中文摘要：

【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p EASY-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 k Da）。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^＋、Hg^（2＋）及SDS抑制,β-巯基乙醇、Pb^（2＋）、Mg^（2＋）、Ca^（2＋）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。

英文摘要：

[Objective] An endoglucanase gene （gluEl） was cloned from a thermalacidophilus （Alicyclobacillus tengchongensis CGMCC 1504） 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 （GluEI： 40.45 kDa）. GluE1 showed the highest identity of 97% with endoglucanase in public databases, and 〈60% identities with other endoglucanase. GluEI efficiently hydrolyzed CMC-Na, soluble starch and barley-13-glucan, which showed apparent optimal at pH 6.5 and 55 ℃. GluEI was stable and active （〉60%） at pH 5.0-10.0, and had a high stability at 37 ℃; and it exhibited Km, Vmax and kcat 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＋, Ca〉 and Na＋, 30% NaC1 still retains more than 64% of the activity. The residual enzyme activity kept 93% after pre-incubation of the enzyme in 30% NaC1. [Conclusion] Endoglucanase gene gluEl from Alicyclobacillus was first reported, and GluE1 showed a good pH stability and strong halo-tolerant property. GluE 1 might have greater potential applications.