芽孢杆菌N1碱性蛋白酶的克隆表达及酶学性质

Cloning, expression and characterization of an alkaline protease from Bacillus sp. N1

摘要:

【背景】蛋白酶广泛应用于制革行业中，酶法脱毛对环境污染较小，但蛋白酶对化学试剂的不稳定性及胶原降解活性限制了其工业应用。【目的】克隆芽孢杆菌(Bacillus sp.) N1基因组的碱性蛋白酶基因，实现其在大肠杆菌中的异源表达，并对重组酶酶学性质及脱毛作用进行研究。【方法】利用基因组文库法克隆获得蛋白酶基因aprG，构建重组大肠杆菌(Escherichia coli) BL21(DE3) pLysS/pET-28a-aprG。异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达该重组酶，以福林酚显色法对其酶学性质进行研究，并将AprG作用于羊皮、兔皮和羽毛。【结果】克隆得到蛋白酶基因aprG，并实现其在大肠杆菌中的表达。重组酶AprG最适反应温度为50 °C，最适反应pH为10.0。各种金属离子对AprG活性影响较小，且AprG对表面活性剂和氧化剂、还原剂的耐受性较强。底物特异性分析表明，该酶胶原活性较低。AprG对羊皮和兔皮作用显著，且降解羽毛效果明显。【结论】蛋白酶AprG在制革行业中具有良好的应用前景。

Abstract:

[Background] Proteases are widely used in the leather industry because the enzymatic dehairing approaches are environment-friendly. However, the instability to chemical reagents and the collagen degradation activity of proteases limit their industrial applications. [Objective] An alkaline protease gene from genome of Bacillus sp. N1 was cloned and expressed in Escherichia coli. The enzymatic properties of the recombinant protease were characterized and its application in dehairing process was discussed. [Methods] Genomic library was constructed to clone the protease gene aprG. The recombinant strain Escherichia coli BL21(DE3) pLysS/pET-28a-aprG was constructed for the protease expression with the induction of isopropyl β-D-thiogalactoside (IPTG). Characterization of the protease was performed through the forint phenol chromogenic method. The dehairing activity to the sheep and rabbit skin as well as feather of the protease was investigated. [Results] An alkaline protease gene aprG was cloned and expressed in E. coli expression system. The characterization of AprG showed that the optimum temperature and pH was 50 °C and 10.0, respectively. The metal ions showed soft influence on the AprG activity. Moreover, the protease AprG exhibited outstanding tolerance to surfactants, oxidants and reducing agents. The investigation on substrate specificity demonstrated that the protease displayed low hydrolysis ability toward collagen. The application study showed that AprG demonstrated effective dehairing on sheep and rabbit skin, and also showed high feather degradation activity. [Conclusion] The protease AprG has significant application potential in the leather industry.