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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
张文彬1,2, 白露1,2, 刘彬3,4, 王朋梅2, 莫照兰5, 李杰2
1.上海海洋大学 水产科学国家级实验教学示范中心 国家水生动物病原库 上海 201306;2.中国水产科学研究院黄海水产研究所 农业农村部海水养殖病害防治重点实验室 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 山东 青岛 266071;3.青岛市精神卫生中心 山东 青岛 266034;4. 中国海洋大学海洋生命学院 山东 青岛 266003;5.中国海洋大学海洋生命学院 山东 青岛 266003
摘要:
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(Yarrowia lipolytica)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℃时表现出最高活力;在pH为5.0~10.0的范围内稳定性较好,孵育后剩余超过60%的活力;0~2.0 mol/L的NaCl能明显激活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,所产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。本研究为低温褐藻胶裂解酶的筛选和开发利用提供了参考数据。
关键词:  褐藻胶裂解酶  分泌表达  编码基因  pH稳定性
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Secretory expression and enzymatic properties of low-temperature-adapted alginate lyase from marine bacteria
ZHANG Wenbin,BAI Lu,LIU Bin,WANG Pengmei,MO Zhaolan,LI Jie
1.Shanghai Ocean University, National Demonstration Center for Experimental Fisheries Science Education, National Pathogen Collection Center for Aquatic Animals, Shanghai 201306, China;2.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao, Shandong 266071, China;3.Qingdao Mental Health Center, Qingdao, Shandong 266034, China;4.College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong 266003, China
Abstract:
Low-temperature-adapted alginate lyase has unique advantages for alginate oligosaccharide preparation and brown algae processing. Producing a low-temperature-adapted alginate lyase with better stability remains an urgent need for industrial applications. In this study, to screen for a low-temperature- adapted alginate lyase with good stability, we identified and isolated marine bacteria, by cloning and analyzing enzyme-encoding gene and secretory expression and purification of enzyme. The effects of different factors on enzyme activity and stability, as well as analysis of enzymatic hydrolysis products were evaluated. Results showed that the marine bacterium SJ-H-12 is capable of secreting low- temperature-adapted alginate lyase when cultivated with alginate as the sole carbon source. An evolutionary tree was constructed based on 16S rDNA sequences, and the strain was identified as Yangia sp. SJ-H-12. Furthermore, the gene encoding the enzyme ALYYA was identified as belonging to the PL5 alginate lyase family. 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 of ALYYA was about 39.0 kDa, and it had a strong PolyM preference. ALYYA showed more than 80% vitality at 25℃~35℃, with peak vitality at 30℃; it demonstrated good stability in the pH range of 5.0~10.0, with more than 60% vitality remaining after incubation. NaCl could activate the activity of ALYYA at a concentration of 0~2.0 mol/L. The products of ALYYA-degraded alginate are mainly disaccharides and small amounts of monosaccharides and trisaccharides, resulting from its endoalginate lyase activity. In summary, a low-temperature-adapted alginate lyase-producing bacterium was screened, and most of the alginate lyases found were mesophilic enzymes. This study showed that ALYYA is a typical low-temperature- adapted alginate lyase with excellent enzyme activity and stability, providing reference data for the screening and industrial development and utilization of low-temperature-adapted alginate lyase.
Key words:  Alginate lyase  Secretory expression  Coding gene  pH stability