Microbulbifer sp.FG5琼胶酶的分离纯化及酶学性质研究  

Isolation,purification and characterization of the agarase from Microbulbifer sp.FG5

作　　者：刘明明[1] 张力雄 龙昊 Mingming Liu;Lixiong Zhang;Hao Long(State Key Laboratory of Marine Resource Utilization in South China Sea,Hainan University,Haikou,Hainan 570228,China;Marine College,Hainan University,Haikou,Hainan 570228,China)

机构地区：[1]海南大学南海海洋资源利用国家重点实验室,海南海口570228 [2]海南大学海洋学院,海南海口570228

出　　处：《日用化学工业》2022年第8期812-818,共7页China Surfactant Detergent & Cosmetics

基　　金：国家自然科学基金资助项目(40666001);海南大学青年基金(qnjj1205)。

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。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.

关 键 词：琼胶降解菌 琼胶酶 葡聚糖凝胶分离 

分 类 号：TQ658[化学工程—精细化工]