毕赤酵母表达食琼脂火色杆菌褐藻胶裂解酶  

Expression of Flammeovirga agarivorans Alginate Lyase in Pichia pastoris

作　　者：程志钱 程晓峰 涂红艳 冯青青 程功[4] CHENG Zhiqian;CHENG Xiaofeng;TU Hongyan;FENG Qingqing;CHENG Gong(Huzhou Inspection&Quarantine Comprehensive Technology Center,Huzhou 313000,China;School of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China;Hefei Center for Animal Disease Control and Prevention,Hefei 230601,China;State Key Laboratory of Biochemical Engineering,Institute of Process Engineering,Chinese Academy of Sciences,Beijing 100190,China)

机构地区：[1]湖州检验检疫综合技术服务中心,浙江湖州313000 [2]安徽农业大学动物科技学院,安徽合肥230036 [3]合肥市动物疫病预防控制中心,安徽合肥230601 [4]中国科学院过程工程研究所,生化工程国家重点实验室,北京100190

出　　处：《食品科技》2024年第6期19-25,共7页Food Science and Technology

基　　金：浙江省基础公益研究计划项目(LGN20C140001);湖州市公益性应用研究项目(2023GZ53)。

摘　　要：选择食琼脂火色杆菌(Flammeovirga agarivorans)的潜在褐藻胶裂解酶基因,对其进行序列优化及全基因合成,在毕赤酵母(Pichia pastoris)中进行分泌表达。摇瓶诱导表达产物的蛋白浓度为0.23 mg/mL。表达的褐藻胶裂解酶最适pH值为9.0,最适温度为40℃,在该条件下的酶活力达400.00 U/mL。该酶在40℃及以下时较稳定。底物特异性分析结果显示,该酶对聚甘露糖醛酸及聚古罗糖醛酸均有水解能力,但对聚古罗糖醛酸的水解能力更强。利用该酶水解褐藻酸钠并使用高效液相色谱及质谱方法对产物进行了分析,结果显示,产物主要为聚合度2~4的褐藻寡糖。研究获得的褐藻胶裂解酶可用于褐藻寡糖的酶法规模制备。Putative alginate lyase encoding gene of Flammeovirga agarivorans was optimized,synthesized and secretory expressed in Pichia pastoris.The protein concentration of the expressed product reached 0.23 mg/mL.The optimum pH and temperature of the expressed alginate lyase was 9.0 and 40℃,respectively,and enzymatic activity reached 400.00 U/mL under these reaction conditions.The enzyme was thermostable at no more than 40℃.Substrate specific analysis showed that the enzyme could hydrolyze both poly-mannuronic acid(polyM)and poly-guluronic acid(polyG),but the hydrolysis ability of polyG was stronger.Sodium alginate was hydrolyzed by this enzyme and the products were analyzed by high performance liquid chromatography and mass spectrometry.The results showed that the main products were alginate oligosaccharides with polymerization degree from 2 to 4.The alginate lyase obtained in this study can be used for the enzymatic scale preparation of alginate oligosaccharides.

关 键 词：食琼脂火色杆菌 褐藻胶裂解酶 毕赤酵母 褐藻寡糖 

分 类 号：TS201.3[轻工技术与工程—食品科学]