文章摘要
海洋细菌来源低温褐藻胶裂解酶的分泌表达和酶学性质研究
Study on the Secreted Expression and Enzymatic Properties of Low Temperature Alginate Lyase from Marine Bacteria
投稿时间:2020-04-27  修订日期:2020-07-06
DOI:
中文关键词: 褐藻胶裂解酶  分泌表达  编码基因  pH稳定性
英文关键词: Alginate lyase  Secretory expression  Coding gene  pH stability
基金项目:国家藻类产业技术体系
作者单位E-mail
白露 国家水生动物病原库上海海洋大学 上海水产养殖工程技术研究中心上海海洋大学 水产科学国家级实验教学示范中心上海海洋大学 上海 bailubai@163.com 
刘彬 青岛市精神卫生中心 青岛
青岛市精神卫生中心 青岛 
 
王朋梅 中国水产科学研究院黄海水产研究所 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 青岛  
张文彬 国家水生动物病原库上海海洋大学 上海水产养殖工程技术研究中心上海海洋大学 水产科学国家级实验教学示范中心上海海洋大学 上海  
莫照兰 中国海洋大学 海洋生命学院  
李杰 中国水产科学研究院黄海水产研究所 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 青岛 lijie@ysfri.ac.cn 
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中文摘要:
      低温褐藻胶裂解酶对于褐藻寡糖制备和褐藻加工具有独特的优势,工业应用迫切需要稳定性较好的低温褐藻胶裂解酶。为了筛选稳定性较好的低温褐藻胶裂解酶,本研究进行了海洋细菌分离鉴定、酶的编码基因克隆与分析、酶分泌表达与纯化、不同因素对酶活力和稳定性的影响以及酶解产物分析实验。结果显示,用以褐藻胶为唯一碳源的平板筛选出一株能分泌低温褐藻胶裂解酶的海洋细菌SJ-H-12,基于16S rDNA序列构建进化树,该菌株鉴定为Yangia sp. SJ-H-12。进而克隆酶的编码基因ALYYA,Alyya属于PL5家族褐藻胶裂解酶。将基因在食品级宿主解脂耶氏酵母中进行分泌表达,重组Alyya的活力达到34.2 U/mL,分子量约为39.0 kDa,具有较强的PolyM偏好性。Alyya在25-35℃时表现出80%以上的活力,且在30℃时表现出最高的活力;在5.0-10.0的pH范围内稳定性较好,孵育后剩余超过60%的活力;NaCl在0至2.0 M的浓度下能够明显激活Alyya的活力。Alyya降解褐藻胶的产物主要是二糖,另有少部分单糖和三糖,该酶是一种内切褐藻胶裂解酶。综上所述,本研究筛选到一株产低温褐藻胶裂解酶细菌,已发现的褐藻胶裂解酶多为中温酶,并且本文研究的Alyya是典型的低温褐藻胶裂解酶,具有优良的酶活力和稳定性,为低温褐藻胶裂解酶的筛选和工业上开发利用提供了参考数据。
英文摘要:
      Low-temperature alginate lyase has unique advantages for alginate oligosaccharide preparation and brown algae processing, and industrial applications urgently need low-temperature alginate lyase with better stability. To screen low-temperature alginate lyase with good stability, in this study, we conducted the isolation and identification of marine bacteria, cloning and analysis of enzyme-encoding genes, secretory expression and purification of enzyme, the effects of different factors on enzyme activity and stability, and analysis of enzymatic hydrolysis products. These results showed that, we screened marine bacteria SJ-H-12 capable of secreting low-temperature alginate lyase on a plate with sole alginate carbon source, based on the 16S rDNA sequence, an evolutionary tree was constructed. the strain was identified as Yangia sp. SJ-H-12. furthermore, the gene encoding the enzyme ALYYA, Alyya belongs to the PL5 family alginate lyase. the gene was secreted and expressed in Yarrowia lipolytica, a food-grade host. the activity of recombinant Alyya reached 34.2 U/mL, the molecular weight was about 39.0 kDa, and it had a strong PolyM preference. Alyya showed that more than 80% vitality at 25-35℃, and the highest vitality at 30℃; it has good stability in the pH range of 5.0-10.0, with more than 60% vitality remaining after incubation; NaCl can obviously activate the activity of Alyya at a concentration of 0 to 2.0 M. The products of Alyya degrading alginate are mainly disaccharides, and a small amount of monosaccharides and trisaccharides, and this enzyme is an endoalginate lyase. In summary, a low-temperature alginate lyase-producing bacteria was screened in this study, most of the alginate lyases found are mesophilic enzymes, and Alyya studied in this paper is a typical low-temperature alginate lyase with excellent enzyme activity and stability, which provided reference data for the screening and industrial development and utilization of low-temperature alginate lyase.
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