J]. China Surfactant Detergent & Cosmetics, 2022, 52(8): 812-818.

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记录编号	5 mmol/L半乳糖标准溶液体积/mL	ddH₂O体积/mL	还原糖含量测定试剂（DNS）体积/mL	冷热加工后 ddH₂O体积/mL	处理后半乳糖浓度/（μmol/L）
1	0	0.53	0.53	4.0	0
2	0.13	0.40	0.50	4.0	0.51
3	0.21	0.30	0.51	4.0	1.01
4	0.31	0.21	0.51	4.0	1.51
5	0.42	0.11	0.52	4.0	2.01
6	0.52	0	0.51	4.0	2.51

代码	pH	w（反应物）/%	温度/℃	酶活力/（U/mL）
（1）	6.0	0.253	41	231.897
（2）	6.0	0.304	51	319.154
（3）	6.0	0.353	46	301.352
（4）	6.4	0.252	51	273.725
（5）	6.4	0.301	46	273.481
（6）	6.4	0.352	41	171.523
（7）	7.3	0.253	46	255.151
（8）	7.3	0.302	41	189.531
（9）	7.3	0.351	51	333.423
D1	149.79	133.51	147.54
D2	150.28	156.72	143.94
D3	152.76	161.60	150.40
K	2.97	27.08	2.85