一株多糖降解菌的分离、鉴定与琼脂糖降解能力

Isolation，identification and agarose degradation of a polysaccharide-degrading marine bacterium Persicobacter sp. JZB09

摘要:

摘要:【目的】筛选海洋来源的多糖降解菌，分析其多糖降解能力并初探机制。【方法】碘液染色法从海泥中初筛琼脂糖降解菌，唯一碳源生长法分析菌株的多糖利用能力，克隆16S rRNA 基因以分析系统分类地位。用硫酸铵沉淀法制备胞外粗酶制剂，DNS-还原糖法测定琼胶酶活性，活性染色法分析胞外琼胶酶系的组成特征。分离、纯化琼脂糖的酶解产物，通过TLC 测定寡糖Rf 值、阳离子质谱测定分子量。【结果】分离到1株能液化琼脂糖的海洋细菌JZB09，鉴定至桃色杆菌属(Persicobacter)。JZB09 能利用11 种不同的多糖为唯一碳源生长，在利用琼脂糖、纤维素和木聚糖时生长较好。胞外粗酶制剂的琼胶酶活力约77.2U/mg，含有至少2 条琼胶酶，大小约45kDa、70kDa。酶制剂降解琼脂糖后的产物是系列新琼寡糖，四糖是主产物，表明β-琼胶酶在胞外琼胶酶系降解琼脂糖时起关键作用。【结论】海洋细菌Persicobacter sp．JZB09 是1 株多能型多糖降解菌，可分泌β-琼胶酶降解琼脂糖且活性显著，具有潜在开发价值。

Abstract:

Abstract:［Objective］To isolate and identify a versatile carbohydrate-degrading bacterium from marine environments，and characterize the extracellular agarase activity.［Methods］The I2 staining method was applied in the isolation of agarosedegrading bacteria from coastal sediments of the Jiaozhou bay nearby Qingdao city，China． The JZB09 strain was cultured in multiple media using various complex polysaccharides as the sole carbon source to test the carbohydrate utilizing abilities． The 16S rRNA gene was cloned，sequenced and analyzed to identify the taxonomic position of the strain． Crude extracellular proteins were prepared using (NH4) 2 SO4 precepitation method． The dialyzed enzyme extract was applied in further studies including activity testing，activity staining，and agarose degrading for oligosaccharides purifiction． Three purified oligosaccharides were individually analyzed using thin layer chromatograph (TLC) and MALDI-TOF MS method．［Results］ The agarolytic marine bacterium，Persicobacter sp． JZB09，could use multiple complex polysaccharides as the sole carbon source and grew well on agarose，cellulose and xylan． The extracellular enzyme extract exhibits efficient and extensive degradation activity on agarose with an activity of 77.2 U/mg proteins． The extracellular agarase system (EAS) in the crude extracellular enzymes contains at least two agarose depolymerases with molecular masses of approximately 45 kDa and 70 kDa，respectively． A series of degradation products from agarose by the EAS was purified and identified as neoagaro-oligosaccharides，among which neoagarotetraose was the major product of the crude enzymatic products，which suggests that β-agarase is the major constituent of the JZB09 EAS．［Conclusion］The polysaccharide-degrading bacterium Persicobacter sp．JZB09 and its polysaccharide-degrading system is promising for the exploration of polysaccharide depolymerase resources including β-agarases．