State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People's Republic of China.
Appl Biochem Biotechnol. 2012 Dec;168(7):1820-33. doi: 10.1007/s12010-012-9899-x. Epub 2012 Sep 28.
A 1,080-bp cDNA (CGMCC 2873) encoding of a cold-active lipase of Aspergillus fumigatus (AFL67) was cloned and expressed in Escherichia coli for the first time. The new lipase, AFL67, was one-step purified by 8.30 folds through Ni-NTA affinity chromatography with a recovery of 86.8 %. The specific activity of purified AFL67 was 449 U mg(-1) on p-NP hexanoate. AFL67 preferentially hydrolyzed p-nitrophenyl esters of short- and medium-chain fatty acids, with p-nitrophenyl hexanoate the maximum. The optimum temperature and pH was 15 °C and 7.5, respectively. The purified AFL67 was stable at 10-25 °C for 30 min, and in the pH range of 6.0-9.0 for 16 h (at 4 °C). Its activity was increased by 47 and 50 %, in the presence of 10 % (v/v) ethanol and isopropanol, respectively. The new lipase AFL67 highly enantioselectively deacylated (S)-α-acetoxyphenylacetic acid (APA) and o-Cl-APA, m-Cl-APA, and p-Cl-APA to (S)-mandelic acid and its derivates. These features render this cold-active novel lipase AFL67 attractive for biotechnological applications in the field of enantioselective synthesis of chiral mandelic acids, o-acylated mandelic acids, and their derivates and detergent additives.
首次在大肠杆菌中克隆并表达了编码烟曲霉（AFL67）冷活性脂肪酶的 1080bp cDNA（CGMCC 2873）。新脂肪酶 AFL67 通过 Ni-NTA 亲和层析一步纯化 8.30 倍，回收率为 86.8%。纯化的 AFL67 在 p-NP 己酸酯上的比活为 449 U·mg-1。AFL67 优先水解短链和中链脂肪酸的 p-硝基苯酯，以 p-硝基苯己酸酯为最大。最适温度和 pH 分别为 15°C 和 7.5。纯化的 AFL67 在 10-25°C 下稳定 30 分钟，在 6.0-9.0 pH 范围内稳定 16 小时（4°C）。在存在 10%（v/v）乙醇和异丙醇的情况下，其活性分别提高了 47%和 50%。新脂肪酶 AFL67 对（S）-α-乙酰氧基苯乙酸（APA）和 o-Cl-APA、m-Cl-APA 和 p-Cl-APA 具有高度对映选择性脱酰基作用，生成（S）-扁桃酸及其衍生物。这些特性使这种冷活性新型脂肪酶 AFL67 在手性扁桃酸、o-酰化扁桃酸及其衍生物和洗涤剂添加剂的对映选择性合成等生物技术领域具有吸引力。
