食品与发酵工业 >

2025 , Vol. 51 >Issue 15: 174 - 184

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.040941

研究报告

海洋弧菌Vibrio sp.L2褐藻胶裂解酶AlgL3199的表达及酶学性质研究

  • 王海英 ,
  • 陈志芳 ,
  • 朱甜甜 ,
  • 孙晶晶 ,
  • 王伟 ,
  • 郝建华
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  • 1(中国水产科学研究院黄海水产研究所,山东 青岛,266071)
    2(大连海洋大学 水产与生命学院, 辽宁 大连,116000)
    3(上海海洋大学 食品学院,上海,200000)
第一作者:博士,副研究员(郝建华研究员为通信作者,E-mail:haojh@ysfri.ac.cn)

收稿日期: 2024-09-03

  修回日期: 2024-11-05

  网络出版日期: 2025-08-22

基金资助

国家重点研究发展计划项目(2022YFC2805101);山东省自然科学基金项目(ZR2022MD084);中国水产科学研究院基本科研业务费项目(2023TD71);青岛市市南区应用基础研究计划项目(2023-2-027-YY);黄海水产研究所基本科研业务费项目(20603022024003)

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Expression and enzymatic properties of alginate lyase AlgL3199 from Vibrio sp.L2

  • WANG Haiying ,
  • CHEN Zhifang ,
  • ZHU Tiantian ,
  • SUN Jingjing ,
  • WANG Wei ,
  • HAO Jianhua
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  • 1(Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China)
    2(College of Fisheries and Life Science, Dalian Ocean University, Dalian 116000, China)
    3(College and Food Sciences and Technology, Shanghai Ocean University, Shanghai 200000, China)

Received date: 2024-09-03

  Revised date: 2024-11-05

  Online published: 2025-08-22

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摘要

褐藻胶通过生物降解法可制备低分子质量的褐藻寡糖,以克服褐藻胶生物利用度低的缺点,广泛应用于医疗、食品、化妆品等行业。该研究克隆表达了一株海洋弧菌Vibrio sp.L2中的褐藻胶裂解酶基因AlgL3199,并对其酶学性质和降解产物进行研究。以Vibrio sp.L2基因组为模板,克隆褐藻胶裂解酶基因,构建重组质粒 pET-24a(＋)/AlgL3199进行异源表达,经HisTrapTM HP柱亲和层析纯化后进行酶学性质及降解产物的研究。结果表明,重组酶AlgL3199最适温度为40 ℃,在10~35 ℃下保温1 h可保留80%以上的活性;最适pH值为10,在pH 8~10孵育12 h可保留70%以上的活性;金属离子终浓度为1 mmol/L时,Cu2+、Mn2+、Co2+对重组酶AlgL3199有明显促进作用,十二烷基硫酸钠(sodium dodecyl sulfate,SDS)及EDTA完全抑制酶的活性;NaCl终浓度为1.125 mol/L时对重组酶AlgL3199激活效应最强;根据底物特异性及薄层层析结果表明,重组酶AlgL3199酶解PolyM、PolyG及褐藻酸钠的主产物均为二糖至四糖,是一种具有G偏好性的内切型双功能酶。以上结果表明,AlgL3199已成功在大肠杆菌中实现了异源表达,在低温和弱碱性环境下比较稳定,是一种双功能褐藻胶裂解酶,这为褐藻胶的降解提供了一种新型工具酶,有利于褐藻工业生物技术的发展和应用。

关键词： 褐藻胶裂解酶; 褐藻寡糖; 酶学性质; 异源表达

本文引用格式

王海英 , 陈志芳 , 朱甜甜 , 孙晶晶 , 王伟 , 郝建华 . 海洋弧菌Vibrio sp.L2褐藻胶裂解酶AlgL3199的表达及酶学性质研究[J]. 食品与发酵工业, 2025 , 51(15) : 174 -184 . DOI: 10.13995/j.cnki.11-1802/ts.040941

Abstract

Alginate oligosaccharides with low molecular weight can be prepared by alginate lyase to overcome its disadvantage of low bioavailability and are widely used in medical, food, and cosmetics industries.AlgL3199, a gene-producing alginate lyase, was cloned from Vibrio sp.L2 genome, recombinant plasmid pET-24a (+)/AlgL3199 was constructed, and it was heterologously expressed.The enzymatic properties and degradation products of recombinant AlgL3199 were studied after purified with His TrapTM HP column affinity chromatography.The optimum temperature was 40 ℃, and the activity was retained more than 80% when it was kept at 10-35 ℃ for 1 h.The optimum pH value was 10, and above 70% activity could be retained after incubation for 12 h in a pH 8-10 buffer.Cu2+、Mn2+、Co2+ in 1 mmol/L could promote the activity of AlgL3199, while SDS and EDTA could inhibit its activity completely.AlgL3199 was most active with 1.125 mol/L NaCl.The thin layer chromatography showed the main products of PolyM, PolyG, and sodium alginate hydrolyzed by AlgL3199 were disaccharides, trisaccharides, and tetrasaccharide, suggesting AlgL3199 was a bifunctional endocytic enzyme with G preference.Study showed that AlgL3199 had been successfully expressed in E.coli and was stable at low temperature and weak alkalinity.It might be a new tool enzyme and an interesting candidate for the alginate industry and biotechnological applications.

Key words： alginate lyase; alginate oligosaccharide; enzymatic property; heterologous expression

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