黑曲霉（Aspergillus niger）F044脂肪酶新型基因lipB的克隆、表达及酶学性质分析

Cloning, expression and characterization of a novel lipase gene lipB from Aspergillus niger F044

摘要:

摘要：【目的】本研究拟克隆新型的黑曲霉（Aspergillus niger）脂肪酶（EC 3.1.1.3）基因，实现其在大肠杆菌（Escherichia coli）的高效表达，并对表达产物进行系统的酶学性质分析，为该脂肪酶的工业化生产及应用奠定基础。【方法】通过PCR和RT-PCR克隆脂肪酶基因，并将其开放式阅读框（ORF）克隆入融合表达载体pET28a；表达产物经Ni-agarose纯化后对LipB进行酶学性质分析，并通过圆二色谱进行结构分析。【结果】成功地从A. niger F044中克隆了一个新型的脂肪酶基因lipB，获得了该基因的全基因组序列和cDNA序列（GenBank: FJ536287、FJ536288），并实现了其在E. coli中的高效表达。LipB分子量约为43.0 kDa，最适底物为pNPC（C8），酶学动力学常数Km=5.98 mmol/L，最适反应温度为50℃，最适pH为6.0；该酶能在40℃条件下保持稳定，在60℃条件下处理1 h后残余酶活仅为18.8%；该酶对Ca2+敏感，当脂肪酶经2 mmol/L Ca2+处理1 h后，酶活提高了2.6倍。圆二色谱分析表明该酶在Ca2+处理前后具有明显的结构变化。【结论】新型A. niger脂肪酶lipB基因的克隆不仅积累了脂肪酶基因资源，而且为高效基因工程菌的构建及规模化应用奠定基础；对LipB的酶学性质分析表明该酶在食品和油酯化工等领域具有广阔的应用前景。

Abstract:

Abstract: [Objective] We cloned, expressed and characterized a novel lipase gene lipB from Aspergillus niger F044, to facilitate the large scale production and application of that enzyme. [Method] We cloned lipB gene and the cDNA sequence by PCR and RT-PCR, and then cloned the open reading frame of lipB into pET28a vector and expressed by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. After Ni-agarose purification, the characteristics were determined and the conformation change was checked by circular dichroism methods. [Results] The novel lipase genes cDNA of lipB were cloned from Aspergillus niger F044 (GenBank: FJ536287, FJ536288) and expressed in Escherichia coli. The molecular weight of LipB was about 43 kDa. The optimal substrate of this enzyme is 4-nitrophenyl octanoate (pNPC-C8) with Km=5.98 mmol/L. The optimal temperature and pH was 50oC and pH 6.0. The enzyme was stable below 40 oC. After incubated at 60 oC for 1 h, only 18.8% activity remained. After treated by 2 mmol/L Ca2+ for 1 h, the activity improved 2.6-fold. [Conclusion] Enzymatic characteristics of LipB determined showed this enzyme might have potential in industrial applications.