College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, Guangdong, People's Republic of China.
World J Microbiol Biotechnol. 2012 Nov;28(11):3227-37. doi: 10.1007/s11274-012-1133-z. Epub 2012 Aug 28.
In order to search for valuable and extremely thermo-stable enzymes that could be used in the protein hydrolysis industry, the gene corresponding to a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2 (GtLAP) was cloned and expressed in E. coli. The recombinant enzyme was purified, and its characteristics were examined. Meanwhile, potential applications of GtLAP in the hydrolysis of anchovy proteins were also investigated. GtLAP was overexpressed in IPTG-induced E. coli BL21 (pET28a-LAP) as a soluble protein, and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 125 ± 8.75 U/mg proteins. The molecular mass of GtLAP was estimated to be 55 kDa by SDS-PAGE analysis. The optimal reaction temperature and pH of GtLAP were 70 °C and 8.0, respectively. Under optimal conditions, GtLAP showed a marked preference for Leu-p-nitroanilide, followed by Met- and Phe-derivatives. Activity of GtLAP was strongly stimulated by Ni²⁺ ions, but was strongly inhibited by Hg²⁺. Conformational studies via circular dichroism spectroscopy indicated that various factors could influence the secondary structure of GtLAP to various extents and further induce changes in enzymatic activity. Results of hydrolytic experiment showed that combining GtLAP with endogenous enzymes could significantly increase the degree of hydrolysis to anchovy proteins and concentrations of free amino acids in hydrolysates. In this regard, GtLAP could potentially be used in the protein hydrolysis industry.
为了寻找可用于蛋白质水解工业的有价值且极耐热的酶，本研究克隆并在大肠杆菌中表达了来自 Geobacillus thermodenitrificans NG80-2 (GtLAP) 的亮氨酸氨肽酶基因。对重组酶进行了纯化，并研究了其特性。同时，还研究了 GtLAP 在凤尾鱼蛋白水解中的潜在应用。在 IPTG 诱导的大肠杆菌 BL21 (pET28a-LAP) 中过表达 GtLAP，作为可溶性蛋白，并通过镍螯合层析进行纯化为比活 125 ± 8.75 U/mg 蛋白。SDS-PAGE 分析估计 GtLAP 的分子量为 55 kDa。GtLAP 的最佳反应温度和 pH 分别为 70°C 和 8.0。在最佳条件下，GtLAP 对 Leu-p-硝基苯胺表现出明显的偏好，其次是 Met-和 Phe 衍生物。Ni²⁺ 离子强烈刺激 GtLAP 的活性，但 Hg²⁺ 强烈抑制其活性。通过圆二色性光谱学进行的构象研究表明，各种因素可以不同程度地影响 GtLAP 的二级结构，并进一步导致酶活性的变化。水解实验结果表明，将 GtLAP 与内源性酶结合使用可以显著提高对凤尾鱼蛋白的水解程度和水解产物中游离氨基酸的浓度。在这方面，GtLAP 可能在蛋白质水解工业中得到应用。
