巨大芽孢杆菌β-淀粉酶在枯草芽孢杆菌中诱导表达及碳代谢去阻遏

Inducible heterologous expression and carbon catabolite repression of β-amylase from Bacillus megaterium in Bacillus subtilis

【背景】β-淀粉酶在食品和医疗领域应用广泛。目前工业上使用的β-淀粉酶主要从植物中提取,生产成本高,限制了β-淀粉酶的应用。微生物生产的β-淀粉酶尽管早有报道,但由于产酶水平低下,因而一直未能实现工业化。【目的】实现巨大芽孢杆菌β-淀粉酶在枯草芽孢杆菌中的高效诱导表达,缓解碳分解代谢物阻遏(Carbon catabolite repression,CCR)对该重组酶表达的影响,并研究其酶学性质。【方法】克隆枯草芽孢杆菌木糖诱导启动子,构建木糖诱导表达载体以介导巨大芽孢杆菌1514的β-淀粉酶编码基因amyM在枯草芽孢杆菌中的异源表达。定点突变位于amyM信号肽编码区的分解代谢物响应元件(Catabolite responsive element,CRE),降低碳源代谢对重组β-淀粉酶施加的阻遏。【结果】构建了诱导表达β-淀粉酶基因的重组枯草芽孢杆菌菌株。同义替换amyM-CRE保守碱基在不同程度上缓解了碳源所施加的CCR效应,重组酶的表达水平得到显著提高。重组酶的分子量为57 kD,水解可溶性淀粉主要生成麦芽糖和少量葡萄糖,其中麦芽糖含量为72%。该酶最适作用温度为50°C,最适反应pH为6.0。Co2+、Ca2+对重组β-淀粉酶具有激活作用。【结论】通过木糖诱导表达系统和碳代谢去阻遏实现了β-淀粉酶在枯草芽孢杆菌中的高效表达,酶活最高可达97.16 U/mL发酵液,比amyM基因来源菌巨大芽孢杆菌1514的β-淀粉酶产量提高了440倍,为β-淀粉酶发酵生产的工业化提供了支撑。

[Background] β-Amylases have been widely used in food and medical fields.Most industrial β-amylases are extracted from plants,hampering the application of β-amylase due to high costs.Microbial production of β-amylase has been reported before but has not been industrialized because of the low yields.[Objective] To achieve an efficient inducible expression of a β-amylase from Bacillus megaterium in Bacillus subtilis,relieve carbon catabolite repression (CCR) exerted on the expression of recombinant β-amylase and characterize the recombinant enzyme.[Methods] A xylose-induced vector was constructed to mediate the expression of the amyM gene from Bacillus megaterium 1514 encoding a β-amylase in Bacillus subtilis.CCR of the recombinant β-amylase was studied by site-directed mutagenesis of the catabolite responsive element (CRE) located within the signal peptide-encoding region of amyM.[Results] The recombinant Bacillus subtilis that inductively expressed the β-amylase was obtained.The yield of the recombinant enzyme was significantly improved by silent mutagenesis of conserved nucleotide within amyM-CRE.The recombinant β-amylase had a molecular size of 57 kD and hydrolyzed soluble starch to yield 72% maltose and a little glucose.The enzyme was optimally active at pH 6.0 and 50 ℃.Co2+ and Ca2+ increased the efficiency of enzymatic hydrolysis.[Conclusion] Highly efficient expression of β-amylase was achieved to provide experimental support for the industrial production of β-amylase from fermentation.