脂环酸芽孢杆菌D-1的耐盐内切葡聚糖酶基因克隆、表达与酶学性质

Gene cloning, heterologous expression and enzyme characterizations of halo-tolerant endoglucanase from Alicyclobacillus D-1

摘要:

[目的]克隆温泉中嗜热嗜酸的脂环酸芽孢杆菌D-1（Alicyclobacillus tengchongensis CGMCC1504）的内切葡聚糖酶基因gluE1，并对该酶进行序列分析和重组酶的酶学特性分析。[方法]通过全基因组测序获得gluE1全长，并对其氨基酸序列（GluE1）进行分析。将gluE1重组到载体pEASY-E2中并转化到大肠杆菌BL21（DE3）中异源表达，利用组氨酸标签纯化GluE1并进行酶学性质分析。[结果]gluE1与NCBI数据库中GH5的内切葡聚糖酶具有较高的相似性，全长1020 bp，GC含量50.5%，编码339个氨基酸（40.45 kDa）。GluE1与数据库中序列的最高一致性为97%，与其余纤维素酶的一致性<60%。GluE1可水解CMC-Na、可溶性淀粉和大麦β-葡聚糖，表观最适pH为6.5，pH 5.0-10.0稳定并维持60%以上的酶活性。GluE1的表观最适温度为55℃，在37℃下稳定。在55℃ pH 6.5条件下，GluE1对大麦β-葡聚糖的K_m、V_max和k_cat分别为8.58 mg/mL、416.67 U/mg和280.90 s^-1。GluE1受Ag⁺、Hg²⁺及SDS抑制，β-巯基乙醇、Pb²⁺、Mg²⁺、Ca²⁺和Na⁺对GluE1有微弱的促进作用，NaCl对GluE1的影响不大，加入30%的NaCl，仍有64%以上的酶活性；经30%的NaCl在37℃下处理60 min，仍能保持93%以上的活性。[结论]首次报道从Alicyclobacillus属的细菌中克隆得到内切葡聚糖酶基因并对其酶学性质进行研究，GluE1具有良好的pH稳定性和有较强的耐盐性，可能具有更大应用潜力。

Abstract:

[Objective] An endoglucanase gene (gluE1) was cloned from a thermalacidophilus (Alicyclobacillus tengchongensis CGMCC1504) isolated from a hot spring, and the sequence and biochemical characterization of enzyme were analyzed. [Methods] The full-length gluE1 was obtained based on genome sequencing, analysis of amino acid sequence of GluE1. gluE1 was ligated into pEASY-E2 vector and expressed in Escherichia coli BL21 (DE3) cells. GluE1 was purified to electrophoretic homogeneity by Ni²⁺-NTA metal chelating affinity chromatography, and then the enzyme characterizations were determined. [Results] The 1020 bp full-length gluE1 (50.5% GC content) encodes a 339 residues polypeptide (GluE1: 40.45 kDa). GluE1 showed the highest identity of 97% with endoglucanase in public databases, and <60% identities with other endoglucanase. GluE1 efficiently hydrolyzed CMC-Na, soluble starch and barley-β-glucan, which showed apparent optimal at pH 6.5 and 55℃. GluE1 was stable and active (>60%) at pH 5.0-10.0, and had a high stability at 37℃; and it exhibited K_m, V_max and k_cat values of 8.58 mg/mL, 416.67 U/mg and 280.90 s^-1 respectively. GluE1 was strongly inhibited by Ag⁺, Hg²⁺ and SDS, partial promoted by β-Mercaptoethanol, Pb²⁺, Mg²⁺, Ca²⁺ and Na⁺, 30% NaCl still retains more than 64% of the activity. The residual enzyme activity kept 93% after pre-incubation of the enzyme in 30% NaCl. [Conclusion] Endoglucanase gene gluE1 from Alicyclobacillus was first reported, and GluE1 showed a good pH stability and strong halo-tolerant property. GluE1 might have greater potential applications.