中村芽胞杆菌壳聚糖酶在大肠杆菌中重组表达及酶学性质

Recombinant Expression and Enzymatic Characterization of Bacillus nakamurai Chitosanase in Escherichia coli

壳聚糖是一类来源丰富的大分子聚合物,在化工、食品、生物等多个领域都有广泛的应用价值,然而,只有降解成小分子的低聚糖才能更好地发挥其活性。随着酶法降解壳聚糖相较于其它方法的优势愈来愈明显,探索新型稳定高效的壳聚糖酶引起研究者的关注。参考GeneBank数据库中中村芽胞杆菌(Bacillus nakamurai)的壳聚糖酶氨基酸序列设计相应的编码基因,利用大肠杆菌原核表达系统表达和纯化重组蛋白。酶学性质研究结果显示:该酶在最适温度37℃,46℃处理30 min时仍有50%的相对酶活,最适pH值4.5。在pH 2.5条件下处理60 min时,仍可保持60%相对酶活。Ag^(+)、Hg^(+)完全抑制酶活性,Mn^(2+)、Mg^(2+)、Ca^(2+)对酶活性有促进作用,K^(+)、Na^(+)、EDTA对酶活性无明显作用。本文首次研究来源于中村芽胞杆菌的壳聚糖酶的酶学性质,以拓展该酶库资源,为该酶后续研究和应用提供理论依据。

Chitosan is a kind of macromolecular polymer with rich source,which has a wide range of application value in many fields,such as chemical industry,food,biology and so on.However,it needs to be degraded into small molecular oligosaccharides to give full play to its activity.As the advantages of enzymatic degradation of chitosan are more and more obvious compared with other methods,the exploration of new stable and efficient chitosanase has attracted the attention of the majority of researchers.According to the chitosanase's amino acid sequence of Bacillus nakamurai in genebank database,we designed the corresponding coding gene and expressed and purified the recombinant protein by E.coli prokaryotic expression system.The results of enzymatic properties showed that the optimum temperature was 37℃and the relative enzyme activity was still 50%after 30 min treatment at 46℃,and the optimum pH was 4.5,and 60%relative enzyme activity could be maintained after 60 min treatment at pH 2.5.Ag^(+)and Hg^(+)completely inhibited the enzyme activity.Mn^(2+),Mg^(2+)and Ca^(2+)promoted the enzyme activity,while K^(+),Na^(+) and EDTA had no obvious effect on the enzyme activity.In this study,the enzymatic properties of chitosanase from B.nakamurai were studied for the first time,which expanded the resources of chitosanase library and provided theoretical basis for the subsequent research and practical application of the enzyme.