硫酸软骨素ABC I酶的融合表达及传感器应用研究

采用MiSeq高通量测序技术对来凤地区盐渍藠头盐水细菌多样性进行解析，并采用PICRUSt技术对其基因功能进行预测。结果表明，99.58%的基因序列长度为425～451 bp，基本覆盖16S rRNA V3-V4区序列；优势细菌门为硬壁菌门（Firmicutes）、变形菌门（Proteobacteria）和放线菌门（Actinobacteria），平均相对含量分别为43.94%、33.12%和18.21%；优势细菌属为乳杆菌属（Lactobacillus）、假单胞菌属（Pseudomonas）和嗜热油菌属（Thermoleophilum），平均相对含量分别为30.28%、18.21%和16.13%；共有219个核心操作分类单元（OTU），包含的序列数占总序列数的75.56%；盐渍藠头盐水中可能共有大量具有较强氨基酸转运和代谢功能的细菌类群。

Fusion expression of chondroitinase ABC I and biosensor application

Chondroitinase (ChSase) is a class of enzymes that can degrade chondroitin sulfate (CS) to small molecular polysaccharides, which can be classified as ChSase ABC, ChSase AC, ChSase B and ChSase C according to the kind of substrates. In this study, the ChSase ABC I derived from Proteus vulgaris KCTC 2579 was integrated and expressed with maltose-binding protein (MBP). The recombinant vector pMAL-c2x-ChSase ABC I was constructed and successfully expressed in Escherichia coli BL21 (DE3). The maltose-binding protein-chondroitinase (MBP-ChSase) ABC I enzyme was immobilized on a polyaniline carrier to prepare a biosensor, which was detected by cyclic voltammetry. The results showed that the enzyme activity and specific activity of MBP-ChSase ABC I were 3 180 IU/L fermentation broth and 76 IU/mg protein, respectively. Using chondroitin sulfate A (CS A) as the reaction substrate of the enzyme, under the optimized conditions, the peak current of the reaction had a linear relationship with the concentration of CS A of 0.1 nmol/L-0.1 μmol/L. The biosensor reacted quickly to the substrate, with high sensitivity, and could perform effective detection.