耐热异淀粉酶的高效表达及其在麦芽糖浆制备中的作用

Overexpression of thermotolerant isoamylase and its application in maltose syrup preparation

淀粉酶法制备高麦芽糖浆工艺中，淀粉液化酶、脱支酶以及β-淀粉酶不可或缺，其中脱支酶是决定淀粉转化为麦芽糖的转化率高低的关键因素。在工业上常用的两种脱支酶中，异淀粉酶比普鲁兰酶能更好地协助β-淀粉酶水解淀粉生成麦芽糖。首先通过PCR扩增获得异淀粉酶的编码基因iso并克隆入表达载体pHY—WZX，在枯草芽胞杆菌1A717中获得重组质粒pHY—ISO，将构建好的重组质粒电转化入地衣芽孢杆菌D402中，其摇瓶发酵酶活力达330U／mL，实现了异淀粉酶的异源高效表达。基本酶学特征分析表明：该重组酶适宜反应条件为50～55℃，pH6．5～9．0；K＋、Ca2＋、Mg2＋对酶活有促进作用，其他离子或化合物强烈抑制酶活。HPLC分析表明，该重组异淀粉酶与普鲁兰酶相比，更有助于极高麦芽糖浆的制备。最后通过在线软件对该酶进行同源结构模拟和分析，进一步确定其为异淀粉酶，为后续对其进行分子改造奠定了基础。

In enzymatic preparation of high maltose syrup from starch, eL-amylase, β-amylase and starch debranching enzyme are indispensable. Among them, debranching enzyme is a key factor which determines the conversion rate of starch to maltose syrup. Of the two commonly used debranching enzymes in the industry, isoamylasc is better than pullulanase in assisting β-amylase to hydrolyze starch into maltose. In this study, gene iso encoding isoamylase was amplified by PCR, cloned into expression vector pHY-WZX and chemically transformed into Bacillus subtilis 1A717. The recombinant plasmid was subsequently electro-transformed into B. licheniformis D402. At the shake-flask level, the activity of isoamylase in the recombinant bacterium reached its maximum of 330 U/mL, suggesting its heterologous overexpression. By investigating the biochemical properties of the enzyme, its optimal reaction conditions were determined to be 50 -55 ℃ and pH 6.5 -9.0. K ＋ , Ca2＋ and Mg2＋ could enhance the enzyme activity, while other metal ions or chemicals strongly inhibited its activity. As analyzed by HPLC, the recombinant isoamylase was better than pullulanase in preparing high-purity maltose syrup. Finally, the 3D structure of this enzyme was constructed and analyzed by online software, which further confirmed that this enzyme was an isoamylse and provided valuable information for its molecular modification in the future.